Anti-CTLA-4 antibodies and uses thereof

Abstract

The present disclosure provides anti-CTLA-4 antibodies and methods of producing and using the antibodies. The present disclosure also provides nucleic acids encoding an anti-CTLA-4 antibody and host cells containing the nucleic acids. In addition, the present disclosure provides polypeptides containing variant Fc regions and methods of producing and using the polypeptides.

Description

[0001] This application is a divisional application of the patent application with international application number PCT / JP2019 / 051447, international application date December 27, 2019, Chinese application number 201980103496.6, and invention name “Anti-CTLA-4 antibodies and their uses”. Technical Field

[0002] The present invention relates to anti-CTLA-4 antibodies and methods of using the same. Background Art

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

[0004] CTLA-4 is a glycoprotein belonging to the immunoglobulin superfamily. Its gene was cloned in 1987 from a cDNA library of a mouse-derived killer T cell clone (see, for example, NPL 2). CTLA-4 is known to suppress T cell immune responses. In 1996, it was reported that tumor regression effects were observed by administering anti-CTLA-4 antibodies to cancer-bearing mice, based on the idea that inhibiting CTLA-4 function promotes T cell activation and leads to cancer regression (see, for example, NPL 3). Evaluation of the efficacy of anti-CTLA-4 antibodies in humans has been ongoing since 2000, and in 2011, the anti-human CTLA-4 monoclonal antibody (ipilimumab) was approved by the US Food and Drug Administration (FDA) as the world's first immunostimulatory therapeutic antibody. In addition to ipilimumab, many anti-CTLA-4 monoclonal antibodies have been produced (see, for example, PTLs 1, 2, 3, and 4), and their development as pharmaceuticals is ongoing. Drugs that inhibit immune checkpoints to abolish immunosuppressive mechanisms and thus enhance immune responsiveness are called immune checkpoint inhibitors.

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

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

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

[0008] The cytotoxic effector functions of IgG antibodies, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP), have attracted attention as promising means of achieving anti-tumor effects through antibodies (see, for example, National Publication Nos. 7 and 8). These effector functions are induced by the binding of the Fc region of IgG antibodies to antibody receptors (FcγRs) or various complement components present on the surfaces of effector cells, such as natural killer cells and macrophages. Numerous studies have been conducted on Fc region variants, and variants have been obtained that exhibit various properties, such as FcγR binding activity, that are higher than those of the wild-type variant (see, for example, Patent Documents 5 and 6, and National Publication Nos. 9 and 10). Furthermore, it has been reported that the Fc region of an antibody binds to FcγRs in a 1:1 ratio and recognizes FcγRs asymmetrically at the lower hinge and CH2 regions (see, for example, National Publication No. 11). Based on this, methods for optimizing the interaction with FcγR by introducing different changes into two polypeptide chains constituting the Fc region of an antibody and generating asymmetric Fc region variants have also been reported (see, for example, PTLs 7, 8, 9, and 10).

[0009] It is hoped that when therapeutic antibodies are administered to living organisms, their target antigens are specifically expressed only at the site of injury. However, in many cases, the same antigen is also expressed in non-lesioned areas and normal tissues, and it may cause undesirable side effects in terms of treatment. For example, although antibodies against tumor antigens can exert cytotoxic activity against tumor cells through ADCC, when the same antigen is also expressed in normal tissues, the antibody may also damage normal cells. To address the above problems, technologies have been developed that focus on the phenomenon that a large amount of a specific compound is present in the target tissue (e.g., tumor tissue) and produces antigen-binding molecules whose antigen-binding activity changes according to the concentration of the compound (see, for example, PTL 11).

[0010] [Citation List]

[0011] [Patent Document]

[0012] [PTL 1] WO 2000 / 037504

[0013] [PTL 2] WO 2001 / 014424

[0014] [PTL 3] WO 2012 / 120125

[0015] [PTL 4] WO 2016 / 196237

[0016] [PTL 5] WO 2000 / 042072

[0017] [PTL 6] WO 2006 / 019447

[0018] [PTL 7] WO 2012 / 058768

[0019] [PTL 8] WO 2012 / 125850

[0020] [PTL 9] WO 2013 / 002362

[0021] [PTL 10] WO 2014 / 104165

[0022] [PTL 11] WO 2013 / 180200

[0023] [Non Patent Literature]

[0024] [NPL 1] Pardoll, Nat Rev Cancer (2012) 12: 252-264

[0025] [NPL 2] Brunet et al., Nature (1987) 328: 267-270

[0026] [NPL 3] Leach et al., Science (1996) 271: 1734-1736

[0027] [NPL 4] Sakaguchi et al., J Immunol (1995) 155: 1151-1164

[0028] [NPL 5] Takahashi et al., J Exp Med (2000) 192: 303-310

[0029] [NPL 6] Nishikawa & Sakaguchi, Int J Cancer (2010) 127: 759-767

[0030] [NPL 7] Clynes et al., Proc Natl Acad Sci U S A (1998) 95: 652-656

[0031] [NPL 8] Clynes et al., Nat Med (2000) 6: 443-446

[0032] [NPL 9] Lazar et al., Proc Natl Acad Sci USA (2006) 103: 4005-4010

[0033] [NPL 10] Chu et al., Mol Immunol (2008) 45: 3926-3933

[0034] [NPL 11] Radaev et al., J Biol Chem (2001) 276: 16469-16477 Summary of the Invention

[0035] [Technical Issues]

[0036] The present invention provides anti-CTLA-4 antibodies and methods of using the same. The present invention also provides polypeptides comprising variant Fc regions and methods of producing the same.

[0037] [Problem Solution]

[0038] More specifically, the present invention provides the following [1] to

[47] .

[0039] [1] An anti-CTLA-4 antibody having CTLA-4 binding activity that is dependent on the concentration of an adenosine-containing compound, wherein the antibody has at least one characteristic selected from the group consisting of (a) to (i):

[0040] (a) The binding activity in the presence of 100 μM adenosine-containing compounds is two or more times greater than that in the absence of adenosine-containing compounds;

[0041] (b) The KD value in the presence of 100 μM adenosine-containing compounds is 5 x 10 -7 M or lower;

[0042] (c) The KD value in the absence of adenosine-containing compounds is 1×10 -6 M or higher;

[0043] (d) forming a ternary complex with adenosine-containing compounds and CTLA-4;

[0044] (e) a region that binds to amino acid 97 to amino acid 106 of human CTLA-4 (extracellular domain, SEQ ID NO: 28);

[0045] (f) competes with ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11) for binding to CTLA-4;

[0046] (g) binds to the same epitope as that bound by ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11);

[0047] (h) exhibit cytotoxic activity against cells expressing CTLA-4; and

[0048] (i) Binds to human and mouse derived CTLA-4.

[0049] [2] The antibody of [1], wherein the antibody is a monoclonal antibody.

[0050] [3] The antibody of [1] or [2], wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0051] [4] The antibody of any one of [1] to [3], wherein the antibody is an antibody fragment that binds to CTLA-4.

[0052] [5] The antibody of any one of [1] to [4], wherein the antibody comprises: (a) HVR-H1 (SEQ ID NO: 223) comprising the amino acid sequence of SX1TMN, wherein X1 is H, A, R, or K; (b) HVR-H2 (SEQ ID NO: 224) comprising the amino acid sequence of SISX1X2SX3YIYYAX4SVX5G, wherein X1 is S or T, X2 is R or Q, X3 is G or H, X4 is D, E or R, and X5 is K or R; and (c) HVR-H3 (SEQ ID NO: 225) comprising the amino acid sequence of YGX1REDMLWVFDY, wherein X1 is K or A.

[0053] [6] The antibody of [5], further comprising: (a) HVR-L1 (SEQ ID NO: 226) comprising the amino acid sequence of X1GX2STX3VGDYX4X5VX6, wherein X1 is T, D, Q or E, X2 is T or P, X3 is D or G, X4 is N or T, X5 is Y or W, and X6 is S or H; (b) HVR-L2 (SEQ ID NO: 227) comprising the amino acid sequence of X1TX2X3KPX4, wherein X1 is E, F or Y, X2 is S or I, X3 is K or S, and X4 is S, E, or K; and (c) HVR-L3 (SEQ ID NO: 228) comprising the amino acid sequence of X1TYAAPLGPX2, wherein X1 is S or Q and X2 is M or T.

[0054] [7][5] The antibody further comprises: a heavy chain variable domain FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 229 to 232; FR2 comprising the amino acid sequence of SEQ ID NO: 233; FR3 comprising the amino acid sequence of SEQ ID NO: 234; and FR4 comprising the amino acid sequence of SEQ ID NO: 235.

[0055] [8][6] The antibody further comprises: a light chain variable domain FR1 comprising an amino acid sequence of any one of SEQ ID NOs: 236 to 238; FR2 comprising an amino acid sequence of any one of SEQ ID NOs: 240 and 241; FR3 comprising an amino acid sequence of any one of SEQ ID NOs: 242 to 244; and FR4 comprising an amino acid sequence of any one of SEQ ID NOs: 245 and 246.

[0056] [9] The antibody of any one of [1] to [4], comprising: (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 83 to 86, 98 and 135 to 141; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 88 to 95, 97, 99, 134 and 144 to 149; or (c) a VH sequence having the amino acid sequence of any one of SEQ ID NOs: 83 to 86, 98 and 135 to 141 and a VL sequence having the amino acid sequence of any one of SEQ ID NOs: 88 to 95, 97, 99, 134 and 144 to 149.

[0057]

[10] The antibody of any one of [1] to [3] and [5] to [9], which is a full-length IgG1 antibody.

[0058]

[11] The antibody of

[10] , wherein the Fc region is a variant Fc region comprising amino acid changes, and wherein the variant Fc region has enhanced binding activity to at least one Fcγ receptor selected from FcγRIa, FcγRIIa, FcγRIIb and FcγRIIIa compared to a native Fc region.

[0059]

[12] An isolated nucleic acid encoding the antibody of any one of [1] to

[11] .

[0060]

[13] A host cell comprising the nucleic acid of

[12] .

[0061]

[14] A method for producing an antibody, wherein the method comprises culturing the host cell of

[13] so as to produce the antibody.

[0062]

[15] A pharmaceutical preparation comprising the antibody of any one of [1] to

[11] and a pharmaceutically acceptable carrier.

[0063]

[16] The pharmaceutical preparation of

[15] , wherein the antibody is an immunoconjugate.

[0064]

[17] The pharmaceutical preparation of

[15] or

[16] , wherein the pharmaceutical preparation is used in combination with at least one selected from an immune checkpoint inhibitor, an EGFR inhibitor, a HER2 inhibitor, and a chemotherapeutic agent.

[0065]

[18] The pharmaceutical preparation of any one of

[15] to

[17] , wherein the pharmaceutical preparation is used for treating a tumor.

[0066]

[19] The pharmaceutical preparation of

[18] , wherein the tumor is a solid tumor into which regulatory T (Treg) cells have infiltrated.

[0067]

[20] The pharmaceutical preparation of any one of

[15] to

[17] , wherein the pharmaceutical preparation is used to damage cells.

[0068]

[21] The pharmaceutical preparation of any one of

[15] to

[17] , wherein the pharmaceutical preparation is used to damage Treg cells.

[0069]

[22] The pharmaceutical preparation of

[20] , wherein the damage to the cells is due to ADCC activity, CDC activity or ADCP activity.

[0070]

[23] The pharmaceutical preparation of

[20] or

[21] , wherein immunity is activated by damaging Treg cells.

[0071]

[24] The pharmaceutical preparation of any one of

[15] to

[17] , wherein the pharmaceutical preparation is used for activating immunity.

[0072]

[25] The pharmaceutical preparation of

[24] , wherein the immune activation is the activation of T cells.

[0073]

[26] The pharmaceutical preparation of

[24] or

[25] , wherein immunity in tumor tissue is activated.

[0074]

[27] The pharmaceutical formulation of any one of

[24] to

[26] , wherein the level of immune activation in non-tumor tissue is lower compared to a pharmaceutical formulation comprising a control anti-CTLA-4 antibody.

[0075]

[28] The pharmaceutical formulation of any one of

[24] to

[27] , wherein the level of side effects is lower compared to a pharmaceutical formulation comprising a control anti-CTLA-4 antibody.

[0076]

[29] The pharmaceutical formulation of any one of

[24] to

[28] , wherein the control anti-CTLA-4 antibody is an anti-CTLA-4 antibody that does not have CTLA-4 binding activity dependent on the concentration of the adenosine-containing compound.

[0077]

[30] The pharmaceutical formulation of

[29] , wherein the side effect is an autoimmune disease.

[0078]

[31] The pharmaceutical formulation of any one of

[18] ,

[19] , and

[26] to

[30] , wherein the tumor is a breast cancer or a liver cancer.

[0079]

[32] A polypeptide comprising a variant Fc region comprising amino acid alterations in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises amino acid alterations in the following positions:

[0080] (i) position 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 in the first polypeptide of the parent Fc region according to EU numbering; and

[0081] (ii) position 236, 250, 270, 298, 307, 326, and 334 in the second polypeptide of the parent Fc region according to EU numbering.

[0082]

[33] The polypeptide of

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

[0083]

[34] The polypeptide of

[32] or

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

[0084]

[35] The polypeptide of any one of

[32] to

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

[0085]

[36] The polypeptide of any one of

[32] to

[35] , wherein the variant Fc region further comprises an amino acid alteration at position 356 in the first polypeptide of the parent Fc region according to EU numbering.

[0086]

[37] The polypeptide of any one of

[32] to

[36] , wherein the variant Fc region further comprises an amino acid alteration at position 366 in the first polypeptide of the parent Fc region according to EU numbering.

[0087]

[38] The polypeptide of any one of

[32] to

[37] , wherein the variant Fc region further comprises an amino acid alteration at position 439 according to EU numbering in the second polypeptide of the parent Fc region.

[0088]

[39] The polypeptide of any one of

[32] to

[38] , wherein the variant Fc region further comprises amino acid alterations at positions 366, 368 and 407 according to EU numbering in the second polypeptide of the parent Fc region.

[0089]

[40] The polypeptide of any one of

[32] to

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

[0090] (i) Tyr or 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, Asp at position 326, Glu at position 332, Cys at position 349, Lys at position 356, Trp at position 366 in the first polypeptide of the parent Fc region according to EU numbering; and

[0091] (ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met or Lys at position 330, Asp or Glu at position 332, Glu at position 334, Cys at position 356, Ser at position 366, Ala at position 368, Val at position 407, Glu at position 439 according to EU numbering in the second polypeptide of the parent Fc region.

[0092]

[41] The polypeptide of any one of

[32] to

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

[0093] (a) Ala at position 434 according to EU numbering;

[0094] (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to EU numbering;

[0095] (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to EU numbering; and

[0096] (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, according to EU numbering.

[0097]

[42] The polypeptide of any one of

[32] to

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

[0098]

[43] The polypeptide of

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

[0099]

[44] The polypeptide of any one of

[32] to

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

[0100]

[45] The polypeptide of

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

[0101]

[46] The polypeptide of any one of

[32] to

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

[0102]

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

[0103] (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 according to EU numbering in the first polypeptide of the parent Fc region; and

[0104] (ii) positions 236, 250, 270, 298, 307, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1The binding activity of the anti-CTLA-4 antibody ABAM004 to CTLA-4 is shown, and the binding activity depends on the concentration of ATP, ADP, or AMP as described in Examples 1-9.

[0106] Figure 2 The binding activity of the anti-CTLA-4 antibody ABAM004 to cells expressing CTLA-4 is shown, and the binding activity is dependent on the AMP concentration described in Examples 1-10.

[0107] Figure 3 ADCC activity of the anti-CTLA-4 antibody ABAM004 against cells expressing CTLA-4 in the presence and absence of AMP as described in Examples 1-11 is shown.

[0108] Figure 4 The binding pattern of the ABAM004 Fab fragment to AMP is shown, as described in Examples 2-13. In the figure, the heavy chain of the antibody is shown in black, the light chain is shown in gray, and AMP is shown as a ball-and-stick model. The amino acid residues that interact with AMP are shown as stick models. The dashed lines and their values ​​represent the distance (Å) between each amino acid residue and AMP.

[0109] Figure 5 The binding pattern of the ABAM004 Fab fragment and AMP to human CTLA4 (hCTLA4) is shown, as described in Examples 2-14. In the figure, the heavy chain of the antibody is shown in black, the light chain is shown in gray, hCTLA4 is shown in white, and the AMP is shown in ball-and-stick models. Amino acid residues of hCTLA4 containing one or more non-hydrogen atoms located within 4.2 Å of any part of the antibody or AMP are considered epitopes and are shown in stick models.

[0110] Figure 6 Mapping of the epitope of the ABAM004 Fab fragment within the hCTLA4 amino acid sequence as described in Example 2-14 is shown. In the figure, the amino acid residues shown in black are those of hCTLA4 that contain one or more non-hydrogen atoms that are located within 4.2 Å of any portion of ABAM004 or AMP in the crystal structure. The amino acid residues shown in gray indicate residues for which the model was not constructed because they are disordered in the crystal structure.

[0111] Figure 7is a superimposition of the antibody and AMP structure extracted from the crystal structure of the ABAM004 Fab fragment alone, the complex of ABAM004 Fab fragment and AMP, and the ternary complex of ABAM004 Fab fragment, AMP and CTLA4, as described in Examples 2-15. In the figure, the heavy chain of the antibody is represented in black, the light chain in grey, and the AMP as a stick model. The structure of the ABAM004 Fab fragment alone is represented with thin lines, the structure of the binary complex with AMP with medium thick lines, and the structure of the ternary complex with thick lines.

[0112] Figure 8 The binding activity of the anti-CTLA-4 antibody ABAM004 and its variant 04H0150 / 04L0072 to CTLA-4 is shown, which is dependent on the concentration of ATP, ADP or AMP described in Example 3-2. As shown, WT and H150L072 represent ABAM004 and 04H0150 / 04L0072, respectively.

[0113] Figure 9 The neutralizing activity of the anti-CTLA-4 antibody SW1077 to CTLA-4 is shown, which is dependent on the concentration of ATP described in Example 3-6.

[0114] Figure 10 The anti-tumor effect of the anti-CTLA-4 antibody mNS-mFa55 (control antibody) in a mouse model grafted with the FM3A cell line is shown, as described in Example 3-7-4. The antibody was administered at 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 by tail vein. Each point represents the average tumor volume of a group, n=4.

[0115] Figure 11 The anti-tumor effect of the anti-CTLA-4 antibody SW1208-mFa55 (switch antibody) in a mouse model grafted with the FM3A cell line is shown, as described in Example 3-7-4. The antibody was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg and 500 mg / kg by tail vein. Each point represents the average tumor volume of a group, n=4.

[0116] Figure 12The figure shows the changes in the proportion of effector Treg cells in the tumor when the anti-CTLA-4 antibody mNS-mFa55 (control antibody) or SW1208-mFa55 (switch antibody) was administered in a mouse model transplanted with FM3A cells, as described in Example 3-7-7. mNS-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 100 mg / kg, and SW1208-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. Tumors were collected six days after administration, and the increase or decrease in effector Tregs was evaluated by FACS analysis. The vertical axis is effector Tregs (CD4 + FoxP3 + KLRG1 + ) and CD45 + The ratio of cells is shown. The mean values ​​of n = 3 are shown.

[0117] Figure 13 The present invention shows changes in the proportion of activated helper T cells in the spleen when the anti-CTLA-4 antibody mNS-mFa55 (control antibody) or SW1208-mFa55 (switch antibody) is administered to a mouse model transplanted with the FM3A cell line, as described in Example 3-7-8. mNS-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 100 mg / kg, and SW1208-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. Spleens were collected six days after administration, and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis is the number of activated helper T cells (CD4 + Foxp3 - ICOS + ) and CD45 + The ratio of cells is shown. The mean values ​​of n = 3 are shown.

[0118] Figure 14 The anti-CTLA-4 antibody SW1389-mFa55 (switch antibody) demonstrates its anti-tumor effect in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 4-3-5. The antibody was administered via the tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 100 mg / kg. Each point represents the mean tumor volume for one group, n = 4.

[0119] Figure 15The anti-CTLA-4 antibody hNS-mFa55 (control antibody) demonstrates its anti-tumor effect in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 4-3-5. The antibody was administered via the tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 30 mg / kg. Each point represents the mean tumor volume for one group, n = 4.

[0120] Figure 16 The figure shows the changes in the proportion of effector Treg cells in the tumor when the anti-CTLA-4 antibody hNS-mFa55 (control antibody) or SW1389-mFa55 (switch antibody) was administered in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 4-3-8. hNS-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 30 mg / kg, and SW1389-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg via the tail vein. Tumors were harvested six days after administration, and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis is effector Tregs (CD4 + FoxP3 + CCR7 low KLRG1 + ) and CD45 + The ratio of cells is shown. The mean values ​​of n = 3 are shown.

[0121] Figure 17 The figure shows changes in the proportion of activated helper T cells in the spleen when the anti-CTLA-4 antibody hNS-mFa55 (control antibody) or SW1389-mFa55 (switch antibody) was administered to a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 4-3-9. hNS-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 30 mg / kg, and SW1389-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. Spleens were collected six days after administration, and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis is the number of activated helper T cells (CD4 + Foxp3 - ICOS + ) and CD45 + The ratio of cells is shown. The mean values ​​of n = 3 are shown.

[0122] Figure 18The anti-CTLA-4 antibody SW1610-mFa55 (switch antibody) demonstrates its anti-tumor effect in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 5-4-5. The antibody was administered via the tail vein at 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Each point represents the mean tumor volume for one group, n = 5.

[0123] Figure 19 The anti-CTLA-4 antibody SW1612-mFa55 (switch antibody) demonstrates its anti-tumor effect in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 5-4-5. The antibody was administered via the tail vein at 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Each point represents the mean tumor volume for one group, n = 5.

[0124] Figure 20 The anti-CTLA-4 antibody SW1615-mFa55 (switch antibody) demonstrates its anti-tumor effect in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 5-4-5. The antibody was administered via the tail vein at 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Each point represents the mean tumor volume for one group, n = 5.

[0125] Figure 21 The changes in the proportion of effector Treg cells in the tumor when the anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55 or SW1615-mFa55 (all switch antibodies) were administered in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 5-4-8. SW1610-mFa55 was administered via the tail vein at 50 mg / kg, 100 mg / kg and 200 mg / kg, SW1612-mFa55 was administered at 50 mg / kg, 100 mg / kg and 200 mg / kg, SW1615-mFa55 was administered at 50 mg / kg, 100 mg / kg, 200 mg / kg and 400 mg / kg, and the negative control antibody KLH-mFa55 was administered at 400 mg / kg. Tumors were collected six days after administration and the increase or decrease in effector Tregs was evaluated by FACS analysis. The vertical axis is effector Tregs (CD4 + FoxP3 + CCR7 low KLRG1 + ) and CD45 + The ratio of cells is shown. The mean values ​​of n = 3 are shown.

[0126] Figure 22The following table shows changes in the proportion of activated helper T cells in the spleen when the anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, or SW1615-mFa55 (all switch antibodies) were administered to a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 5-4-9. SW1610-mFa55 was administered via the tail vein at 50 mg / kg, 100 mg / kg, and 200 mg / kg, SW1612-mFa55 was administered at 50 mg / kg, 100 mg / kg, and 200 mg / kg, and SW1615-mFa55 was administered at 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg. A negative control antibody, KLH-mFa55, was administered at 400 mg / kg. Spleens were harvested six days after administration, and the increase or decrease in activated helper T cells was assessed by FACS analysis. The vertical axis is activated helper T cells (CD4 + Foxp3 - ICOS + ) and CD45 + The ratio of cells is shown. The mean values ​​of n = 3 are shown.

[0127] Figure 23 A comparison of in vitro ADCC activity of antibodies with various altered constant regions that enhance FcγR binding, as described in Example 6-2, is shown. As shown, IgG1 represents MDX10D1H-G1m / MDX10D1L-k0MT, GASDALIE represents MDX10D1H-GASDALIE / MDX10D1L-k0MT, ART6 represents MDX10D1H-Kn462 / MDX10D1H-H1445 / MDX10D1L-k0MT, and ART8 represents MDX10D1H-Kn461 / MDX10D1H-H1443 / MDX10D1L-k0MT. Here, IgG1 is an antibody with a control constant region, GASDALIE is an antibody with a constant region described in prior art literature, and ART6 and ART8 are antibodies with altered constant regions generated in Example 6-1.

[0128] Figure 24A comparison of in vitro ADCP activity of antibodies with various altered constant regions that enhance FcγR binding, as described in Example 6-3, is shown. As shown, IgG1 represents MDX10D1H-G1m / MDX10D1L-k0MT, GASDIE represents MDX10D1H-GASDIE / MDX10D1L-k0MT, ART6 represents MDX10D1H-Kn462 / MDX10D1H-H1445 / MDX10D1L-k0MT, and ART8 represents MDX10D1H-Kn461 / MDX10D1H-H1443 / MDX10D1L-k0MT. Here, IgG1 is an antibody with a control constant region, GASDIE is an antibody with a constant region described in prior art literature, and ART6 and ART8 are antibodies with altered constant regions generated in Example 6-1.

[0129] Figure 25 In vitro ADCC activity of the anti-CTLA4 switch antibody SW1389-ART6 with altered constant regions and enhanced binding to FcγRs was demonstrated, as described in Example 6-4.

[0130] Figure 26 In vitro ADCC activity of the anti-CTLA4 switch antibody SW1610-ART6 with altered constant regions and enhanced binding to FcγRs was demonstrated, as described in Example 6-4.

[0131] Figure 27 In vitro ADCC activity of the anti-CTLA4 switch antibody SW1612-ART6 with altered constant regions and enhanced binding to FcγRs was demonstrated, as described in Example 6-4.

[0132] Figure 28 The neutralizing activity of the anti-CTLA4 switch antibody SW1389 on CTLA4 (activity that eliminates CTLA4 signaling that acts in an inhibitory manner on effector cell activation) was demonstrated, as described in Example 6-5.

[0133] Figure 29 The neutralizing activity of the anti-CTLA4 switch antibody SW1610 on CTLA4 (activity that eliminates CTLA4 signaling that acts in an inhibitory manner on effector cell activation) was demonstrated, as described in Example 6-5.

[0134] Figure 30 The neutralizing activity of the anti-CTLA4 switch antibody SW1612 on CTLA4 (activity that eliminates CTLA4 signaling that acts in an inhibitory manner on effector cell activation) was demonstrated, as described in Example 6-5.

[0135] Figure 31The neutralizing activity of the anti-CTLA4 switch antibody SW1615 on CTLA4 (activity that eliminates CTLA4 signaling that acts in an inhibitory manner on effector cell activation) was demonstrated, as described in Example 6-5.

[0136] Figure 32 The in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1389-ART5+ACT1 against CTLA4-positive regulatory T cells was shown, as described in Example 6-6.

[0137] Figure 33 The in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1389-ART6+ACT1 against CTLA4-positive regulatory T cells was shown, as described in Example 6-6.

[0138] Figure 34 The in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1610-ART5+ACT1 against CTLA4-positive regulatory T cells was shown, as described in Example 6-6.

[0139] Figure 35 The in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1610-ART6+ACT1 against CTLA4-positive regulatory T cells was shown, as described in Example 6-6. DETAILED DESCRIPTION

[0140] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are routinely used using conventional methodology, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames, and G. R. Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press, Inc. Press; Animal Cell Culture (RI Freshney, eds., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MPCalos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, ed., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0141] 1. Definition

[0142] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd edition, J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th edition, John Wiley & Sons (New York, NY 1992) provide those skilled in the art with a general guide to many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0143] For the purposes of interpreting this specification, the following definitions apply, and whenever appropriate, terms used in the singular also include the plural, and vice versa. It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. If any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall prevail.

[0144] For the purposes of this paper, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence, or it can comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0145] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins that bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay, such as those described in U.S. Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), can be performed. Useful effector cells for such assays include PBMCs and NK cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0146] "Cytotoxic activity" includes, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity as described above, complement-dependent cytotoxicity (CDC) activity as described below, and T cell-mediated cytotoxicity. CDC activity refers to the cytotoxic activity of the complement system. On the other hand, ADCC activity refers to an activity in which an antibody binds to an antigen present on the cell surface of a target cell, and effector cells further bind to the antibody, thereby damaging the target cell. Whether the target antibody has ADCC activity and whether the target antibody has CDC activity can be determined by known methods (e.g., Current Protocols in Immunology, Chapter 7, Immunologic studies in humans, edited by Coligan et al. (1993)).

[0147] "Neutralizing activity" refers to the ability of an antibody to inhibit a biological activity by binding to a molecule involved in the biological activity. In some embodiments, the biological activity is caused by the binding of a ligand to a receptor. In certain embodiments, the antibody inhibits the binding of a ligand to a receptor by binding to a ligand or receptor. Antibodies with this neutralizing activity are called neutralizing antibodies. The neutralizing activity of a test substance can be determined by comparing the biological activity of the ligand in the presence and absence of the test substance.

[0148] The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to a process in which whole or parts of a cell coated with antibody are engulfed into a phagocytic immune cell (e.g., macrophages, neutrophils, and dendritic cells) that binds the Fc region of the immunoglobulin.

[0149] The term "binding activity" refers to the strength of the sum total of noncovalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Herein, "binding activity" is not strictly limited to 1 : 1 interactions between members of a binding pair (e.g., an antibody and an antigen). For example, when members of a binding pair reflect monovalent 1 : 1 interactions, binding activity refers to intrinsic binding affinity ("affinity"). When members of a binding pair are capable of monovalent and multivalent binding, binding activity is the sum of each binding strength. The binding activity of a molecule X to its partner Y can be generally expressed in terms of a dissociation constant (KD) or "amount of analyte bound per unit amount of ligand." Binding activity can be determined by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for determining binding activity are described below.

[0150] An "affinity matured" antibody refers to an antibody with one or more alterations which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess such alterations.

[0151] The terms "anti-CTLA-4 antibody" and "antibody that binds CTLA-4" refer to an antibody that is capable of binding CTLA-4 with sufficient affinity to be useful as a diagnostic and / or therapeutic agent in targeting CTLA-4. In one embodiment, the extent of binding of an anti-CTLA-4 antibody to an unrelated, non-CTLA-4 protein is less than about 10% of the binding of the antibody to CTLA-4 as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds CTLA-4 has a dissociation constant (KD) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M). In certain embodiments, an anti-CTLA-4 antibody binds the same epitope of CTLA-4 as an antibody selected from the group consisting of ipilimumab, tremelimumab, and BMS- 986013.

[0152] The term "antibody" herein is used in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0153] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0154] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen, e.g., by 50% or more, in a competition assay, and / or the reference antibody blocks binding of the antibody to its antigen, e.g., by 50% or more, in a competition assay. Exemplary competition assays are provided herein.

[0155] "Autoimmune disease" refers to a non-malignant disease or condition that is caused by and directed against an individual's own tissues. Autoimmune diseases herein specifically exclude malignant or cancerous diseases or conditions, and particularly exclude B-cell lymphomas, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloid leukemia. Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses, such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic diseases, such as eczema and asthma, and other conditions involving T cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion defects; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and autoimmune thyroiditis caused by cytokines and T cells. Immune reactions involving acute and delayed hypersensitivity reactions mediated by lymphocytes, typically seen in tuberculosis, sarcoidosis, polymyositis, granulomas, and vasculitis; pernicious anemia (Addison's disease); disorders involving leukocytic diapedesis; inflammatory disorders of the central nervous system (CNS); multiple organ injury syndrome; hemolytic anemias (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated disorders; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; stiff-person syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0156] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include breast cancer and liver cancer.

[0157] The term "complement dependent cytotoxicity" or "CDC" refers to a mechanism of inducing cell death in which the Fc effector domain of an antibody bound to a target activates a series of enzymatic reactions that result in the formation of pores in the cell membrane of the target cell. Typically, an antigen-antibody complex formed on a target cell binds and activates complement component Clq, which in turn activates the complement cascade and leads to target cell death. In addition, complement activation can also result in deposition of complement components on the surface of the target cell, which leads to binding to complement receptors (e.g., CR3) on leukocytes, thereby facilitating ADCC.

[0158] "Chemotherapeutic agent" refers to a compound that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN (registered trademark)); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and hydroxymethylmelamines, including melamine, triethylene melamine, and hydroxymethyl cyanamide. , triethylenephosphamide, triethylenethiophosphamide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL (registered trademark)); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including the synthetic analogue topotecan (HYCAMTIN (registered trademark)), C PT-11 (irinotecan, CAMPTOSAR (registered trademark)), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs of adozelesin, carzelesin, and bizelesin); podophyllotoxin n); podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards, such as chlorambucil, chlornaphthyl mustard, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, nitrogen oxide mustard hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, in particular calicheamicin gamma and calicheamicin omega (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994); CDP323, an oral α-4 integrin inhibitor; dynemicin, including dynemicin A; esperamicin;and new carcinostaticin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin bicin), 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN (registered trademark)), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin hydrochloride liposome injection (DOXIL (registered trademark)), liposomal doxorubicin TLC D-99 (MYOCET (registered trademark)), pegylated liposomal doxorubicin (CAELYX (registered trademark) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid (MYCOPHOS), acid), nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;antimetabolites such as methotrexate, gemcitabine (GEMZAR), tegafur (UFTORAL), capecitabine (XELODA), epothilone, and 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide; aminolevulinic acid; amsacrine; antineoplastons; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamide; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin;losoxantrone; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and roridin B). A) and anguidine); urethane; vindesine (ELDISINE (registered trademark), FILDESIN (registered trademark)); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; cytosine; cytarabine ("Ara-C"); thiotepa; taxoids, such as paclitaxel (TAXOL (registered trademark)), albumin-modified nanoparticle formulations of paclitaxel (ABRAXANE; TM) and docetaxel (TAXOTERE (registered trademark)); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin (e.g., ELOXATIN (registered trademark)), and carboplatin; vincas, which prevent tubulin from polymerizing to form microtubules, including vinblastine (VELBAN (registered trademark)), vincristine (ONCOVIN (registered trademark)), and oxaliplatin (e.g., ELOXATIN (registered trademark)). (Registered Trademark), vindesine (ELDISINE (Registered Trademark), FILDESIN (Registered Trademark)), and vinorelbine (NAVELBINE (Registered Trademark)); etoposide (VP-16); ifosfamide; mitoxantrone; folinic acid; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid, including bexarotene (TARGRETIN (Registered Trademark)); bisphosphonates, such as clodronate (e.g., BONEFOS (Registered Trademark) or OSTAC (Registered Trademark)), etidronate (DIDROCAL (Registered Trademark)), NE-58095, zoledronic acid (Registered Trademark), and bisphosphonates. acid / zoledronate (ZOMETA(registered trademark)), alendronate (FOSAMAX(registered trademark)), pamidronate (AREDIA(registered trademark)), tiludronate (SKELID(registered trademark)), or risedronate (ACTONEL(registered trademark)); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE(registered trademark) vaccine, and gene therapy vaccines, such as ALLOVECTIN(registered trademark) vaccine, LEUVECTIN(registered trademark) vaccine, and VAXID(registered trademark) vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN(registered trademark)); rmRH (e.g., ABARELIX(registered trademark));BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT (registered trademark), Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); bortezomib (VELCADE (registered trademark)); CCI-779; tipifarnib (R11577); sorafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium (GENASEENSE (registered trademark)); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors, such as rapamycin (sirolimus, RAPAMUNE (registered trademark)); farnesyltransferase inhibitors, such as lonafarnib (SCH 6636, SARASAR; TM ); and pharmaceutically acceptable salts, acids or derivatives of any of the above substances; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combined therapy); and FOLFOX (oxaliplatin (ELOXATIN TM ) is an abbreviation for the combined 5-FU and folinic acid treatment regimen.

[0159] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0160] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and some of these are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0161] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211 At 131 I. 125 I. 90 Y. 186Re、 188 Re、 153 Sm, 212 Bi, 32 P. 212 radioisotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and a variety of chemotherapeutic agents disclosed above.

[0162] "Effecter cells" refer to leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcγRIII and perform ADCC effector functions. Examples of leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources such as blood. In certain embodiments, effector cells can be human effector cells.

[0163] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cell-mediated phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0164] The term "epitope" includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen, including specific amino acids that directly contact the antibody. Epitope determinants may include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Typically, antibodies specific for a particular target antigen preferentially recognize epitopes on the target antigen in a complex mixture of proteins and / or macromolecules.

[0165] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, FcR is a natural human FcR. In some embodiments, FcR is a receptor that binds to IgG antibodies (gamma receptors) and includes FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants of these receptors and one of alternative splicing forms. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are generally described, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-492 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-341 (1995). The term "FcR" herein encompasses additional FcRs, including those to be identified in the future.

[0166] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976); and Kim et al., J. Immunol. 24: 249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7): 637-640 (1997); Hinton et al., J. Biol. Chem. 279(8): 6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).

[0167] In vivo binding to human FcRn and the serum half-life of high-affinity human FcRn binding polypeptides can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides having variant Fc regions are administered. WO 2000 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcRs. See also, for example, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0168] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) in the Fc region may or may not be present. Unless otherwise indicated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0169] The term "antibody comprising an Fc region" refers to an antibody comprising an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Thus, a composition comprising an antibody having an Fc region according to the present invention can comprise an antibody having G446-K447, an antibody having G446 and not having K447, an antibody with all of G446-K447 removed, or a mixture of the three types of antibodies.

[0170] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using VH or VL domains from an antigen-binding antibody to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0171] "Framework" or "FR" refers to variable domain residues excluding the hypervariable region (HVR) residues. The FR of a variable domain is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences in VH (or VL) generally appear in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0172] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that comprise an Fc region as defined herein.

[0173] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be associated with a binding domain (e.g., an antibody variable domain) and can be assessed using, for example, the various assays disclosed in the definitions herein.

[0174] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell or derived from a non-human source utilizing human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0175] A "human consensus framework" is a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the sequence subset is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as described in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al., supra.

[0176] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0177] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen-contacting residues ("antigen contacts"). Generally, antibodies comprise six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0178] (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));

[0179] (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));

[0180] (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)); and

[0181] (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).

[0182] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0183] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0184] An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, such as 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 antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0185] An "isolated" nucleic acid is one that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0186] "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules are present at one or more locations in a host cell.

[0187] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0188] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny need not be identical in nucleic acid content to the parent cell but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0189] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies, which, for example, contain naturally occurring mutations or arise during the production process of the monoclonal antibody preparation, wherein these variants are generally present in small amounts. Compared to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous antibody population and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. Such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0190] A "naked antibody" is an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0191] “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. Depending on the amino acid sequence of the constant domain of their light chains, antibodies are assigned to one of two types, called kappa (K) and lambda (l).

[0192] A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of a Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0193] A “variant Fc region” comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification (change), preferably one or more amino acid substitutions. Preferably, a variant Fc region has at least one amino acid substitution, e.g., from about 1 to about 10, preferably from about 1 to about 5, amino acid substitutions in a native sequence Fc region or Fc region of a parent polypeptide, as compared to a native sequence Fc region or Fc region of a parent polypeptide. A variant Fc region herein is preferably at least about 80% homologous to, more preferably at least about 90% homologous to, and most preferably at least about 95% homologous to, a native sequence Fc region and / or to a Fc region of a parent polypeptide.

[0194] "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing spaces (if necessary) to achieve maximum sequence identity percentage and disregarding any conservative substitutions as part of sequence identity. Alignment for determining percent amino acid sequence identity purposes can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0195] The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code and user documentation have been filed with the U.S. Copyright Office, Washington, D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0196] In cases where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a specific amino acid sequence identity with a given amino acid sequence B) is calculated as follows:

[0197] Multiply 100 by the fraction X / Y

[0198] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the preceding paragraph using the ALIGN-2 computer program.

[0199] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to the subject to which the formulation will be administered.

[0200] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0201] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that does not cause unacceptable toxicity to the subject to which the formulation will be administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0202] An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

[0203] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0204] The term "CTLA-4" as used herein, refers to any native CTLA-4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CTLA-4 as well as any form of CTLA-4 that results from processing in cells. The term also encompasses naturally occurring variants of CTLA-4, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human CTLA-4 is set forth in SEQ ID NO: 214, the amino acid sequence of a mouse CTLA-4 is set forth in SEQ ID NO: 247, the amino acid sequence of a monkey CTLA-4 is set forth in SEQ ID NO: 248, and the amino acid sequence of the extracellular domain of human CTLA-4 is set forth in SEQ ID NO: 28. CTLA-4 can also be described herein as CTLA4.

[0205] The term "regulatory T (Treg) cell" refers to a subset of T cells that modulate the immune system, maintain tolerance to self-antigens, and suppress autoimmune disease. These cells generally suppress or down-regulate the induction and proliferation of effector T cells. The best understood Treg cells are CD4 + CD25 +Treg cells are cells that are not T cells. These Treg cells are different from helper T cells. Several different methods are used to identify and monitor Treg cells. + CD25 + Treg cells constitute mature CD4 + T cell subsets are about 5% to about 10%, while Tregs can be detected in whole blood at about 1% to about 2%. + CD25 + Foxp3 + Treg cells can be identified and monitored using the methods described in the following examples. Furthermore, the absence or low expression of CD127 can be used in combination with the presence of CD4 and CD25 as another marker. Treg cells also express high levels of CTLA-4 and GITR. Tregs can also be identified using the methods described in the following examples.

[0206] As used herein, the terms "substantially similar," "substantially equal," or "substantially identical" refer to a sufficiently high degree of similarity between two values ​​(e.g., one associated with an antibody of the invention and the other associated with a reference / comparator antibody) such that one of skill in the art would consider the difference between the two values ​​to have little or no biological and / or statistical significance in the context of the biological characteristic being measured by the value (e.g., KD value).

[0207] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention to attempt to alter the natural course of the individual being treated, and can be performed either prophylactically or during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or relieving the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0208] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive herein.

[0209] The term "tumor tissue" refers to tissue containing at least one type of tumor cell. Tumor tissue typically consists of a population of tumor cells that form the main mass of the tumor (parenchyma), and connective tissue and blood vessels that exist between these cells and support the tumor ("stroma"). In some cases, the distinction between the two is clear, while in others, they are intermingled. Tumor tissue may be infiltrated by immune cells and other cells. On the other hand, "non-tumor tissue" refers to tissue other than tumor tissue within a living organism. Healthy / normal tissue that is not in a diseased state is a typical example of non-tumor tissue.

[0210] II. Compositions and Methods

[0211] In one aspect, the present invention is based in part on anti-CTLA-4 antibodies and their uses. In certain embodiments, antibodies that bind to CTLA-4 are provided. The antibodies of the present invention can be used, for example, in the diagnosis or treatment of cancer.

[0212] A. Exemplary Anti-CTLA-4 Antibodies

[0213] In one aspect, the invention provides isolated antibodies that bind to CTLA-4. In certain embodiments, the anti-CTLA-4 antibodies of the invention have CTLA-4 binding activity that is dependent on the concentration of an adenosine-containing compound. In some embodiments, the binding activity to CTLA-4 is higher in the presence of an adenosine-containing compound than in the absence of an adenosine-containing compound. In another embodiment, the binding activity to CTLA-4 is higher in the presence of a high concentration of an adenosine-containing compound than in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in binding activity to CTLA-4 is, for example, 2-fold or higher, 3-fold or higher, 5-fold or higher, 10-fold or higher, 20-fold or higher, 30-fold or higher, 50-fold or higher, 100-fold or higher, 200-fold or higher, 300-fold or higher, 500-fold or higher, 1×10 3 times or higher, 2×10 3 times or higher, 3×10 3 times or higher, 5×10 3 times or higher, 1×10 4 or higher, 2×10 4 or higher, 3×10 4 times or higher, 5×10 4 times or higher or 1×10 5 times or higher.

[0214] In some embodiments, the binding activity of an anti-CTLA-4 antibody can be represented by a KD (dissociation constant) value. In further embodiments, the KD value of the anti-CTLA-4 antibody in the presence of an adenosine-containing compound is less than the KD value in the absence of the adenosine-containing compound. Alternatively, in another embodiment, the KD value of the anti-CTLA-4 antibody in the presence of a high concentration of an adenosine-containing compound is less than the KD value in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in KD value of the anti-CTLA-4 antibody is, for example, 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 500-fold or more, 1 x 10 3 times or higher, 2 x 10 3 times or higher, 3 x 10 3 times or higher, 5 x 10 3 times or higher, 1 x 10 4 times or higher, 2 x 10 4 times or higher, 3 x 10 4 times or higher, 5 x 10 4 times or higher or 1 x 10 5 In the presence of adenosine-containing compounds or in the presence of high concentrations of adenosine-containing compounds, the KD value of the anti-CTLA-4 antibody can be, for example, 9 x 10 -7 M or smaller, 8 x 10 -7 M or lower, 7 x 10 -7 M or smaller, 6 x 10 -7 M or lower, 5 x 10 -7 M or lower, 4 x 10 -7 M or lower, 3 x 10 -7 M or lower, 2 x 10 -7 M or lower, 1 x 10 -7 M or smaller, 9 x 10 -8 M or smaller, 8 x 10 -8 M or lower, 7 x 10 -8 M or smaller, 6 x 10 -8 M or lower, 5 x 10 -8 M or smaller, 4 x10 -8 M or lower, 3 x 10 -8 M or lower, 2 x 10 -8 M or lower, 1 x 10 -8 M or smaller, 9 x 10 -9M or smaller, 8 x 10 -9 M or lower, 7 x 10 -9 M or smaller, 6 x 10 -9 M or lower, 5 x 10 -9 M or lower, 4 x 10 -9 M or lower, 3 x 10 -9 M or lower, 2 x 10 -9 M or lower, 1 x 10 -9 M or smaller, 9 x 10 -10 M or smaller, 8 x10 -10 M or lower, 7 x 10 -10 M or smaller, 6 x 10 -10 M or lower, 5 x 10 -10 M or lower, 4 x 10 -10 M or lower, 3 x 10 -10 M or lower, 2 x 10 -10 M or lower or 1 x 10 -10 M or less. In the absence of adenosine-containing compounds or in the presence of low concentrations of adenosine-containing compounds, the KD value of the anti-CTLA-4 antibody can be, for example, 1 x 10 -8 M or higher, 2 x 10 -8 M or higher, 3 x 10 -8 M or higher, 4 x 10 -8 M or higher, 5 x 10 -8 M or higher, 6 x 10 -8 M or higher, 7 x 10 -8 M or higher, 8 x 10 -8 M or higher, 9 x 10 -8 M or higher, 1 x 10 -7 M or higher, 2 x10 -7 M or higher, 3 x 10 -7 M or higher, 4 x 10 -7 M or higher, 5 x 10 -7 M or higher, 6 x 10 -7 M or higher, 7 x 10 -7 M or higher, 8 x 10 -7 M or higher, 9 x 10 -7 M or higher, 1 x 10 -6 M or higher, 2 x 10 -6 M or higher, 3 x 10 -6M or higher, 4 x 10 -6 M or higher, 5 x 10 -6 M or higher, 6 x 10 -6 M or higher, 7 x10 -6 M or higher, 8 x 10 -6 M or higher, or 9 x 10 -6 M or higher.

[0215] In another embodiment, the binding activity of an anti-CTLA-4 antibody can be expressed using a kd (dissociation rate constant) value instead of a KD value.

[0216] In another embodiment, the binding activity of an anti-CTLA-4 antibody can be expressed as the amount of CTLA-4 bound per unit amount of antibody. For example, in a surface plasmon resonance assay, the amount of antibody bound to a sensor chip and the amount of antigen bound thereto are each measured as resonance units (RU). The value obtained by dividing the amount of antigen bound by the amount of antibody bound can be defined as the amount of antigen bound per unit amount of antibody. Specific methods for determining and calculating such binding are described in the examples below. In some embodiments, the amount of CTLA-4 bound in the presence of an adenosine-containing compound is greater than the amount bound in the absence of the adenosine-containing compound. Alternatively, in another embodiment, the amount of CTLA-4 bound in the presence of a high concentration of an adenosine-containing compound is greater than the amount bound in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in the amount of CTLA-4 bound is, for example, 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 500-fold or more, 1 x 10 3 times or higher, 2 x 10 3 times or higher, 3 x 10 3 times or higher, 5 x 10 3 times or higher, 1 x10 4 times or higher, 2 x 10 4 times or higher, 3 x 10 4 times or higher, 5 x 10 4 times or higher, or 1 x 10 5The value of the amount of CTLA-4 bound in the presence of an adenosine-containing compound or in the presence of a high concentration of an adenosine-containing compound can be, for example, 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, or 1 or more. The value of the amount of CTLA-4 bound in the absence of an adenosine-containing compound or in the presence of a low concentration of an adenosine-containing compound can be, for example, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less, 0.006 or less, 0.005 or less, 0.004 or less, 0.003 or less, 0.002 or less, or 0.001 or less.

[0217] In some embodiments, KD values, kd values, binding values, etc. described herein are determined or calculated by performing a surface plasmon resonance assay at 25°C or 37°C (see, e.g., Example 3 herein).

[0218] Any concentration of adenosine-containing compound can be selected so long as a difference in the binding activity of the anti-CTLA-4 antibody is detected. In certain embodiments, a high concentration can include, for example, 1 nM or higher, 3 nM or higher, 10 nM or higher, 30 nM or higher, 100 nM or higher, 300 nM or higher, 1 μM or higher, 3 μM or higher, 10 μM or higher, 30 μM or higher, 100 μM or higher, 300 μM or higher, 100 μM or higher, 300 μM or higher, 100 μM or higher, 300 μM or higher, 1 mM or higher, 1 mM or higher, 3 mM or higher, 10 mM or higher, 30 mM or higher, 100 mM or higher, 300 mM or higher, and 1 M or higher. Alternatively, the high concentration herein can be an amount sufficient for each anti-CTLA-4 antibody to exhibit maximal binding activity. In one embodiment, 1 μM, 10 μM, 100 μM, 1 mM, or an amount sufficient for each anti-CTLA-4 antibody to exhibit maximal binding activity can be selected as the high concentration herein. In certain embodiments, low concentrations can include, for example, 1 mM or less, 300 μM or less, 100 μM or less, 30 μM or less, 10 μM or less, 3 μM or less, 1 μM or less, 300 nM or less, 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, 1 nM or less, 300 pM or less, 100 pM or less, 30 pM or less, 10 pM or less, 3 pM or less, and 1 pM or less. Alternatively, the low concentration herein can be the concentration at which the respective anti-CTLA-4 antibody exhibits minimal binding activity. Alternatively, the embodiment in which the substantial concentration is zero (absence of an adenosine-containing compound) can be selected as the low concentration. In one embodiment, the concentration at which each anti-CTLA-4 antibody exhibits minimal binding activity can be 1 mM, 100 μM, 10 μM, 1 μM, or the absence of an adenosine compound. In another embodiment, the ratio of the high concentration to the low concentration can be selected as follows: for example, 3 times or more, 10 times or more, 30 times or more, 100 times or more, 300 times or more, 1 x 10 3 times or higher, 3 x 10 3 times or higher, 1 x 104 times or higher, 3 x 10 4 times or higher, 1 x 10 5 times or higher, 3 x10 5 times or higher, 1 x 10 6 times or higher, 3 x 10 6 times or higher, 1 x 10 7 times or higher, 3 x 10 7 times or higher, 1 x10 8 times or higher, 3 x 10 8 times or higher, 1 x 10 9 times or higher, 3 x 10 9 times or higher, 1 x 10 10 times or higher, 3x 10 10 times or higher, 1 x 10 11 times or higher, 3 x 10 11 times or higher, or 1 x 10 12 times or higher.

[0219] In another embodiment, the anti-CTLA-4 antibodies of the present invention also have binding activity with adenosine-containing compounds. The amount of adenosine-containing compound bound per unit amount of the anti-CTLA-4 antibody can be calculated using the above-described method and used as the binding activity of the antibody with adenosine-containing compounds. Specific methods for determining and calculating such binding amount are described in the following examples. The value of the amount of adenosine-containing compound bound per unit amount of the anti-CTLA-4 antibody of the present invention can be, for example, 0.0001 or more, 0.0002 or more, 0.0003 or more, 0.0004 or more, 0.0005 or more, 0.0006 or more, 0.0007 or more, 0.0008 or more, 0.0009 or more, 0.001 or more, 0.002 or more, 0.003 or more, 0.004 or more, 0.005 or more, 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more, or 0.01 or more.

[0220] In another embodiment, an anti-CTLA-4 antibody of the invention forms a ternary complex with an adenosine-containing compound and CTLA-4. In one embodiment, the anti-CTLA-4 antibody binds to the adenosine-containing compound via the heavy chain CDR1, CDR2, and CDR3. In one embodiment, the anti-CTLA-4 antibody has a binding motif for an adenosine-containing compound. The binding motif for an adenosine-containing compound can consist of, for example, at least one amino acid present at positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c according to Kabat numbering. In a further embodiment, the anti-CTLA-4 antibody binds to the adenosine-containing compound, for example, via at least one amino acid selected from positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c according to Kabat numbering. In certain embodiments, the anti-CTLA-4 antibody has at least one amino acid selected from the group consisting of Thr at position 33, Ser at position 52, Ser at position 52a, Arg at position 53, Tyr at position 56, Tyr at position 58, Tyr at position 95, Gly at position 96, Met at position 100a, Leu at position 100b, and Trp at position 100c, according to Kabat numbering. CTLA-4 can further bind to a complex formed by the anti-CTLA-4 antibody and the adenosine-containing compound. Furthermore, the adenosine-containing compound can be present at the interface between the anti-CTLA-4 antibody and CTLA-4 and can bind to both. The formation of a ternary complex between the anti-CTLA-4 antibody, the adenosine-containing compound, and CTLA-4 can be confirmed by techniques such as crystal structure analysis described below (see Examples).

[0221] In another embodiment, an anti-CTLA-4 antibody of the present invention binds to at least one amino acid selected from amino acids 3 (Met), 33 (Glu), 35 (Arg), 53 (Thr), 97 (Glu), 99 (Met), 100 (Tyr), 101 (Pro), 102 (Pro), 103 (Pro), 104 (Tyr), 105 (Tyr), and 106 (Leu) of human CTLA-4 (extracellular domain; SEQ ID NO: 28). These amino acids may constitute an epitope of the anti-CTLA-4 antibody of the present invention. In another embodiment, an anti-CTLA-4 antibody of the present invention binds to a region from amino acids 97 (Glu) to amino acids 106 (Leu) of human CTLA-4 (extracellular domain; SEQ ID NO: 28). In another embodiment, an anti-CTLA-4 antibody of the invention binds to a region from amino acid 99 (Met) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain; SEQ ID NO: 28).

[0222] In another embodiment, the anti-CTLA-4 antibodies of the invention compete for CTLA-4 binding with ABAM004 (VH, SEQ ID NO: 10; VL, SEQ ID NO: 11; HVR-H1, SEQ ID NO: 100; HVR-H2, SEQ ID NO: 101; HVR-H3, SEQ ID NO: 102; HVR-L1, SEQ ID NO: 113; HVR-L2, SEQ ID NO: 114; HVR-L3, SEQ ID NO: 115). In another embodiment, the anti-CTLA-4 antibodies of the invention bind to the same epitope as ABAM004. When an excess of anti-CTLA-4 antibody is present, the binding of ABAM004 to CTLA-4 can be reduced, for example, by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Exemplary competition assays are provided herein.

[0223] In another embodiment, the anti-CTLA-4 antibodies of the present invention exhibit cytotoxic activity against cells expressing CTLA-4. When CTLA-4 is expressed on the surface of target cells and the anti-CTLA-4 antibody binds thereto, the cells are damaged. The cell damage may be caused by effector cells bound to the antibody, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), or by complement bound to the antibody, such as complement-dependent cytotoxicity (CDC). Alternatively, the damage may be caused by a cytotoxic agent (e.g., a radioisotope or chemotherapeutic agent) conjugated to the antibody, such as an immunoconjugate. Cytotoxicity in this context may include inducing cell death, inhibiting cell proliferation, and impairing cell function. When an anti-CTLA-4 antibody is present in sufficient amounts, it can cause damage to, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the cells expressing CTLA-4. Such cytotoxic activity can be measured by comparing it to an assay in the absence of the antibody or in the presence of a negative control antibody. Exemplary cytotoxicity assays are provided herein.

[0224] In another embodiment, the anti-CTLA-4 antibodies of the present invention exhibit neutralizing activity against CTLA-4. CTLA-4 is known to exert its effects by interacting with its ligands CD80 (B7-1) or CD86 (B7-2). In certain embodiments, the anti-CTLA-4 antibodies inhibit the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2). When the anti-CTLA-4 antibody is present in a sufficient amount, it can inhibit the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2), for example, by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. This determination of inhibitory activity can be compared to that in the absence of the antibody or in the presence of a negative control antibody. Specific methods for determining neutralizing activity are provided herein.

[0225] In another embodiment, the anti-CTLA-4 antibodies of the present invention bind to CTLA-4 derived from a variety of animal species. Exemplary animal species can include mammals, such as humans, monkeys, mice, rats, hamsters, guinea pigs, rabbits, pigs, cattle, goats, horses, sheep, camels, dogs and cats. In certain embodiments, anti-CTLA-4 antibodies bind to CTLA-4 derived from humans and non-humans (e.g., monkeys, mice and rats). The amino acid sequence of human CTLA-4 is shown in SEQ ID NO: 214, the amino acid sequence of ape CTLA-4 is shown in SEQ ID NO: 247, and the amino acid sequence of mouse CTLA-4 is shown in SEQ ID NO: 248. The amino acid sequence of CTLA-4 derived from other animal species can also be appropriately determined by methods known to those skilled in the art.

[0226] In certain embodiments, adenosine-containing compounds of the present invention may include, for example, adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP), deoxyadenosine (dADO), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), deoxyadenosine monophosphate (dAMP), and adenosine (γ-thio) triphosphate (ATPγS).

[0227] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225.

[0228] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 228.

[0229] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 228.

[0230] 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 an amino acid sequence selected from SEQ ID NO: 228.

[0231] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0232] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0233] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0234] 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 an amino acid sequence selected from SEQ ID NO: 115.

[0235] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0236] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0237] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0238] 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 an amino acid sequence selected from SEQ ID NO: 115.

[0239] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0240] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0241] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0242] 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 an amino acid sequence selected from SEQ ID NO: 133.

[0243] In one aspect, the application provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153.

[0244] In another aspect, the application provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153.

[0245] In another aspect, the antibody of the application comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153.

[0246] 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 an amino acid sequence selected from SEQ ID NO: 153.

[0247] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0248] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0249] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0250] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133.

[0251] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0252] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0253] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0254] 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 an amino acid sequence selected from SEQ ID NO: 133.

[0255] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152.

[0256] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0257] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0258] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133.

[0259] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0260] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0261] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0262] 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 an amino acid sequence selected from SEQ ID NO: 133.

[0263] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152.

[0264] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0265] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0266] 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 an amino acid sequence selected from SEQ ID NO: 133.

[0267] In one aspect, the application 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.

[0268] In another aspect, the application 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.

[0269] In another aspect, the antibody of the application 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 (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0270] In another aspect, the application 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 selected from SEQ ID NO: 133.

[0271] In certain embodiments, any one or more amino acids of the anti-CTLA-4 antibodies provided above are substituted at the following HVR positions:

[0272] - in HVR-H1 (SEQ ID NO: 223): position 2

[0273] - in HVR-H2 (SEQ ID NO: 224): positions 4, 5, 7, 13, and 16

[0274] - in HVR-H3 (SEQ ID NO: 225): position 3

[0275] - in HVR-L1 (SEQ ID NO: 226): positions 1, 3, 6, 11, 12, and 14

[0276] - in HVR-L2 (SEQ ID NO: 227): positions 1, 3, 4, and 7

[0277] - in HVR-L3 (SEQ ID NO: 228): positions 1 and 10

[0278] In certain embodiments, the substitutions are conservative substitutions as provided herein. In certain embodiments, any one or more of the following substitutions can be made in any combination:

[0279] - in HVR-H1 (SEQ ID NO: 100): H2A, R, or K

[0280] - in HVR-H2 (SEQ ID NO: 101): S4T; R5Q; G7H; D13E or R; K16R

[0281] - in HVR-H3 (SEQ ID NO: 102): K3A

[0282] - In HVR-L1 (SEQ ID NO: 113): T1D, Q, or E; T3P; D6G; N11T; Y12W; S14H

[0283] - In HVR-L2 (SEQ ID NO: 114): E1F or Y; S3I; K4S; S7E or K

[0284] - In HVR-L3 (SEQ ID NO: 115): S1Q; M10T

[0285] For HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3, all possible combinations of the above substitutions are contained in the consensus sequences of SEQ ID NOs: 223, 224, 225, 226, 227 and 228, respectively.

[0286] In any of the above embodiments, the anti-CTLA-4 antibody is humanized. In one embodiment, the anti-CTLA-4 antibody comprises HVRs as in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-CTLA-4 antibody comprises HVRs as in any of the above embodiments and further comprises a VH or VL comprising a FR sequence. In a further embodiment, the anti-CTLA-4 antibody comprises the following heavy and / or light chain variable domain FR sequences: for the heavy chain variable domain, FR1 comprises the amino acid sequence of any one of SEQ ID NOs: 229 to 232, FR2 comprises the amino acid sequence of SEQ ID NO: 233, FR3 comprises the amino acid sequence of SEQ ID NO: 234, and FR4 comprises the amino acid sequence of SEQ ID NO: 235; for the light chain variable domain, FR1 comprises the amino acid sequence of any one of SEQ ID NOs: 236 to 238, FR2 comprises the amino acid sequence of any one of SEQ ID NOs: 240 to 241, FR3 comprises the amino acid sequence of any one of SEQ ID NOs: 242 to 244, and FR4 comprises the amino acid sequence of any one of SEQ ID NOs: 245 to 246.

[0287] In another aspect, the anti-CTLA-4 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-CTLA-4 antibody comprising that sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10, to 11, to 12, to 13, to 14, or to 15 amino acids in SEQ ID NO: 10 are substituted, inserted and / or deleted. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CTLA-4 antibody comprises the VH sequence in SEQ ID NO: 10, including post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. Post-translational modifications include, but are not limited to, modification of a glutamine or a glutamic acid at the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

[0288] In another aspect, an anti-CTLA-4 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CTLA-4 antibody comprising the sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 11. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CTLA-4 antibody comprises the VL sequence of SEQ ID NO: 11, including post-translational modifications of that sequence. In specific embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0289] In another aspect, an anti-CTLA-4 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 149. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CTLA-4 antibody comprising the sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 149. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CTLA-4 antibody comprises the VL sequence of SEQ ID NO: 149, including post-translational modifications thereof. In specific embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0290] In another aspect, an anti-CTLA-4 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 10 and SEQ ID NO: 11, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 98 and SEQ ID NO: 99, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 83 and SEQ ID NO: 97, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 86 and SEQ ID NO: 134, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 136 and SEQ ID NO: 95, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 140 and SEQ ID NO: 146, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 140 and SEQ ID NO: 147, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 136 and SEQ ID NO: 149, respectively, including post-translational modifications of those sequences. In another aspect, a heteromeric anti-CTLA-4 antibody is provided, wherein the antibody comprises at least two different variable regions selected from the variable regions comprising the VH and VL sequences provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 146, respectively, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of those sequences.In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 147, respectively, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0291] When the N-terminal amino acid of the heavy or light chain of an anti-CTLA-4 antibody provided herein is glutamine, the amino acid may be substituted with glutamic acid. When the N-terminal amino acid of the heavy or light chain of an anti-CTLA-4 antibody provided herein is glutamic acid, the amino acid may be substituted with glutamine.

[0292] In another aspect, the present invention provides antibodies that bind to the same epitope as the anti-CTLA-4 antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitope as any of the antibodies listed in Table 4, Table 9, Table 14, and Table 19 are provided. In certain embodiments, antibodies are provided that bind to an epitope within a CTLA-4 fragment comprising at least one amino acid selected from the group consisting of amino acids at position 3 (Met), position 33 (Glu), position 35 (Arg), position 53 (Thr), position 97 (Glu), position 99 (Met), position 100 (Tyr), position 101 (Pro), position 102 (Pro), position 103 (Pro), position 104 (Tyr), position 105 (Tyr), and position 106 (Leu) of SEQ ID NO: 28. In certain embodiments, antibodies are provided that bind to an epitope within a CTLA-4 fragment consisting of amino acids at positions 97 (Glu) to 106 (Leu) of SEQ ID NO: 28. In certain embodiments, antibodies are provided that bind to an epitope within a CTLA-4 fragment consisting of amino acids from position 99 (Met) to position 106 (Leu) of SEQ ID NO: 28.

[0293] In other aspects of the invention, the anti-CTLA-4 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-CTLA-4 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as an intact IgG1 antibody, an intact IgG4 antibody, or other antibody classes or isotypes as defined herein.

[0294] In other aspects, the anti-CTLA-4 antibodies of the present invention comprise an Fc region. In other aspects, the anti-CTLA-4 antibodies of the present invention comprise a constant region. The constant region can be a heavy chain constant region (including the Fc region), a light chain constant region, or both. In some embodiments, the Fc region is a native sequence Fc region. Exemplary heavy chain constant regions derived from natural antibodies can include, for example, heavy chain constant regions such as human IgG1 (SEQ ID NO: 249), human IgG2 (SEQ ID NO: 250), human IgG3 (SEQ ID NO: 251), and human IgG4 (SEQ ID NO: 252). In addition, other exemplary heavy chain constant regions can include the heavy chain constant regions of SEQ ID NOs: 82 and 158. Exemplary light chain constant regions derived from natural antibodies can include, for example, light chain constant regions such as human kappa chain (SEQ ID NOs: 33, 63, and 159) and human lambda chain (SEQ ID NOs: 53 and 87).

[0295] In another embodiment, the Fc region is a variant Fc region generated by adding amino acid changes to the native sequence Fc region. In certain embodiments, the variant Fc region has enhanced binding activity to at least one Fcγ receptor selected from FcγRIa, FcγRIIa, FcγRIIb and FcγRIIIa compared to the native sequence Fc region. In other embodiments, the variant Fc region has enhanced binding activity to FcγRIIa and FcγRIIIa compared to the native sequence Fc region. Examples of heavy chain constant regions comprising such variant Fc regions include, for example, the heavy chain constant regions listed in Tables 26 to 30 and the heavy chain constant regions of SEQ ID NOs: 31, 32, 41 to 46, 65, 66, 81, 207, 239, 253 to 271, 276, 277 and 278.

[0296] Native sequence Fc regions are generally composed of homodimers consisting of two identical polypeptide chains. In certain embodiments, variant Fc regions may be homodimers composed of polypeptide chains having identical sequences, or heterodimers composed of polypeptide chains having mutually different sequences. Similarly, the heavy chain constant region comprising the Fc region may be a homodimer composed of polypeptide chains having identical sequences, or a heterodimer composed of polypeptide chains having mutually different sequences. Examples of heteromeric heavy chain constant regions include, for example, a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 31 and 32; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 43 and 44; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 45 and 46; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 254 and 256; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 257 and 258; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 259 and 260; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 261 and 263; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 262 and 264; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 265 and 267; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 266 and 268; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 239 and 207; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 259 and 276; and the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 65 and 278.

[0297] In other aspects, an anti-CTLA-4 antibody according to any of the above embodiments can incorporate any of the features, alone or in combination, as described in Sections 1-7 below:

[0298] 1. Antibody Binding Activity

[0299] In certain embodiments, the binding activity of an antibody provided herein is 10 μM or less, 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13The dissociation constant (KD) of

[0300] In one embodiment, the binding activity of the antibody is determined by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined by titrating the antibody with a minimal concentration of ( 125 I) labeled antigen equilibrated Fab, and then the bound antigen was captured using an anti-Fab antibody coated plate for measurement (see, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the assay conditions, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml capture anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w / v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 The antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57: 4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. Once the plate is dry, 150 μl / well of scintillant (MICROSCINT-20) is added. TM ; Packard), and in TOPCOUNT TM Plates were counted for 10 minutes on a gamma counter (Packard).Concentrations of each Fab that gave less than or equal to 20% of maximal binding were selected for competitive binding assays.

[0301] In one embodiment, the binding activity of the antibody is measured using a ligand capture assay, for example, using a BIACORE (registered trademark) T200 or BIACORE (registered trademark) 4000 (GE Healthcare, Uppsala, Sweden), using surface plasmon resonance as the assay principle. BIACORE (registered trademark) control software is used for instrument operation. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions, and a sensor chip coated with carboxymethyl dextran (GE Healthcare, Uppsala, Sweden) is immobilized using a ligand capture molecule, such as an anti-tag antibody, an anti-IgG antibody, and protein A. The ligand capture molecule is diluted with 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate and injection time. The binding activity assay is performed using a buffer containing 0.05% polysorbate 20 (also known as TWEEN (registered trademark)-20) as the assay buffer, at a flow rate of 10-30 μL / min, and at an assay temperature preferably of 25°C or 37°C. When the assay is performed by allowing the antibody serving as the ligand to be captured by a molecule for ligand capture, serial dilutions of the antigen or Fc receptor prepared in the assay buffer (analyte) are injected after the target amount of the antibody is captured by the antibody injection. When the assay is performed by allowing the antigen or Fc receptor serving as the ligand to be captured by a molecule for ligand capture, serial dilutions of the antibody prepared in the assay buffer (analyte) are injected after the target amount of the antigen or Fc receptor is captured by the antigen or Fc receptor injection.

[0302] In one embodiment, the assay results are analyzed using BIACORE (registered trademark) evaluation software. Kinetic parameters are calculated by simultaneously fitting the association and dissociation sensorgrams using a 1:1 binding model, and the association rate (k or ka), dissociation rate (k or k), and equilibrium dissociation constant (KD) can be calculated. When the binding activity is weak, especially when dissociation is rapid and kinetic parameters are difficult to calculate, the equilibrium dissociation constant (KD) can be calculated using a steady-state model. As another parameter of binding activity, the "analyte binding amount per unit amount of ligand" can be calculated by dividing the amount of analyte bound (RU) at a specific concentration by the amount of captured ligand (RU).

[0303] 2. Antibody fragments

[0304] In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0305] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0306] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

[0307] Antibody fragments, as described herein, can be made by various techniques including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0308] 3. Chimeric and Humanized Antibodies

[0309] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In other examples, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0310] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains, wherein HVR, such as CDR, (or a portion thereof) is derived from a non-human antibody, and FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody optionally also comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0311] Humanized antibodies and methods for their preparation are generally described in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991). (describing "resurfacing"); Dall'Acqua et al., Methods 36: 43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Cancer, 83: 252-260 (2000) (describing "guided selection" technology to FR shuffling).

[0312] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al., J. Immunol. 151: 2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J. Immunol., 151: 2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Immunol., 151: 2623 (1993)). et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0313] 4. Human Antibodies

[0314] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0315] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. Such animals typically contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or are present outside the chromosomes or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, the description of the XENOMOUSE TMThe human variable regions of intact antibodies generated from such animals can be further modified, for example, by combining with different human constant regions.

[0316] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-191 (2005).

[0317] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. The technology of selecting human antibodies from antibody libraries is described below.

[0318] 5. Library-derived antibodies

[0319] Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity.For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are summarized in, e.g., Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001) and further described in, e.g., McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0320] In some phage display methods, repertoires of VH and VL genes are cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without the need for any immunization. Finally, as Hoogenboom and Winter, J.Mol.Biol., 227:381-388 (1992) described, also by not rearranging V gene fragments from stem cell clones, and use the PCR primer synthesis preparation original library that contains random sequence, to encode highly variable CDR3 and realize external rearrangement.The patent publication of describing people's antibody phage library comprises, for example: U.S. Patent number 5,750,373, and U.S. Patent Publication No. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0321] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0322] 6. Multispecific Antibodies

[0323] In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one binding specificity is for CTLA-4, and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of CTLA-4. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CTLA-4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0324] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs of different specificities (see Milstein and Cuello, Nature, 305: 537 (1983)), WO 93 / 08829, and Trauneckerd et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for preparing antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152: 5368-5376). (1994)); and the preparation of trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 147:60 (1991).

[0325] Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576A1).

[0326] The antibodies or fragments herein also include "dual-acting Fabs" or "DAFs," which comprise an antigen binding site that binds CTLA-4 as well as another, different antigen (see, eg, US 2008 / 0069820).

[0327] 7. Antibody variants

[0328] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct has desired characteristics, such as antigen binding.

[0329] a) Substitution, insertion, and deletion variants

[0330] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The target sites for substitution mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More essential changes are provided in Table 1 under the heading "Exemplary Substitutions," and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the target antibody, and the desired activity of the product can be screened, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0331] [Table 1]

[0332]

[0333] Amino acids can be divided into several groups based on the common properties of their side chains:

[0334] (1) Hydrophobicity: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu) and isoleucine (Ile);

[0335] (2) Neutral and hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn) and glutamine (Gln);

[0336] (3) Acidic: aspartic acid (Asp) and glutamic acid (Glu);

[0337] (4) Basic: histidine (His), lysine (Lys) and arginine (Arg);

[0338] (5) residues that affect chain orientation: glycine (Gly) and proline (Pro); and

[0339] (6) Aromatic: tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe).

[0340] Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0341] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties relative to the parent antibody from which they are derived. For example, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in one or more of the following: affinity for an antigen, reduced immunogenicity, and / or substantially retaining certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0342] Alterations (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such alterations can be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigen, where the binding affinity of the resulting variant VHand / or VLis tested. Affinity maturation by construction and selection from secondary libraries has been described, e.g., in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which a few HVR residues (e.g., 4-6 residues at a time) are randomized. For example, HVR residues involved in antigen binding can be specifically identified using, e.g., alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly frequently targeted.

[0343] In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) can be made in HVRs, which do not substantially reduce binding affinity. Such alterations can be outside of antigen contacting residues in the HVRs, for example. In certain embodiments of the variant VHand VLsequences provided above, each HVR is either unaltered, or contains no more than one, two, or three amino acid substitutions.

[0344] As described in Cunningham and Wells (1989) Science, 244: 1081-1085, a useful method for identifying residues or regions of antibodies that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis." In this method, a residue or group of target residues (e.g., charged residues, such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further replacements can be introduced at the amino acid positions that demonstrate functional sensitivity to the initial replacement. Alternatively or in addition, the crystal structure of the antigen-antibody complex can be analyzed to identify the contact points between the antibody and the antigen. This type of contact residue and adjacent residue can be targeted or eliminated as the candidate for replacement. Variants can be screened to determine whether they contain desired characteristics.

[0345] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion of an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antibody to the N- or C-terminus of the antibody.

[0346] b) Glycosylation variants

[0347] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0348] Where an antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides, which are typically attached to Asn297 of the CH2 domain of the Fc region via an N-linked bond. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to generate antibody variants with certain improved properties.

[0349] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the carbohydrate chain at Asn297 relative to the sum of all carbohydrate structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297 as determined by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variation in antibodies, Asn297 may also be located approximately + / - 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO 2003 / 085107).

[0350] Antibody variants are also provided with bisected oligosaccharides, for example, wherein a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0351] c) Fc region variants

[0352] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. An Fc region variant can be included in a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0353] In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACT1 for flow cytometry). TMNon-radioactive cell toxicity assays (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® (Registered Trademark) Non-radioactive cell toxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). Clq binding assays can also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., WO 2006 / 029879 and WO 2005 / 100402 for Clq and C3c binding ELISA. To assess complement activation, a CDC assay can be carried out (see, for example, Gazzano-Santoro et al. J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al. Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / kidney half-life determinations can also be carried out using methods known in the art (see, e.g., Petkova, S.B. et al. Int'l. Immunol. 18 (12): 1759-1769 (2006)).

[0354] Antibodies with reduced effector function include those in which one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0355] Certain antibody variants described that have increased or decreased binding to FcRs (see, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0356] In certain embodiments, antibody variants comprise one or more amino acid substitutions in the Fc region that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0357] In some embodiments, alterations are made in the Fc region that result in increased or decreased Clq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0358] Antibodies with increased half lives and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al. J. Immunol. 117: 587 (1976); and Kim et al. J. Immunol. 24:249 (1994)), are described in US 2005 / 0014934 Al (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0359] See also Duncan & Winter, Nature 322: 738-740 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for further examples of Fc region variants.

[0360] d) Cysteine engineered antibody variants

[0361] In certain embodiments, it is desirable to produce cysteine ​​engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In specific embodiments, the substituted residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be produced as described, for example, in U.S. Patent No. 7,521,541.

[0362] e) Antibody derivatives

[0363] In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties that are known and readily available in the art. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymers can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody may vary; if more than one polymer is attached, they may be the same or different molecules. In general, the amount and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under specific conditions, etc.

[0364] In another embodiment, a conjugate of an antibody and a non-protein moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein moiety to a temperature close to that at which the antibody-non-protein moiety cell is killed.

[0365] B. Recombinant Methods and Compositions

[0366] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acids encoding the anti-CTLA-4 antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of making an anti-CTLA-4 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody provided above under conditions suitable for expression of the anti-CTLA-4 antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0367] For recombinant production of anti-CTLA-4 antibodies, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell, e.g., as described above. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0368] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not desired. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli). After expression, the antibodies can be isolated from the bacterial cell pellet as a soluble fraction and can be further purified.

[0369] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0370] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0371] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing plant cell cultures for producing antibodies in transgenic plants). TM technology).

[0372] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be important. Other examples of important mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney line (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells as described in Matherd et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0373] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Bifunctional or derivatizing agents such as maleimidobenzoylsulfosuccinimide ester (conjugated through cysteine ​​residues), N-hydroxysuccinimide (conjugated through lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR (where R and R 1 It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

[0374] Animals (typically non-human mammals) are immunized against the antigen, immunogenic conjugate, or derivative by combining, for example, 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5 to 1 / 10 the initial amount of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animals are bled and the serum is assayed for antibody titers. The animals are boosted until the titer plateaus. Preferably, the animals are boosted with the same antigen, but conjugated to a different protein and / or via a different cross-linking agent. Conjugates can also be prepared as protein fusions in recombinant cell culture. In addition, agglutinating agents such as alum are also suitable for enhancing the immune response.

[0375] A monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0376] For example, monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature 256(5517): 495-497 (1975). In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0377] Immunizing agents typically include antigenic proteins or fusion variants thereof. Typically, if cells of human origin are required, peripheral blood lymphocytes (PBLs) are used, and if non-human mammalian origin is required, spleen cells or lymph node cells are used. Lymphocytes are then fused with immortalized cell lines using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0378] Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, cattle, and human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus prepared are inoculated and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium used for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT culture medium), which is a substance that prevents the growth of HGPRT-deficient cells.

[0379] Preferred immortalized myeloma cells are those that effectively fuse, support the selected antibody-producing cells to stably produce antibodies at high levels, and are sensitive to culture media such as HAT culture media. Among them, preferred are mouse myeloma lines, such as MOPC-21 and MPC-11 mouse tumors derived from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) available from the American Type Culture Collection, Manassas, Virginia USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for producing human monoclonal antibodies (Kozbor et al., J. Immunol. 133 (6): 3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0380] The production of monoclonal antibodies against the antigen in the culture medium in which the hybridoma cells are grown is measured. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis of Munson, Anal. Biochem. 107(1): 220-239 (1980).

[0381] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown as tumors in mammals.

[0382] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0383] Antibodies can be produced by immunizing an appropriate host animal against an antigen. In one embodiment, the antigen is a polypeptide comprising full-length CTLA-4. In one embodiment, the antigen is a polypeptide comprising soluble CTLA-4. In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acids from position 97 (Glu) to position 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acids from position 99 (Met) to position 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). Also included in the present invention are antibodies produced by immunizing an animal against an antigen. As described above in "Exemplary anti-CTLA-4 antibodies," the antibodies may bind to any of the features alone or in combination.

[0384] C. Determination

[0385] The anti-CTLA-4 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0386] 1. Binding Assays and Other Assays

[0387] In one aspect, the antigen binding activity of the antibodies of the invention is tested, for example, by known methods such as ELISA, Western blot, surface plasmon resonance assay, and the like.

[0388] In another aspect, competition assays can be used to identify antibodies that compete with the anti-CTLA-4 antibodies described herein (e.g., the anti-CTLA-4 antibodies described in Tables 4, 9, 14, and 19) for binding to CTLA-4. In certain embodiments, if such competing antibodies are present in excess, binding of the reference antibody to CTLA-4 is prevented (e.g., reduced) by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some examples, binding is prevented by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the epitope bound by the anti-CTLA-4 antibodies described herein (e.g., the anti-CTLA-4 antibodies described in Tables 4, 9, 14, and 19). Detailed exemplary methods for mapping epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology vol 66 (Humana Press, Totowa, NJ).

[0389] In an exemplary competition assay, immobilized CTLA-4 is incubated in a solution containing a first labeled antibody that binds to CTLA-4 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to CTLA-4. The second antibody can be present in the hybridoma supernatant. As a control, immobilized CTLA-4 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. Following incubation under conditions permissive for binding of the first antibody to CTLA-4, excess unbound antibody is removed, and the amount of label bound to the immobilized CTLA-4 is determined. If the amount of label bound to the immobilized CTLA-4 in the test sample is significantly reduced compared to the control sample, this indicates that the second antibody is competing with the first antibody for binding to CTLA-4. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0390] 2. Activity Assay

[0391] In one aspect, assays are provided for identifying anti-CTLA-4 antibodies that have biological activity. Biological activity can include, for example, cell proliferation inhibition activity, cytotoxic activity (e.g., ADCC / CDC activity and ADCP activity), immunostimulatory activity, and CTLA-4 inhibitory activity. Antibodies that have such biological activity in vivo and / or in vitro are also provided.

[0392] In certain embodiments, the antibodies of the invention are tested for such biological activities.

[0393] In certain embodiments, the antibodies of the invention are tested for their ability to inhibit cell growth or proliferation in vitro. Assays for inhibiting cell growth or proliferation are well known in the art. Certain assays for cell proliferation measure cell viability, as exemplified by the "cell killing" assay described herein. One such assay is the CellTiter-Glo TM Luminescent Cell Viability Assay, which is commercially available from Promega (Madison, WI). This assay determines the number of viable cells in the culture based on the quantification of the ATP present, which is an indicator of metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160: 81-88, US Pat. No. 6602677. The assay can be performed in 96-well or 384-well formats, making it suitable for automated high-throughput screening (HTS). See Cree et al. (1995) Anticancer Drugs 6: 398-404. The assay procedure involves adding a single reagent (CellTiter-Glo (registered trademark) reagent) directly to the cultured cells. This results in cell lysis and the generation of a luminescent signal generated by the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is proportional to the number of viable cells present in the culture. The data can be recorded by a luminometer or CCD camera imaging device. The luminescent output is expressed as relative light units (RLU).

[0394] Another assay for cell proliferation is the "MTT" assay, which is a colorimetric assay that measures the oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by mitochondrial reductases. TM As with the CAR T cell assay, this assay indicates the number of metabolically active cells present in a cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65: 55-63, and Zhang et al. (2005) Cancer Res. 65: 3877-3882.

[0395] Cells used in any of the above-described in vitro assays include cells or cell lines that naturally express CTLA-4 or that have been engineered to express CTLA-4. Such cells also include cell lines that express CTLA-4 and cell lines that do not normally express CTLA-4 but have been transfected with a nucleic acid encoding CTLA-4.

[0396] In one aspect, an anti-CTLA-4 antibody is tested for its ability to inhibit cell growth or proliferation in vivo. In certain embodiments, an anti-CTLA-4 antibody is tested for its ability to inhibit tumor growth in vivo. In vivo model systems, such as xenograft models, can be used for such testing. In exemplary xenograft systems, human tumor cells are introduced into a suitably immunocompromised non-human animal, such as an athymic "nude" mouse. An antibody of the application is administered to the animal. The ability of the antibody to inhibit or reduce tumor growth is determined. In certain embodiments of the above-described xenograft systems, the human tumor cells are tumor cells from a human patient. Such xenograft models are commercially available from Oncotest GmbH (Frieberg, Germany). In certain embodiments, the human tumor cells are introduced into the suitably immunocompromised non-human animal by subcutaneous injection or by implantation into a suitable site, such as the mammary fat pad.

[0397] It will be appreciated that any of the above-described assays can be performed using an immunoconjugate of the application instead of or in addition to an anti-CTLA-4 antibody.

[0398] A typical assay for determining the ADCC activity of a therapeutic antibody is based on a 51 Cr release assay and includes the following steps: labeling target cells with 51 Cr]Na2CrO4; opsonizing target cells expressing an antigen on their cell surface with an antibody; combining the opsonized, radiolabeled target cells with effector cells in the presence or absence of a test antibody in a microtiter plate at an appropriate ratio; incubating the cell mixture, preferably at 37°C, for a period of time, preferably 16 to 18 hours; collecting the supernatant; and analyzing the supernatant sample for radioactivity. The cytotoxicity of the test antibody is then determined, for example, by the following equation: % specific cytotoxicity = (radioactivity in the presence of antibody - radioactivity in the absence of antibody) / (maximum radioactivity - radioactivity in the absence of antibody) x 100. A graph can be generated by varying the ratio of target cells to effector cells or the concentration of antibody.

[0399] To assess complement activation, a complement-dependent cytotoxicity (CDC) assay can be performed as described, for example, in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Briefly, various concentrations of polypeptide variants and human complement are diluted with buffer. Cells expressing an antigen that binds to the polypeptide variant are diluted to approximately 1×10 6 The density of cells / ml was 200 μg / ml. A mixture of polypeptide variants, diluted human complement, and antigen-expressing cells was added to a flat-bottomed 96-well tissue culture plate and incubated at 37°C and 5% CO2 for 2 hours to promote complement-mediated cell lysis. Then, 50 μl of Alamar Blue (Accumed International) was added to each well and incubated overnight at 37°C. Absorbance was measured using a 96-well fluorimeter with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The results are expressed in relative fluorescence units (RFU). Sample concentrations can be calculated from the standard curve, and the percentage of activity compared to the non-variant polypeptide is reported for the polypeptide variant of interest.

[0400] An exemplary assay for ADCP activity may include the following: coating target bioparticles, such as E. coli labeled with FITC (Molecular Probes) or Staphylococcus aureus-FITC, with a test antibody; forming opsonized particles; adding the opsonized particles to THP-1 effector cells (a monocytic cell line available from ATCC) at a ratio of 1:1, 10:1, 30:1, 60:1, 75:1, or 100:1 to induce FcγR-mediated phagocytosis; preferably incubating the cells and E. coli-FITC / antibody at 37°C for 1.5 hours; following the incubation, adding trypan blue to the cells (preferably at room temperature for two to three minutes) to quench the fluorescence of bacteria that have not yet incorporated into the cells and are attached to the exterior of the cell surface; transferring the cells to FACS buffer (e.g., 0.1% BSA and 0.1% sodium azide in PBS) to measure the fluorescence of the THP-1 cells using FACS (e.g., BD FACS Calibur). To determine the extent of ADCP, a gate is preferably set on THP-1 cells and the median fluorescence intensity is determined. In a most preferred embodiment, ADCP assays are performed using E. coli-FITC in culture medium (control); E. coli-FITC and THP-1 cells (for FcγR-independent ADCP activity); and E. coli-FITC, THP-1 cells, and a test antibody (for FcγR-dependent ADCP activity).

[0401] The cytotoxic activity of an antibody generally involves binding of the antibody to the cell surface. Whether the antigen is expressed on the surface of the target cell can be appropriately confirmed by methods known to those skilled in the art, such as FACS.

[0402] The activation of immunity can be detected by using cellular or humoral immune responses as indicators. The activation of immunity specifically includes increased expression levels of cytokines (e.g., IL-6, G-CSF, IL-12, TNFα and IFNγ) or their receptors, promoting the proliferation of immune cells (e.g., B cells, T cells, NK cells, macrophages and monocytes), increased activation state, increased function and enhanced cytotoxic activity. In particular, T cell activation can be detected by measuring the increased expression of activation markers such as CD25, CD69 and ICOS. For example, it is known that patients administered with the anti-CTLA-4 antibody ipilimumab have increased ICOS in peripheral blood after administration. + CD4 + T cells, which is considered to be the activation effect of systemic immune status by administration of anti-CTLA-4 antibodies (Cancer Immunol. Res. (2013) 1(4): 229-234).

[0403] T cell activation requires not only stimulation via the antigen receptor (TCR) but also auxiliary stimulation via CD28. When CD28 on the T cell surface binds to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells, the auxiliary signal is transmitted to the T cell, leading to T cell activation. On the other hand, CTLA-4 is expressed on the surface of activated T cells. Because CTLA-4 binds to CD80 and CD86 with a stronger affinity than CD28, it interacts with CD80 and CD86 in preference to CD28, resulting in inhibition of T cell activation.

[0404] Based on this mechanism of action, inhibitory activity against CTLA-4 can be measured as the activity of inhibiting CTLA-4 binding to CD80 or CD86. In one embodiment, an assay for measuring inhibitory activity against CTLA-4 comprises the following steps: binding purified CTLA-4 protein to a support such as a microtiter plate or magnetic beads; adding a test antibody and labeled soluble CD80 or CD86; washing away unbound components; and quantifying bound labeled CD80 or CD86. Whether the test antibody cross-reacts with CD28 can be confirmed by using a similar assay in which CD28 replaces CTLA-4. In another embodiment, a functional assay for detecting T cell activation as described above can also be used to measure inhibitory activity against CTLA-4. For example, when a test antibody with CTLA-4 inhibitory activity is added to a system in which T cell activation is measured by stimulating a T cell population with cells expressing CD80 or CD86, T cell activation is further enhanced.

[0405] D. Immunoconjugates

[0406] The invention also provides immunoconjugates comprising an anti-CTLA-4 antibody herein conjugated to one or more cytotoxic agents, e.g., chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0407] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansine (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatin, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53: 3336-3342 (1993); and Lode et al., Cancer Res. 58: 2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chem. 16: 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tadalafil, and ortataxel; trichothecenes; and CC1065.

[0408] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0409] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce radioconjugates. Examples include 211 At 131 I. 125 I. 90 Y. 186 Re、 188 Re、 153 Sm, 212 Bi, 32 P. 212 When the radioconjugate is used for detection, it may contain a radioactive atom such as Tc-99m or 123 I, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0410] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl diimidoadipate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that promotes release of the cytotoxic drug. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0411] The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with cross-linking reagents including, but not limited to, commercially available BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0412] E. Methods and Compositions for Diagnosis and Detection

[0413] In certain embodiments, any of the anti-CTLA-4 antibodies provided herein can be used to detect the presence of CTLA-4 in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as serum, whole blood, plasma, biopsy samples, tissue samples, cell suspensions, saliva, sputum, oral fluid, cerebral fluid, amniotic fluid, ascites, milk, colostrum, mammary secretions, lymph, urine, sweat, tears, gastric fluid, synovial fluid, ascites, ocular fluid, and mucus.

[0414] In one embodiment, an anti-CTLA-4 antibody for use in a diagnostic or detection method is provided. In other aspects, a method for detecting the presence of CTLA-4 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-CTLA-4 antibody as described herein under conditions that allow the anti-CTLA-4 antibody to bind to CTLA-4, and detecting whether a complex is formed between the anti-CTLA-4 antibody and CTLA-4. This method can be an in vitro or in vivo method. In one embodiment, the anti-CTLA-4 antibody is used to select a subject suitable for treatment with the anti-CTLA-4 antibody, for example, where CTLA-4 is a biomarker for selecting a patient.

[0415] The antibodies of the present invention can be used, for example, to examine the state of immune response and diagnose immune system dysfunction.

[0416] In certain embodiments, labeled anti-CTLA-4 antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent, chromophore, electron-dense, chemiluminescent, and radiolabeled), as well as indirect detection, for example, moieties detected by enzymatic reactions or molecular interactions, such as enzymes or ligands. Exemplary labels include, but are not limited to, radioisotopes. 32 P. 14 C. 125 I. 3 H and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, those coupled to enzymes that use hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase or microperoxidase), biotin / avidin, spin labels, phage labels, stable free radicals, and the like.

[0417] F. Pharmaceutical Preparations

[0418] Pharmaceutical formulations of the anti-CTLA-4 antibodies described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 Residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0419] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0420] As necessary for the particular indication being treated, the formulations herein may also contain more than one active ingredient, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose.

[0421] The active ingredient can be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

[0422] Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0423] Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0424] G. Methods of Treatment and Compositions

[0425] Any of the anti-CTLA-4 antibodies provided herein can be used in therapeutic methods.

[0426] In one aspect, an anti-CTLA-4 antibody for use as a medicament is provided. In other aspects, an anti-CTLA-4 antibody for use in treating tumors is provided. In certain embodiments, an anti-CTLA-4 antibody for use in a method of treating a tumor is provided. In certain embodiments, the present invention provides an anti-CTLA-4 antibody for use in a method of treating a subject having a tumor, the method comprising administering to the subject an effective amount of the anti-CTLA-4 antibody. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, e.g., as described below. In other embodiments, the present invention provides an anti-CTLA-4 antibody for use in destroying cells. In certain embodiments, the present invention provides an anti-CTLA-4 antibody for use in a method of destroying cells in a subject, the method comprising administering to the subject an effective amount of the anti-CTLA-4 antibody to destroy the cells. In other embodiments, the present invention provides an anti-CTLA-4 antibody for use in immune activation. In certain embodiments, the present invention provides an anti-CTLA-4 antibody for use in a method of activating immunity in a subject, the method comprising administering to the subject an effective amount of the anti-CTLA-4 antibody to activate immunity. The "subject" according to any of the above embodiments is preferably a human.

[0427] In some embodiments, the tumor is a solid tumor. In solid tumors, tumor cells usually proliferate to form colonies, and tumor tissue is mainly formed by these cells. In addition, tumor tissue in living organisms is often infiltrated by immune cells such as lymphocytes, which also constitute a part of tumor tissue. In some embodiments, tumor tissue is infiltrated by immune cells, particularly regulatory T (Treg) cells. In one embodiment, damage to cells is caused by ADCC activity, CDC activity or ADCP activity. In one embodiment, cells expressing CTLA-4 on their cell surface are damaged. In other embodiments, cells to be damaged are Treg cells. In certain embodiments, Treg cells that have infiltrated into tumor tissue are damaged. In one embodiment, immunity is activated by damage to Treg cells (immunosuppression of Treg cells is cancelled). In other embodiments, immunity (particularly anti-tumor immunity) in tumor tissue is activated. In some embodiments, immune activation is T cell activation.

[0428] In other aspects, the extent of the pharmaceutical effect produced by the anti-CTLA-4 antibodies of the invention varies depending on the tissue of the individual. In certain embodiments, the extent of the effect varies depending on the concentration of the adenosine-containing compound in the tissue. In other embodiments, the effect is increased in tissues with high concentrations of the adenosine-containing compound compared to tissues with low concentrations of the adenosine-containing compound. Tissues with high concentrations of the adenosine-containing compound include, for example, tumor tissue. Tissues with low concentrations of the adenosine-containing compound include, for example, non-tumor tissues such as normal tissue. In some embodiments, immunity is more strongly activated in tumor tissue than in non-tumor tissue. This difference in response need not be observed for all doses of the anti-CTLA-4 antibody, but only for a specific dose range. In another embodiment, immunity is activated in tumor tissue at a lower dose than in non-tumor tissue. Furthermore, in another embodiment, a therapeutic effect is observed at a dose below that at which side effects are observed. In certain embodiments, the therapeutic effect is the expression of an anti-tumor effect (e.g., tumor regression and induction of cell death or inhibition of tumor cell growth), while the side effect is the development of an autoimmune disease (including damage to normal tissue due to an excessive immune response).

[0429] On the other hand, the extent of the drug effect produced by an anti-CTLA-4 antibody of the invention varies depending on whether it has an adenosine-containing compound-dependent binding activity to CTLA-4 (i.e., varies depending on the concentration of the adenosine-containing compound). In some embodiments, the anti-CTLA-4 antibody of the invention is an antibody whose binding activity to CTLA-4 increases with increasing concentration of the adenosine-containing compound. In some embodiments, a control anti-CTLA-4 antibody is an antibody that does not have CTLA-4 binding activity that is dependent on the concentration of the adenosine-containing compound. In certain embodiments, an antibody that does not have CTLA-4 binding activity that is dependent on the concentration of the adenosine-containing compound is an antibody in which the difference in CTLA-4 binding activity in the presence and absence of the compound is, for example, less than 2-fold, less than 1.8-fold, less than 1.5-fold, less than 1.3-fold, less than 1.2-fold, or less than 1.1-fold. It is desirable that the anti-CTLA-4 antibody of the invention and the control anti-CTLA-4 antibody have approximately the same CTLA-4 binding activity as each other in the presence of a sufficient amount of the adenosine-containing compound.

[0430] In certain aspects, the anti-CTLA-4 antibodies of the present invention and a control anti-CTLA-4 antibody differ in their effectiveness as drugs produced by each antibody. In certain embodiments, they differ in their effectiveness as drugs in tissues with low concentrations of adenosine-containing compounds. Tissues with low concentrations of adenosine-containing compounds include, for example, non-tumor tissues such as normal tissue. The anti-CTLA-4 antibodies can also be provided as pharmaceutical formulations containing the antibodies. In some embodiments, in tissues with low concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the present invention exhibit lower levels of immune activation compared to the control anti-CTLA-4 antibody. In some embodiments, in tissues with low concentrations of adenosine-containing compounds, the dose of the anti-CTLA-4 antibody of the present invention required for immune activation is higher than that of the control anti-CTLA-4 antibody. In some embodiments, in tissues with low concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the present invention exhibit lower levels of side effects compared to the control anti-CTLA-4 antibody. In some embodiments, side effects are observed at higher doses of an anti-CTLA-4 antibody of the invention compared to a control anti-CTLA-4 antibody in tissues with low concentrations of adenosine-containing compounds. This difference in response need not be observed in all tissues (e.g., all tissues with low concentrations of adenosine-containing compounds), but only in some tissues. In certain embodiments, the side effect is an autoimmune disorder (including damage to normal tissues due to an excessive immune response).

[0431] In certain aspects, the anti-CTLA-4 antibodies of the application and the control anti-CTLA-4 antibodies each produce essentially the same effect as a drug. In certain embodiments, they produce essentially the same effect as a drug in tissues having a high concentration of adenosine-containing compounds. Tissues having a high concentration of adenosine-containing compounds include, for example, tumor tissue. The anti-CTLA-4 antibodies can also be provided as a pharmaceutical formulation comprising the antibody. In some embodiments, the anti-CTLA-4 antibodies of the application and the control anti-CTLA-4 antibodies exhibit essentially equal levels of immune activation in tissues having a high concentration of adenosine-containing compounds. In some embodiments, the anti-CTLA-4 antibodies of the application and the control anti-CTLA-4 antibodies are essentially equal in the dose required to activate immunity in tissues having a high concentration of adenosine-containing compounds. In some embodiments, the anti-CTLA-4 antibodies of the application and the control anti-CTLA-4 antibodies have essentially the same level of therapeutic effect in tissues having a high concentration of adenosine-containing compounds. In some embodiments, the anti-CTLA-4 antibodies of the application and the control anti-CTLA-4 antibodies are essentially equal in the dose at which a therapeutic effect is observed in tissues having a high concentration of adenosine-containing compounds. In certain embodiments, the therapeutic effect is an expression of anti-tumor effect (e.g., tumor regression and induction of cell death or inhibition of growth of tumor cells).

[0432] In certain embodiments, the tumor is selected from the group consisting of breast cancer and liver cancer.

[0433] In other aspects, the application provides use of an anti-CTLA-4 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating a tumor. In other embodiments, the medicament is for use in a method of treating a tumor, the method comprising administering to an individual having a tumor an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In other embodiments, the medicament is for destroying a cell. In other embodiments, the medicament is for use in a method of destroying a cell in an individual, the method comprising administering to the individual an effective amount of the medicament to destroy the cell. In other embodiments, the medicament is for activating immunity. In other embodiments, the medicament is for use in a method of activating immunity in an individual, the method comprising administering to the individual an effective amount of the medicament to activate immunity. The “individual” according to any of the above embodiments can be a human.

[0434] In other aspects, the application provides a method for treating a tumor. In one embodiment, the method comprises administering to an individual having such a tumor an effective amount of an anti-CTLA-4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. The “individual” according to any of the above embodiments can be a human.

[0435] In other aspects, the present invention provides a method for destroying cells in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-CTLA-4 antibody to destroy the cells. In other aspects, the present invention provides a method for activating immunity in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-CTLA-4 antibody to activate immunity. In one embodiment, the "individual" is a human.

[0436] In other aspects, the present invention provides pharmaceutical formulations comprising any of the anti-CTLA-4 antibodies provided herein, e.g., for use in any of the above-described treatment methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-CTLA-4 antibodies provided herein and a pharmaceutically acceptable carrier. In one embodiment, the present invention provides pharmaceutical formulations for treating tumors. In one embodiment, the present invention provides pharmaceutical formulations for destroying cells. In one embodiment, the present invention provides pharmaceutical formulations for activating immunity. In another embodiment, the pharmaceutical formulation comprises any of the anti-CTLA-4 antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0437] In other aspects, the present invention provides methods for preparing a medicament or pharmaceutical formulation (e.g., for use in any of the above-described treatment methods), comprising mixing any of the anti-CTLA-4 antibodies provided herein with a pharmaceutically acceptable carrier. In one embodiment, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

[0438] The antibodies of the present invention can be used alone or in combination with other agents in treatment. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an immune checkpoint inhibitor, an EGFR inhibitor, a HER2 inhibitor, or a chemotherapeutic agent. Immune checkpoint inhibitors can include, for example, anti-CTLA-4 inhibitors, anti-PD-1 inhibitors, anti-PD-L1 inhibitors, anti-PD-L2 inhibitors, anti-TIM-3 inhibitors, anti-LAG-3 inhibitors, anti-TIGIT inhibitors, anti-BTLA inhibitors, and anti-VISTA inhibitors. Anti-CTLA-4 inhibitors can include, for example, ipilimumab and tremelimumab. Anti-PD-1 inhibitors can include, for example, nivolumab and pembrolizumab. Anti-PD-L1 inhibitors can include, for example, atezolizumab, durvalumab, and avelumab. Anti-PD-L2 inhibitors can include, for example, anti-PD-L2 inhibitory antibodies. Anti-TIM-3 inhibitors may include, for example, anti-TIM-3 inhibitory antibodies. Anti-LAG-3 inhibitors may include, for example, anti-LAG-3 inhibitory antibodies. Anti-TIGIT inhibitors may include, for example, anti-TIGIT inhibitory antibodies. Anti-BTLA inhibitors may include, for example, anti-BTLA inhibitory antibodies. Anti-VISTA inhibitors may include, for example, anti-VISTA inhibitory antibodies. EGFR inhibitors may include, for example, cetuximab, panitumumab, nimotuzumab, necitumumab, and zalutumumab. HER2 inhibitors may include, for example, trastuzumab, trastuzumab emtansine, and pertuzumab.

[0439] Such combination therapies include combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case the administration of the antibody of the invention can be prior to, concurrently with, and / or subsequent to the administration of the additional therapeutic agent or agents. In one embodiment, the administration of the anti-CTLA-4 antibody and the additional therapeutic agent occurs within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days. The antibodies of the invention can also be used in combination with radiation therapy.

[0440] The antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if necessary for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations, bolus administration and pulse infusions at multiple time points.

[0441] The antibodies of the present invention will be formulated, dosed, and applied in a manner consistent with good medical practice. Factors to consider in this case include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the medicament, the method of administration, the administration time arrangement, and other factors known to the doctor. The antibodies do not need to be formulated together with one or more medicaments currently used to prevent or treat the condition in question. The effective amount of such other medicaments depends on the amount of antibody present in the formulation, the type of condition or treatment, and other factors discussed above. These are typically used in the same dosage and route of administration as described herein, or in about 1% to 99% of the dosage described herein, or in any dosage and any route determined to be suitable by experience / clinical practice.

[0442] For the prevention or treatment of disease, the appropriate dosage of the antibodies of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is used for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitable for administration to the patient at one time or in a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) can be an initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. Depending on the above factors, a typical daily dose may vary in the range of about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dosage of the antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about 2 to about 20, or for example, about 6 doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. The progress of this treatment is easily monitored by conventional techniques and assays.

[0443] It will be understood that any of the above-described formulations or treatment methods can be performed using the immunoconjugates of the invention in place of or in addition to anti-CTLA-4 antibodies.

[0444] H. Products

[0445] In another aspect of the application, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the above disorders is provided. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition, which is by itself or in combination with another composition effective for treating, preventing and / or diagnosing the condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is an antibody of the application. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture can further comprise (a) a first container wherein there is a composition contained, which comprises an antibody of the application; and (b) a second container wherein there is a composition contained, which comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture of this embodiment of the application can further include a package insert indicating that the compositions can be used to treat the particular condition. Alternatively or additionally, the article of manufacture can further include a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and

[0446] It is understood that any of the above articles of manufacture can include an immunoconjugate of the application in place of or in addition to an anti-CTLA-4 antibody.

[0447] <Polypeptides comprising variant Fc regions>

[0448] In one aspect, the application provides an isolated polypeptide comprising a variant Fc region. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the variant Fc region comprises at least one amino acid residue alteration (e.g., substitution) as compared to the corresponding sequence in a Fc region of a native sequence or a reference variant sequence (which can be collectively referred to herein as a "parental" Fc region). A Fc region of a native sequence typically consists of a homodimer of two identical polypeptide chains. The amino acid alterations in the variant Fc regions of the application can be introduced into either of the two polypeptide chains of the parental Fc region, or into both of the two polypeptide chains.

[0449] In some aspects, the present invention provides variant Fc regions whose function has been modified compared to the parent Fc region. In some aspects, the variant Fc region of the present invention has enhanced binding activity to Fcγ receptors compared to the parent Fc region. In certain embodiments, the variant Fc region of the present invention has enhanced binding activity to at least one Fcγ receptor selected from FcγRIa, FcγRIIa, FcγRIIb and FcγRIIIa compared to the parent Fc region. In some embodiments, the variant Fc region of the present invention has enhanced binding activity to FcγRIIa. In some embodiments, the variant Fc region of the present invention has enhanced binding activity to FcγRIIIa. In other embodiments, the variant Fc region of the present invention has enhanced binding activity to FcγRIIa and FcγRIIIa.

[0450] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration at at least one position selected from EU numbering positions 234, 235, 236, 298, 330, 332, and 334. Alternatively, amino acid alterations such as those described in WO 2013 / 002362 and WO 2014 / 104165 can similarly be used in the present invention.

[0451] In certain embodiments, the binding activity of the parent Fc region and the variant Fc region can be expressed as a KD (dissociation constant) value. In one embodiment, the ratio of [KD value of the parent Fc region for FcγRIIa] / [KD value of the variant Fc region for FcγRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In other embodiments, FcγRIIa can be FcγRIIa R or FcγRIIa H, or both. In one embodiment, the ratio of [binding activity of the parent Fc region to FcγRIIIa] / [binding activity of the variant Fc region to FcγRIIIa] has a value of, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1 x 10 3 or higher, 2 x 10 3 or higher, 3 x 10 3 or higher, or 5 x 10 3 In other embodiments, the FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both.

[0452] In one embodiment, the variant Fc region has a KD value for FcγRIIa of, for example, 1.0 x 10 -6 M or lower, 5.0x 10 -7 M or lower, 3.0 x 10 -7 M or lower, 2.0 x 10 -7 M or lower, 1.0 x 10 -7 M or smaller, 5.0 x10 -8 M or lower, 3.0 x 10 -8 M or lower, 2.0 x 10 -8 M or lower, 1.0 x 10 -8 M or smaller, 5.0 x 10 -9 M or lower, 3.0 x 10 -9 M or lower, 2.0 x 10 -9 M or lower, or 1.0 x 10 -9 M or less. In other embodiments, FcγRIIa can be FcγRIIa R or FcγRIIa H, or both. In one embodiment, the variant Fc region has a KD value for FcγRIIIa of, for example, 1.0 x 10 -6 M or smaller, 5.0 x 10 -7 M or lower, 3.0 x10 -7 M or lower, 2.0 x 10 -7 M or lower, 1.0 x 10 -7 M or smaller, 5.0 x 10 -8 M or lower, 3.0 x 10 -8 M or lower, 2.0 x 10 -8 M or lower, 1.0 x10 -8 M or smaller, 5.0 x 10 -9 M or lower, 3.0 x 10 -9 M or lower, 2.0 x 10 -9 M or lower, 1.0 x 10 -9 M or smaller, 5.0 x 10 -10 M or lower, 3.0 x 10 -10 M or lower, 2.0 x 10 -10 M or lower, or 1.0 x 10 -10 M or less. In other embodiments, the FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both.

[0453] In another embodiment, the binding activity of the parent and variant Fc regions may be expressed in terms of kd (dissociation rate constant) values ​​rather than KD values.

[0454] In another embodiment, the binding activity of the parent and variant Fc regions can be represented by the amount of binding of the Fc region to the Fcγ receptor per unit amount. For example, in a surface plasmon resonance assay, the binding amount of the Fc region fixed on the sensor chip and the binding amount of the Fcγ receptor further bound thereto are each measured as resonance units (RU). The value obtained by dividing the binding amount of the Fcγ receptor by the binding amount of the Fc region is defined as the binding amount of the Fc region to the Fcγ receptor per unit amount. Specific methods for measuring and calculating such binding amounts are described in the following examples. In some embodiments, the value of the ratio of [binding amount of variant Fc region to FcγRIIa] / [binding amount of parent Fc region to FcγRIIa] is, for example, 1.5 or higher, 2 or higher, 3 or higher, 4 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher, 10 or higher, 15 or higher, 20 or higher, 25 or higher, 30 or higher, or 40 or higher, or 50 or higher. In some embodiments, the ratio of [the amount of binding of the variant Fc region to FcγRIIIa] / [the amount of binding of the parent Fc region to FcγRIIIa] has a value of, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1 x 10 3 or higher, 2 x 10 3 or higher, 3 x 10 3 or higher, or 5 x 10 3 or higher.

[0455] In certain embodiments, KD values, kd values, binding magnitude values, etc. described herein are determined or calculated by surface plasmon resonance assays performed at 25°C or 37°C (see, eg, Example 6 herein).

[0456] In certain aspects, the variant Fc regions of the present invention have improved selectivity between activating and inhibitory Fcγ receptors compared to the parent Fc region. In other words, the variant Fc regions of the present invention have significantly improved binding activity for activating Fcγ receptors compared to inhibitory Fcγ receptors compared to the parent Fc region. In certain embodiments, the activating Fcγ receptor is at least one Fcγ receptor selected from FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, and FcγRIIIa V, and the inhibitory Fcγ receptor is FcγRIIb. In some embodiments, the variant Fc regions of the present invention have improved selectivity between FcγRIIa and FcγRIIb. In some embodiments, the variant Fc regions of the present invention have improved selectivity between FcγRIIIa and FcγRIIb. In other embodiments, the variant Fc regions of the invention have improved selectivity between FcγRIIa and FcγRIIb, and between FcγRIIIa and FcγRIIb.

[0457] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration at at least one position selected from positions 236, 239, 268, 270, and 326 according to EU numbering. Alternatively, the amino acid alterations described in WO 2013 / 002362 and WO 2014 / 104165 can be similarly used in the present invention.

[0458] In certain embodiments, the binding activity of the parent and variant Fc regions can be represented by KD (dissociation constant) values. Embodiments of binding activity to FcγRIIa and FcγRIIIa are described above. In one embodiment, the ratio of [KD value of the parent Fc region to FcγRIIb] / [KD value of the variant Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In another embodiment, the binding activity of the parent and variant Fc regions can be represented by kd (dissociation rate constant) values ​​instead of KD values.

[0459] In another embodiment, the binding activity of the parent and variant Fc regions can be represented by the above-mentioned binding amount of the Fc region to the Fcγ receptor per unit amount. In some embodiments, the value of the ratio of [the amount of binding of the variant Fc region to FcγRIIb] / [the amount of binding of the parent Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In some embodiments, the amount of binding of the variant Fc region to FcγRIIb is, for example, 0.5 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.005 or less, 0.003 or less, 0.002 or less, or 0.001 or less.

[0460] In some aspects, the variant Fc district of the present invention has improved stability compared to the parent Fc district. In certain embodiments, stability is thermodynamic stability. For example, the thermodynamic stability of a polypeptide can be determined by using a Tm value as an indicator. The Tm value can be determined using techniques known to those skilled in the art, such as circular dichroism (CD), differential scanning calorimetry (DSC), and differential scanning fluorimetry (DSF). In one embodiment, the Tm value in the CH2 district is increased by 0.1 degree or higher, 0.2 degree or higher, 0.3 degree or higher, 0.4 degree or higher, 0.5 degree or higher, 1 degree or higher, 2 degrees or higher, 3 degrees or higher, 4 degrees or higher, 5 degrees or higher, or 10 degrees or higher compared to the parent Fc district.

[0461] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration at at least one position selected from positions 250 and 307 according to EU numbering. Alternatively, the amino acid alterations described in WO 2013 / 118858 can be similarly used in the present invention.

[0462] In certain aspects, the variant Fc region of the present invention is composed of two polypeptide chains having different sequences. In other aspects, the variant Fc region of the present invention promotes heterodimerization between the first polypeptide and the second polypeptide. When using recombinant methods to produce heterodimeric proteins, it is preferred that different peptide chains preferentially associate to form heterodimers, rather than identical polypeptide chains associate to form homodimers. For example, by separating homodimers and heterodimers from the produced variant Fc region using techniques such as chromatography, and determining the ratio of each component, it can be determined whether heterodimerization is promoted in the variant Fc region.

[0463] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration at at least one position selected from positions 349, 356, 366, 368, 407, and 439 according to EU numbering. Alternatively, the amino acid alterations described in WO 2006 / 106905 and WO 1996 / 027011 can be similarly used in the present invention.

[0464] In certain aspects, the variant Fc regions of the present invention have enhanced binding activity to FcRn at acidic pH. In some embodiments, acidic pH refers to pH 4.0 to 6.5. In other embodiments, acidic pH is at least one selected from 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.

[0465] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration at at least one position selected from positions 428, 434, 436, 438, and 440 according to EU numbering. Alternatively, the amino acid alterations described in WO 2016 / 125495 can be similarly used in the present invention.

[0466] In one aspect, the variant Fc region of the invention comprises at least one amino acid alteration 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 according to EU numbering.

[0467] In certain aspects, the variant Fc regions of the invention comprise amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 334, according to EU numbering. In other aspects, the variant Fc regions comprise amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, according to EU numbering, in (i) a first polypeptide of a parent Fc region, and (ii) amino acid alterations at positions 236, 250, 270, 298, 307, 326, and 334, according to EU numbering, in a second polypeptide of a parent Fc region.

[0468] In another aspect, the variant Fc region of the invention further comprises an amino acid alteration at EU numbering position 332. In other aspects, the variant Fc region comprises an amino acid alteration at EU numbering position 332 in the first polypeptide of the parent Fc region.

[0469] In another aspect, the variant Fc region of the invention further comprises an amino acid alteration at position 356 according to EU numbering. In other aspects, the variant Fc region comprises an amino acid alteration at position 356 according to EU numbering in the first polypeptide of the parent Fc region.

[0470] In another aspect, the variant Fc region of the invention further comprises an amino acid alteration at position 366 according to EU numbering. In other aspects, the variant Fc region comprises an amino acid alteration at position 366 according to EU numbering in the first polypeptide of the parent Fc region.

[0471] In another aspect, the variant Fc region of the invention further comprises an amino acid alteration at position 349 according to EU numbering. In other aspects, the variant Fc region comprises an amino acid alteration at position 349 according to EU numbering in the first polypeptide of the parent Fc region.

[0472] In another aspect, the variant Fc region of the invention further comprises an amino acid alteration at EU numbering position 332. In other aspects, the variant Fc region comprises an amino acid alteration at EU numbering position 332 in the second polypeptide of the parent Fc region.

[0473] In another aspect, the variant Fc region of the invention further comprises an amino acid alteration at position 330 according to EU numbering. In other aspects, the variant Fc region comprises an amino acid alteration at position 330 according to EU numbering in the second polypeptide of the parent Fc region.

[0474] In another aspect, the variant Fc region of the invention further comprises an amino acid change at EU numbering position 439. In other aspects, the variant Fc region comprises an amino acid change at EU numbering position 439 in the second polypeptide of the parent Fc region.

[0475] In another aspect, the variant Fc region of the invention further comprises amino acid alterations at positions 366, 368, and 407 according to EU numbering. In other aspects, the variant Fc region comprises amino acid alterations at positions 366, 368, and 407 according to EU numbering in the second polypeptide of the parent Fc region.

[0476] In another aspect, the variant Fc region of the invention further comprises an amino acid alteration at EU numbering position 356. In other aspects, the variant Fc region comprises an amino acid alteration at EU numbering position 356 in the second polypeptide of the parent Fc region.

[0477] In some aspects, the variant Fc regions of the invention comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 334, 349, 356, 366, 368, and 407. In other aspects, the variant Fc regions comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366 in (i) a first polypeptide of a parent Fc region and (ii) at EU numbering positions 236, 250, 270, 298, 307, 326, 334, 356, 366, 368, and 407 in a second polypeptide of a parent Fc region.

[0478] In some aspects, the variant Fc regions of the invention comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 334, 356, and 439. In other aspects, the variant Fc regions comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 356 in (i) a first polypeptide of a parent Fc region, and (ii) at EU numbering positions 236, 250, 270, 298, 307, 326, 334, and 439 in a second polypeptide of a parent Fc region.

[0479] In some aspects, the variant Fc regions of the invention comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, and 407. In other aspects, the variant Fc regions comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366 in (i) a first polypeptide of a parent Fc region and (ii) at EU numbering positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 356, 366, 368, and 407 in a second polypeptide of a parent Fc region.

[0480] In some aspects, the variant Fc regions of the invention comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 356, and 439. In other aspects, the variant Fc regions comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 356 in (i) a first polypeptide of a parent Fc region and (ii) at EU numbering positions 236, 250, 270, 298, 307, 326, 330, 332, 334, and 439 in a second polypeptide of a parent Fc region.

[0481] In some aspects, the variant Fc regions of the invention comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 349, 356, 366, 368, and 407. In other aspects, the variant Fc regions comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 349, and 366 in (i) a first polypeptide of a parent Fc region and (ii) at EU numbering positions 236, 250, 270, 298, 307, 326, 332, 334, 356, 366, 368, and 407 in a second polypeptide of a parent Fc region.

[0482] In some aspects, the variant Fc regions of the invention comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 356, and 439. In other aspects, the variant Fc regions comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 356 in (i) a first polypeptide of a parent Fc region and (ii) at EU numbering positions 236, 250, 270, 298, 307, 326, 332, 334, and 439 in a second polypeptide of a parent Fc region.

[0483] In some aspects, the variant Fc regions of the invention comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 366, 368, and 407. In other aspects, the variant Fc regions comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 366 in (i) a first polypeptide of a parent Fc region and (ii) at EU numbering positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 366, 368, and 407 in a second polypeptide of a parent Fc region.

[0484] In some aspects, the Fc regions of the invention comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 366, 368, and 407. In other aspects, the variant Fc regions comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 366 in (i) a first polypeptide of a parent Fc region and (ii) at EU numbering positions 236, 250, 270, 298, 307, 326, 332, 334, 366, 368, and 407 in a second polypeptide of a parent Fc region.

[0485] In some aspects, the variant Fc regions of the invention comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, and 407. In other aspects, the variant Fc regions comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366 in (i) a first polypeptide of a parent Fc region and (ii) at EU numbering positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 356, 366, 368, and 407 in a second polypeptide of a parent Fc region.

[0486] In some aspects, the variant Fc regions of the invention comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 349, 356, 366, 368, and 407. In other aspects, the variant Fc regions comprise amino acid alterations at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 349, and 366 in (i) a first polypeptide of a parent Fc region and (ii) at EU numbering positions 236, 250, 270, 298, 307, 326, 332, 334, 356, 366, 368, and 407 in a second polypeptide of a parent Fc region.

[0487] In other embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration selected from the group consisting of: (i) Tyr or 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, Asp at position 326, Glu at position 332, Cys at position 349, Lys at position 356, and Trp at position 366 in the first polypeptide of the parent Fc region according to EU numbering; and (ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met or Lys at position 330, Asp or Glu at position 332, Glu at position 334, Cys at position 356, Ser at position 366, Ala at position 368, Val at position 407, and Glu at position 439 in the second polypeptide of the parent Fc region according to EU numbering.

[0488] In other aspects, the variant Fc region of the present invention further comprises any one of the following amino acid changes (a) to (d):

[0489] (a) Ala at position 434 according to EU numbering;

[0490] (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440 according to EU numbering;

[0491] (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to EU numbering; and

[0492] (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 according to EU numbering.

[0493] In other embodiments, the present invention provides a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 43 to 46, 65, 66, 81, 207, 239, 253 to 271, 276, 277 and 278.

[0494] "Fcγ receptor" (referred to herein as Fcγ receptor, FcγR, or FcgR) refers to a receptor that can bind the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and refers to virtually any member of the protein family encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 (H-type) and R131 (R-type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as, but not limited to, any human FcγR, FcγR isoform, or allotype yet to be discovered. FcγRIIb1 and FcγRIIb2 have been reported as splice variants of human FcγRIIb. In addition, a splice variant named FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splice variants, human FcγRIIb also includes all splice variants registered in NCBI, namely NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. Furthermore, human FcγRIIb includes each previously reported genetic polymorphism, as well as FcγRIIb (Arthritis Rheum. 48: 3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14: 2881-2892 (2005); and Kyogoju et al., Arthritis Rheum. 46: 1242-1254 (2002)), as well as each genetic polymorphism to be reported in the future.

[0495] FcγRIIa has two isoforms, one in which the amino acid at position 131 of FcγRIIa is histidine (H type) and the other in which the amino acid at position 131 is substituted with arginine (R type) (Warrmerdam, J. Exp. Med. 172: 19-25 (1990)).

[0496] FcγRs include, but are not limited to, those derived from humans, mice, rats, rabbits, and monkeys, and may be derived from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isotype.

[0497] The amino acid sequence of human FcγRI is shown in SEQ ID NO: 131 (NP_000557.1); the amino acid sequence of human FcγRIIa is shown in SEQ ID NO: 132 (AAH20823.1), SEQ ID NO: 142, SEQ ID NO: 143 or SEQ ID NO: 150; the amino acid sequence of human FcγRIIb is shown in SEQ ID NO: 151 (AAI46679.1), SEQ ID NO: 169 or SEQ ID NO: 172; the amino acid sequence of human FcγRIIIa is shown in SEQ ID NO: 174 (AAH33678.1), SEQ ID NO: 175, SEQ ID NO: 176 or SEQ ID NO: 177; and the amino acid sequence of human FcγRIIIb is shown in SEQ ID NO: 178 (AAI28563.1).

[0498] Unlike Fcγ receptors, which belong to the immunoglobulin superfamily, human FcRn is structurally similar to polypeptides of the class I major histocompatibility complex (MHC), displaying 22% to 29% sequence identity with class I MHC molecules (Ghetie et al., Immunol. Today (1997) 18 (12): 592-598). FcRn is expressed as a heterodimer composed of a soluble β chain, or light chain (β2 microglobulin), and a transmembrane α chain, or heavy chain. Like MHC, the FcRn α chain contains three extracellular domains (α1, α2, and α3), with a short cytoplasmic domain anchoring the protein to the cell surface. The α1 and α2 domains interact with the FcRn binding domain of the antibody Fc region (Raghavan et al., Immunity (1994) 1: 303-315). The amino acid sequence of human FcRn is shown in SEQ ID NO: 179 (NP_004098.1), and the amino acid sequence of β2 microglobulin is shown in SEQ ID NO: 180.

[0499] As used herein, "parent Fc region" refers to the Fc region prior to the introduction of the amino acid changes described herein. In some embodiments, the parent Fc region is a native sequence Fc region (or the Fc region of a native antibody). Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM. Antibodies can be derived from humans or monkeys (e.g., cynomolgus monkeys, rhesus macaques, marmosets, chimpanzees, or baboons). Natural antibodies can include naturally occurring mutations. Multiple allotype sequences of IgG resulting from genetic polymorphisms are described in "Sequences of protein of immunological interest," NIH Publication No. 91-3242, and any of these can be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356 to 358 (EU numbering) can be DEL or EEM. In certain embodiments, the parent Fc region is an Fc region derived from the heavy chain constant region of human IgG1 (SEQ ID NO: 249), human IgG2 (SEQ ID NO: 250), human IgG3 (SEQ ID NO: 251), or human IgG4 (SEQ ID NO: 252). In another embodiment, the parent Fc region is an Fc region derived from the heavy chain constant region of SEQ ID NO: 82 or SEQ ID NO: 158. In other embodiments, the parent Fc region may be an Fc region generated by adding amino acid alterations other than those described herein to a native sequence Fc region (referenced to the Fc region of the variant sequence). Native sequence Fc regions are typically organized as homodimers composed of two identical polypeptide chains.

[0500] Furthermore, amino acid changes made for other purposes may be combined in the variant Fc regions described herein. For example, amino acid substitutions that increase FcRn binding activity can be added (Hinton et al., J. Immunol. 176(1): 346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33): 23514-23524 (2006); Petkova et al., Intl. Immunol. 18(12): 1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2): 157-159 (2010); WO 2006 / 019447; WO 2006 / 053301; and WO 2009 / 086320), as well as amino acid substitutions for improving antibody heterogeneity or stability (WO 2009 / 041613). Alternatively, the polypeptides described in WO 2011 / 122011, WO 2012 / 132067, WO 2013 / 046704 or WO 2013 / 180201 with the characteristics of promoting antigen clearance, the polypeptides described in WO 2013 / 180200 with specific binding characteristics to target tissues, the polypeptides described in WO 2009 / 125825, WO 2012 / 073992 or WO 2013 / 047752 with the characteristics of repeating binding to multiple antigen molecules can be combined with variant Fc regions as described herein. Alternatively, for the purpose of imparting binding ability with other antigens, the amino acid changes disclosed in EP1752471 and EP1772465 can be combined in the CH3 of variant Fc regions as described herein. Alternatively, for the purpose of increasing plasma retention, the amino acid changes (WO 2012 / 016227) that reduce the pI of the constant region can be combined in variant Fc regions as described herein. Alternatively, for the purpose of promoting uptake into cells, amino acid changes that increase the pI of the constant region (WO 2014 / 145159) can be combined in the variant Fc regions described herein. Alternatively, for the purpose of promoting elimination of the target molecule from plasma, amino acid changes that increase the pI of the constant region (WO 2016 / 125495) can be combined in the variant Fc regions described herein. In one embodiment, such changes may include, for example, substitutions at at least one position selected from positions 311, 343, 384, 399, 400, and 413 according to EU numbering. In other embodiments, such substitutions may be amino acid replacements with Lys or Arg at each position.

[0501] Furthermore, the heterodimerization antibody production technology using the association of antibody CH1 and CL and the association of VH and VL described in WO 2011 / 028952 can also be used.

[0502] As using the methods described in WO 2008 / 119353 and WO 2011 / 131746, a technique for producing heterodimeric antibodies by previously producing two types of homodimeric antibodies, incubating the antibodies under reducing conditions to dissociate them, and allowing them to associate again can also be used.

[0503] Furthermore, techniques for generating heterodimeric antibodies by adding changes to the CH2 and CH3 domains, such as using the methods described in WO 2012 / 058768, can also be used.

[0504] When expressing two kinds of polypeptides comprising variant Fc districts with different amino acid sequences simultaneously, in order to produce the polypeptide comprising heterologous variant Fc district, the polypeptide comprising homologous variant Fc district is also usually produced as impurity.In this case, the polypeptide comprising heterologous variant Fc district can be effectively obtained by using known technology to separate and purify them from the polypeptide comprising homologous variant Fc district.It has been reported that ion exchange chromatography is used to effectively separate and purify the method for heterodimerization antibody from homodimerization antibody, and this method is introduced into the variable region of two types of antibody heavy chains by amino acid changes to produce isoelectric point difference (WO 2007 / 114325) between homodimerization antibody and heterodimerization antibody.Another method has been reported to comprise the heterodimerization antibody of two types of heavy chains of mouse IgG2a and rat IgG2b not in conjunction with protein A by building, and protein A chromatography is used to purify heterodimerization antibody (WO 1998 / 050431 and WO1995 / 033844).

[0505] Furthermore, heterodimeric antibodies can be efficiently purified using Protein A chromatography by substituting amino acid residues at positions 435 and 436 (EU numbering) of the Protein A binding site of the antibody heavy chain with amino acids such as Tyr or His to generate different Protein A binding affinities.

[0506] In the present invention, amino acid changes refer to any one of substitution, deletion, addition, insertion and modification, or a combination thereof. In the present invention, amino acid changes can be restated as amino acid mutations.

[0507] The number of amino acid changes introduced into the Fc region is not limited. In certain embodiments, it can be 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 8 or less, 10 or less, 12 or less, 14 or less, 16 or less, 18 or less, or 20 or less.

[0508] In one aspect, the present invention provides methods for producing polypeptides comprising variant Fc regions. In other aspects, the present invention provides methods for producing polypeptides comprising variant Fc regions whose functions have been modified. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, those methods include introducing at least one amino acid change into a parent Fc region. In certain embodiments, those methods include: (i) providing a polypeptide comprising a parent Fc region; and (ii) introducing at least one amino acid change into a parent Fc region. In certain embodiments, those methods may further include (iii) determining the function of the polypeptide comprising the variant Fc region. A native Fc region is typically composed of two identical polypeptide chains. Amino acid changes in the parent Fc region can be introduced into one of the two polypeptide chains of the parent Fc region, or into both polypeptide chains.

[0509] In another embodiment, a method for producing a polypeptide comprising a variant Fc region comprises: (i) providing one or more nucleic acids encoding a polypeptide comprising a parent Fc region; (ii) introducing at least one mutation into a region of the parent Fc region in the nucleic acid encoding the nucleic acid; (iii) introducing the nucleic acid produced in (ii) into a host cell; and (iv) culturing the cell described in (iii) to express the polypeptide comprising the variant Fc region. In certain embodiments, the above method may further comprise (v) collecting the polypeptide comprising the variant Fc region from the host cell culture described in (iv).

[0510] In certain embodiments, the nucleic acid produced in (ii) can be included in one or more vectors (eg, expression vectors).

[0511] In some embodiments, the amino acid alterations used in the production methods of the present invention are selected from any single alteration, combination of single alterations, or combination alterations selected from the amino acid alterations that can be contained in the variant Fc regions described above.

[0512] The Fc region can be obtained by re-eluting the fraction adsorbed on a protein A column after partial digestion of IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc., using a protease such as pepsin. The protease is not particularly limited as long as it can digest the full-length antibody to produce Fab and F(ab')2 in a restricted manner by appropriately setting the enzyme reaction conditions such as pH, and examples include pepsin and papain.

[0513] In addition to the above-mentioned production methods, the polypeptides comprising variant Fc regions of the present invention can also be produced by other methods known in the art. Polypeptides comprising variant Fc regions produced by the production methods described herein are also included in the present invention.

[0514] The assays described herein or various assays known in the art can be used to identify or screen the variant Fc regions provided herein, or to elucidate their physical or chemical properties or biological activities.

[0515] Assays for determining the binding activity of polypeptides containing variant Fc regions to one or more FcR family members are described herein or otherwise known in the art. Such binding assays include, but are not limited to, surface plasmon resonance assays, amplified luminescent proximity homogeneous assay (ALPHA) screening, ELISA, and fluorescence activated cell sorting (FACS) (Lazar et al., Proc. Natl. Acad. Sci. USA (2006) 103(11): 4005-4010).

[0516] In one embodiment, the binding activity of polypeptides comprising a variant Fc region to FcR family members can be measured using surface plasmon resonance (SPR) assays. For example, various FcRs can be used as analytes to interact with polypeptides comprising a variant Fc region, which have been immobilized or captured on a sensor chip using known methods and reagents (e.g., Protein A, Protein L, Protein A / G, Protein G, anti-λ chain antibodies, anti-κ chain antibodies, antigenic peptides, and antigenic proteins). Alternatively, FcRs can be immobilized or captured on a sensor chip, and polypeptides comprising a variant Fc region can be used as analytes. Binding sensorgrams are obtained as a result of this interaction, and by analyzing these, the dissociation constant (KD) value for this binding can be calculated. Furthermore, the difference in resonance unit (RU) values ​​in the sensorgrams before and after interaction with the FcR (i.e., the amount of FcR bound) can be used as an indicator of the binding activity of the polypeptide comprising a variant Fc region to the FcR. Furthermore, a correction value obtained by dividing the above-mentioned amount of FcR binding (i.e., the amount of binding of a polypeptide comprising a variant Fc region) by the difference in RU values ​​in the sensorgrams before and after the polypeptide comprising a variant Fc region is immobilized or captured on the sensor chip (i.e., the correction value is the amount of FcR binding per unit amount of the polypeptide comprising a variant Fc region) can be used as an indicator of binding activity.

[0517] In other aspects, the present invention provides a pharmaceutical formulation comprising a polypeptide comprising a variant Fc region provided herein. In one embodiment, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier.

[0518] Example

[0519] Examples of methods and compositions of the present invention are shown below.It is understood that various other embodiments may be performed, given the above general description.

[0520] [Example 0] Concept of a switch antibody that exerts antibody-dependent cellular cytotoxicity activity against cell surface markers of regulatory T cells only in the cancer microenvironment

[0521] Ipilimumab is thought to exert its anti-tumor effects by inhibiting effector T cell activation through CTLA4 expressed on the surface of effector T cells; however, antibody-dependent cellular cytotoxicity (ADCC) activity against CTLA4-expressing T cells has recently been reported to be important, and depletion of regulatory T cells in tumors and ADCC activity have been found to be important mechanisms of the anti-tumor effects of anti-CTLA4 antibodies.

[0522] Furthermore, it is known that the ADCC activity of IgG1 antibodies is the result of the binding of the antibody constant region to FcγR of NK cells and macrophages, thereby inducing cytotoxic activity. Antibodies having constant regions modified to enhance this binding induce stronger cytotoxic activity and exert anti-tumor effects.

[0523] On the other hand, it has been reported that systemic depletion of regulatory T cells causes autoimmune disease-like systemic reactions, and it is believed that regulation of the balance between cytotoxic activity and systemic reactions for exerting antitumor effects is important.

[0524] More specifically, antibodies that can strongly bind to regulatory T cells or T cells depleted in the cancer microenvironment are expected to exert more effective cytotoxic activity and suppress systemic responses, thereby achieving effective anti-tumor effects by removing regulatory T cells or T cells depleted of cytotoxic activity and limiting responses to the cancer microenvironment. Antibodies with this mechanism of action have not yet been reported. Therefore, we have generated and validated an antibody (CTLA4 switch antibody) that acts only locally on CTLA4 in tumors and contains a constant region that enhances binding to FcγR(s) expressed on NK cells and macrophages.

[0525] [Example 1] Obtaining Antibodies Binding to Antigens in the Presence of ATP or Its Metabolites from Naive and Rationally Designed Antibody Libraries Using Phage Display Technology

[0526] (1-1) Preparing antigens to obtain antibodies that bind to the antigen in the presence of small molecules

[0527] Biotinylated mouse CTLA4 extracellular region (mCTLA4), human CTLA4 extracellular region (hCTLA4) and Abatacept were prepared as antigens. Specifically, with respect to the hCTLA4 extracellular region, the gene of hCTLA4-His-BAP (SEQ ID NO: 1) was synthesized, wherein the His-tag and BAP-tag were fused to the C-terminus of the hCTLA4 extracellular region, and the gene was inserted into an animal expression plasmid. The antigen protein was expressed and purified using the following method. The prepared plasmid was introduced into the FreeStyle 293-F line (Invitrogen) derived from human embryonic kidney cells by lipofection and cultured in FreeStyle 293 expression medium (Invitrogen) at a concentration of 1.33 x 10 6 After suspending the cell line at a cell density of 100 cells / mL, it was inoculated into a flask. 3 hours after the introduction of the plasmid, biotin was added to a final concentration of 100 μM, cultured for 4 days in a CO2 incubator (37°C, 8% CO2, 125 rpm), and the antigen was purified from the culture supernatant by methods known to those skilled in the art. The absorbance of the purified antigen solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antigen was calculated from the obtained measured values ​​using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423). On the other hand, mCTLA4-His (Sino Biologics Inc. 50503-M08H, Accession No. NP_033973.2) (in which the His-tag was fused to the extracellular region of mCTLA4) and Abatacept (Alfresa Corporation) (in which the human IgG1 constant region was fused to hCTLA4) were biotinylated by the amine coupling method (PIERCE Cat. No. 21329).

[0528] (1-2) In the presence of small molecules, beads were used to select antibodies that are similar to those of mice from the initial human antibody library. CTLA4-binding antibodies

[0529] According to methods known to those skilled in the art, poly A RNA prepared from human PBMCs, commercially available human poly A RNA, etc. are used as templates to construct a human antibody phage display library consisting of multiple phages that display Fab domains of human antibody sequences different from each other.

[0530] From a constructed initial human antibody phage display library, antibodies whose binding activity to the extracellular region of mouse CTLA4 (mCTLA4) changes in the presence and absence of small molecules are screened. More specifically, phage presenting antibodies that exhibit binding activity to mCTLA4 captured on beads in the presence of small molecules are collected. Phage are then recovered from the phage eluate eluted from the beads in the absence of small molecules. In this acquisition method, biotin-labeled mCTLA4 (mCTLA4-His-Biotin) is used as the antigen.

[0531] Phage produced from E. coli carrying the constructed phagemid for phage display were purified using standard methods. The phage library solution was then dialyzed against TBS. Panning was performed using antigen immobilized on magnetic beads. NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptavidin-coated beads (Dynabeads M-280 Streptavidin-coated) were used as magnetic beads.

[0532] To efficiently obtain small molecule-dependent small molecule switch antibodies that can act as switches in cancer tissues, panning was performed based on the method described in the prior art patent document WO 2013 / 180200. This panning method enriches for antibodies that bind to the antigen in the presence of adenosine 5'-triphosphate (ATP) and ATP metabolites, but not in the absence of ATP.

[0533] (1-3) Evaluation of binding activity by phage ELISA in the presence and absence of small molecules

[0534] According to conventional methods (Methods Mol. Biol. (2002) 178, 133-145), the culture supernatant containing phage was recovered from the single E. coli colony obtained in (1-2). The culture supernatant recovered using NucleoFast 96 (MACHERY-NAGEL) was subjected to ultrafiltration. 100 μL of each culture supernatant was added to each well of the NucleoFast 96 and centrifuged at 4,500 g for 45 minutes to remove the flow-through. 100 μL of HO was added and the mixture was washed again by centrifugation at 4,500 g for 30 minutes. 100 μL of TBS was then added, and the mixture was allowed to stand at room temperature for 5 minutes, after which the phage solution contained in the supernatant was recovered.

[0535] ELISA was performed using purified phage supplemented with TBS using the following procedure. StreptaWell 96 microtiter plates (Roche) were coated overnight with 100 μL of TBS containing mCTLA4-His-Biotin. Each well was washed with TBST to remove unbound mCTLA4-His-Biotin, and then blocked with 250 μL of 2% skim milk-TBS for 1 hour or longer. After removing the 2% skim milk-TBS, the purified phage was added to each well, and the plates were incubated at room temperature for 1 hour to allow the antibody-presenting phage to bind to the mCTLA4-His-Biotin present in each well in the absence or presence of ATP. After washing each well with TBST or ATP / TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted in TBS or ATP / TBS was added, and the plates were incubated for 1 hour. After washing with TBST or ATP / TBST, the color development reaction in each well containing TMB single solution (ZYMED) was terminated by adding sulfuric acid, and the color development was then measured by absorbance at 450 nm. The results confirmed that various antibodies bound to mCTLA4 only in the presence of ATP. The results of the phage ELISA are shown in Table 2. Clones with an absorbance higher than 0.2 in the presence of ATP were identified as positive, and clones with an absorbance ratio higher than 2 in the presence / absence of ATP were identified as clones with ATP-dependent antigen-binding ability (switch clones). In this example, SM is used as an abbreviation for small molecules / low-weight molecules (such as ATP).

[0536] [Table 2]

[0537]

[0538] (1-4) Using ATP or its metabolites to obtain proteins from rationally designed libraries that bind to antigens in the presence of small molecules Bound antibodies

[0539] Antibodies that exhibit antigen-binding activity in the presence of ATP or ATP metabolites (e.g., ADP, AMP, adenosine (ADO), etc.) were obtained from a rationally designed antibody phage display library constructed in prior patent document WO 2015 / 083764. To obtain antibodies, phage-presenting antibodies that exhibit binding to antigens captured on beads in the presence of ATP or ATP metabolites are collected, and then the phage are recovered from the beads in the eluate after elution in the absence of ATP or ATP metabolites.

[0540] Phage were generated from E. coli carrying the phagemid construct used for phage display using conventional methods. A phage library solution was obtained by diluting the phage swarm with TBS, where the phage swarm was precipitated by adding 2.5 M NaCl / 10% PEG to a culture solution of the phage-producing E. coli. Next, BSA was added to the phage library solution to a final concentration of 4%. Panning was performed using antigens immobilized on magnetic beads. NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptavidin-coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads. Biotinylated Abatacept (Abatacept-Biotin) was used as the antigen.

[0541] In order to effectively obtain small molecule-dependent small molecule switch antibodies that can act as switches in cancer tissues, panning was performed with reference to the method shown in the existing patent document WO 2015 / 083674 to enrich antibodies that bind to the antigen in the presence of 5'-adenosine triphosphate (ATP) or ATP metabolites, but not in the absence of ATP or ATP metabolites.

[0542] (1-5) Evaluation of binding activity in the presence and absence of ATP or its metabolites by phage ELISA

[0543] The culture supernatant containing phage was recovered from the single E. coli colony obtained by the above method according to conventional methods (Methods Mol. Biol. (2002) 178, 133-145). The recovered culture supernatant was ultrafiltered using NucleoFast 96 (MACHEREY-NAGEL). 100 μL of culture supernatant was added to each well of the NucleoFast 96 and centrifuged (4,500 g, 45 minutes) to remove the flow-through. 100 μL of HO was added to each well of the NucleoFast 96 and washed again by centrifugation (4,500 g, 30 minutes). Finally, 100 μL of TBS was added, and the phage solution contained in the supernatant from each well of the NucleoFast 96, which had been allowed to stand at room temperature for 5 minutes, was recovered.

[0544] TBS or TBS containing ATP or its metabolites (SM / TBS) is added to the purified phage, and the phage is subjected to ELISA by the following procedure. StreptaWell 96 microtiter plates (Roche) are coated overnight with 100 μL TBS containing the biotin-labeled antigen (Abatacept-Biotin) prepared in Example 1-1. After removing free Abatacept-Biotin by washing each well of the plate with TBST, the wells are blocked with 250 μL of 2% skim milk-TBS for 1 hour or longer. After removing the 2% skim milk-TBS, the prepared purified phage is added to each well, and the plate is left to stand at 37°C for 1 hour, thereby allowing the phage-presented antibodies to bind to the Abatacept-Biotin present in each well in the absence or presence of ATP or its metabolites. After washing each well with TBST or TBST containing ATP or its metabolites (SM / TBST), an HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted in TBS or SM / TBST was added, and the plate was incubated for 1 hour. After washing the wells with TBST or SM / TBST, the color reaction in each well, to which TMB single solution (ZYMED) was added, was terminated by adding sulfuric acid, and the color development was measured by absorbance at 450 nm. The results confirmed that several antibodies exhibited altered binding activity to abatacept in the presence and absence of ATP or its metabolites. The results of the phage ELISA are shown in Table 3. Clones with an absorbance S / N ratio greater than 2 in the presence of ATP or its metabolites were identified as positive, while clones with an absorbance ratio greater than 2 in the presence / absence of ATP or its metabolites were identified as clones with antigen-binding activity dependent on ATP or its metabolites (switch clones).

[0545] [Table 3]

[0546]

[0547] (1-6) Switchable antibodies whose antigen-binding activity changes depending on the presence or absence of ATP and its metabolites Sequence analysis

[0548] The nucleotide sequences of genes amplified using the specific primers lacPF (SEQ ID NO: 2) and G1seqR (SEQ ID NO: 3) from clones that had been confirmed to have antigen-binding activity in the presence of ATP and its metabolites by phage ELISA were analyzed. As a result of this analysis, clones ABADh11-4_020, ABADh11-4_086, ABADh12-4_014, ABADh12-5_001, ABADh12-5_046, and ABADh5_041 were obtained, which were determined to have binding activity for biotinylated abatacept in the presence of ATP and its metabolites. The clones were renamed ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, respectively (Table 4).

[0549] [Table 4]

[0550]

[0551] (1-7) Switchable antibodies whose antigen-binding activity changes depending on the presence or absence of ATP and its metabolites Expression and purification

[0552] The genes encoding the variable regions of ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006 obtained from a human rationally designed phage library were inserted into the animal expression plasmid human IgG1 / Lambda. The antibodies were expressed using the following method. The prepared plasmids were introduced into FreeStyle 293-F cells (Invitrogen) derived from human embryonic kidney cells by lipofection. The cells were cultured at 1.33 x 10 6 The cells were suspended in FreeStyle 293 expression medium (Invitrogen) at a density of 10 cells / mL and seeded in each well of a 6-well plate at 3 ml / well. TM Antibodies were purified using Fast Flow (Amersham Biosciences) from the supernatant of cultures grown for 4 days in a CO2 incubator (37°C, 8% CO2, 90 rpm). The absorbance of the purified antibody solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antibody was calculated from the obtained measured value using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

[0553] (1-8) The binding of antibodies to hCTLA4 obtained in the presence and absence of AMP was evaluated by IgG ELISA. Synergistic activity

[0554] The six antibodies obtained, ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, were subjected to IgG ELISA. The buffers shown in Table 5 were appropriately prepared. Biotin-labeled human CTLA4 (hCTLA4-His-Biotin) was used as an antigen.

[0555] [Table 5]

[0556]

[0557] First, a StreptaWell 96 microtiter plate (Roche) was coated with 100 μL of TBS containing hCTLA4-His-Biotin at room temperature for 1 hour or longer. After washing each well with wash buffer to remove unbound hCTLA4-His-Biotin, the wells were blocked with 250 μL of blocking buffer for 1 hour or longer. To each well after removing the blocking buffer, 100 μL of each purified IgG prepared at 2.5 μg / mL in sample buffer containing a final concentration of 1 mM AMP was added, and the plate was left to stand at room temperature for 1 hour to allow each IgG to bind to the hCTLA4-His-Biotin present in each well. After washing with wash buffer containing a final concentration of 1 mM AMP, an HRP-conjugated anti-human IgG antibody (BIOSOURCE) diluted in sample buffer was added to each well and the plate was incubated for 1 hour. After washing with a washing buffer containing each small molecule, the color reaction of the solution in each well to which TMB single solution (ZYMED) was added was terminated by adding sulfuric acid, and then the color reaction was measured by absorbance at 450 nm. The buffer containing the composition shown in Table 5 was used as the buffer.

[0558] The measurement results are shown in Table 6. Wells with overflow values ​​were assumed to be 5.00. The results showed that for all clones of ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, the absorbance in the absence of AMP was significantly lower than that in the presence of AMP. These results confirmed that all clones of ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006 have the property of altering antigen binding depending on the presence or absence of small molecules.

[0559] [Table 6]

[0560]

[0561] (1-9) Surface plasmon resonance evaluation of the effects of ATP and its metabolites on binding to human CTLA4

[0562] ABAM004 was further evaluated as a CTLA4 switch antibody.

[0563] The antigen-antibody interaction between ABAM004 and hCTLA4-His-BAP was analyzed using a Biacore T200 (GE Healthcare). ABAM004 was captured by amine coupling onto a sensor chip CM5 (GE Healthcare) immobilized with an appropriate amount of Protein A / G (Pierce) and allowed to interact with the hCTLA4-His-BAP antigen prepared in Example 1-1. TBS was used as the running buffer, and 10 mM Glycine-HCl (pH 1.5) was used as the regeneration solution.

[0564] After capturing 1 μg / mL of ABAM004 suspended in TBS, a solution containing 500 nM hCTLA4-His-BAP and 10 concentrations of ATP, ADP, or AMP diluted in a common ratio of 4 from 4000 μM, and 2 mM MgCl2 was injected into each flow cell at a flow rate of 10 μL / min for 3 minutes. This 3-minute period served as the hCTLA4-His-BAP association phase. After the association phase, the injection was switched to running buffer for 2 minutes, which served as the hCTLA4-His-BAP dissociation phase. After the dissociation phase, regeneration solution was injected for 30 seconds at a flow rate of 30 μL / min. This constituted the ABAM004 binding activity measurement cycle. The amount of hCTLA-4-His-BAP bound to ABAM004 during the association phase was corrected for the amount of captured antibody. Biacore T200 Evaluation Software version: 2.0 and Microsoft Excel 2013 (Microsoft) were used to analyze and plot the data.

[0565] FIG1 shows the amount of binding of ABAM004 and hCTLA4-His-BAP in the presence of ATP and its metabolites obtained by this assay.

[0566] like Figure 1 This confirms that ABAM004 uses not only ATP but also ATP metabolites as a switch to bind to hCTLA4. Furthermore, this antibody exhibits the strongest binding activity, particularly in the presence of AMP.

[0567] (1-10) Evaluation of Antibody Binding to Human CTLA4-Expressing Cells

[0568] Flow cytometry was used to assess how the antigen-antibody interaction between ABAM004 and human CTLA4 changes in the presence and absence of AMP. CHO cells stably expressing human CTLA4 (hCTLA4-CHO cells) were prepared at appropriate concentrations. PBS containing 0.1% BSA (FACS buffer) was used for suspension. Antibody was added to 100 μL of the cell solution to a final concentration of 10 mg / mL, followed by AMP at final concentrations of 0, 0.4, 4, 40, 200, and 1000 μM. The cells were then incubated at 4°C for 30 minutes. The cells were then washed with FACS buffer containing AMP at final concentrations of 0, 0.4, 4, 40, 200, and 1000 μM. A FITC-labeled secondary antibody (Goat F(ab'2) Anti-Human IgG Mouseads-FITC, Beckman, 732598) was then added and incubated again at 4°C in the dark for 30 minutes. After washing again, the cells were analyzed using a flow cytometer (FACS CyAn TM ADP) was measured and analyzed. The results are shown in Figure 2 .

[0569] The above results indicate that ABAM004 exhibits AMP concentration-dependent binding activity to hCTLA4-expressing cells, and exhibits AMP concentration-dependent binding activity not only to soluble antigens but also to membrane-type antigens.

[0570] (1-11) ADCC activity of test antibodies using human peripheral blood mononuclear cells as effector cells

[0571] The antibody concentration-dependent ADCC activity of antibodies that bind to antigens in an ATP-dependent manner was measured according to the following method: Here, human peripheral blood mononuclear cells (hereinafter referred to as human PBMCs) were used as effector cells, and the ADCC activity of the test antibody was measured as follows.

[0572] First, prepare a human PBMC solution. Collect 50 mL of peripheral blood from a healthy volunteer (adult male) using a syringe containing 200 μL of a 1000 unit / mL heparin solution (Novo-heparin injection 5000 units, Novo Nordisk). Dilute the peripheral blood 2-fold with PBS(-) into four equal portions and add them to a Leucosep lymphocyte separation tube (Greiner Bio-One) that has been centrifuged after pre-injection with 15 ml of Ficoll-PaquePLUS. Centrifuge the separation tube containing the peripheral blood at 2150 rpm for 10 minutes at room temperature to separate the mononuclear cell fraction. Wash the cells contained in the fraction once with RPMI-1640 (Nacalai Tesque) containing 10% FBS (hereinafter referred to as 10% FBS / RPMI) and then suspend the cells in 10% FBS / RPMI to a concentration of 1 x 10 7 The cell density was 100 cells / mL. The cell suspension was used as the human PBMC solution in subsequent experiments.

[0573] Next, hCTLA4-CHO cells prepared by forced expression of the extracellular region of human CTLA4 in CHO cells were suspended as target cells and prepared in 10% FBS / RPMI to a concentration of 2×10 5 In addition, AMP (Sigma) diluted to 4 mM in RPMI was used as the AMP solution in subsequent tests.

[0574] ADCC activity was assessed by LDH (lactate dehydrogenase) release. First, 50 μL of antibody solution prepared at various concentrations (0, 0.04, 0.4, 4, 40 μg / mL) was added to each well of a 96-well U-bottom plate, in which 50 μL of target cells (1 x 10 4 cells / well). In addition, 50 μL of AMP solution was added to each well, and the mixture was allowed to stand at room temperature for 15 minutes. 50 μL (5 x 10 5 A human PBMC solution (100 μL cells / well) was added to each well, the plate was centrifuged, and then incubated at 37°C in a 5% CO2 incubator for 4 hours. After the reaction was complete, 100 μL of the culture supernatant was collected and transferred to a 96-well plate for measurement. A 1:45 mixture of catalyst (C) and dye solution (D) attached to the LDH detection kit (TaKaRa) was added, and 100 μL of this mixture was added. After incubation at room temperature for 15 minutes, 50 μL of 1N HCl was added to stop the reaction. Absorbance at 492 nm was measured, and ADCC activity was determined by LDH release. ADCC activity was determined using the following formula.

[0575] ADCC activity (%) = {(AD) - (CD)} x 100 / {(BD) - (CD)}

[0576] In the above formula, A represents the average value of LDH activity (OD 492 nm) in the wells to which each test antibody was added. B represents the average value of LDH activity (OD 492 nm) in the wells to which 10 μL of a 20% Triton-X aqueous solution was added after the reaction. C represents the average value of LDH activity (OD 492 nm) in the wells to which 150 μL of 10% FBS / RPMI or 100 μL of 10% FBS / RPMI and 50 μL of AMP solution were added to the target cells. D represents the average value of LDH activity (OD 492 nm) in the wells containing only 10% FBS / RPMI. The test was performed in duplicate, and the average value of ADCC activity (%) in the test, which reflects the ADCC activity of the test antibody, was calculated. The results are shown in Figure 3 middle.

[0577] The above results show that the antibody ABAM004 has antigen binding activity in the presence of AMP and has the ability to kill target cells by exerting ADCC activity.

[0578] [Example 2] Crystal structure analysis of anti-CTLA4 antibodies with ATP-dependent binding properties

[0579] (2-1) X-ray crystal structure analysis of the anti-CTLA4 binding antibody ABAM004 using AMP as a switch

[0580] For the hCTLA4-binding antibody ABAM004 using AMP as a switch and obtained from the library of Example 1, the crystal structures of the Fab fragment of ABAM004 alone, the complex of the Fab fragment of ABAM004 and AMP, and the complex of the Fab fragment of ABAM004, AMP and the hCTLA4 extracellular domain were analyzed.

[0581] (2-2) Preparation of the full-length ABAM004 antibody for crystallization

[0582] Preparation and purification of the ABAM004 full-length antibody for crystallization were performed by methods known to those skilled in the art.

[0583] (2-3) Preparation of Fab Fragments for Crystal Structure Analysis of ABAM004 Fab Fragments

[0584] ABAM004 Fab fragments were prepared by a routine method of restriction digestion with rLys-C (Promega, Cat. No. V1671) and then loaded to a protein A column (MabSelect SuRe, GE Healthcare), a cation exchange column (HiTrap SP HP, GE ...

Claims

1. A polypeptide comprising a variant Fc region comprising an amino acid alteration in a parent Fc region, wherein the parent Fc region consists of two polypeptide chains, and wherein the variant Fc region comprises an amino acid alteration at: (i) Phe at position 234, Gln at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, and Asp at position 326 in the first polypeptide of the parent Fc region according to EU numbering; and (ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326 and Glu at position 334 in the second polypeptide of the parent Fc region according to EU numbering.

2. The polypeptide of claim 1, wherein the variant Fc region further comprises an amino acid change of Asp or Glu at position 332 according to EU numbering in the second polypeptide of the parent Fc region.

3. The polypeptide of claim 2, wherein the variant Fc region further comprises an amino acid change of Glu at position 332 according to EU numbering in the first polypeptide of the parent Fc region.

4. The polypeptide of claim 2, wherein the variant Fc region further comprises an amino acid change of Lys at position 330 according to EU numbering in the second polypeptide of the parent Fc region. 5 . The polypeptide of claim 1 , wherein the variant Fc region has enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to the parent Fc region. The polypeptide of claim 5 , wherein the variant Fc region has enhanced binding activity to FcγRIIa and FcγRIIIa compared to the parent Fc region.

7. The polypeptide of claim 1, wherein the variant Fc region has improved selectivity between activating and inhibitory Fcγ receptors compared to the parent Fc region.

8. The polypeptide of claim 7, wherein the activating Fcγ receptor is at least one Fcγ receptor selected from FcγRIa, FcγRIIa, and FcγRIIIa, and wherein the inhibitory Fcγ receptor is FcγRIIb.

9. The polypeptide of any one of claims 1 to 8, wherein the polypeptide comprising a variant Fc region is an antibody.

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

11. The antibody of claim 10, wherein the antibody comprises: (a) HVR-H1 (SEQ ID NO: 223) comprising the amino acid sequence of SX1TMN, wherein X1 is H, A, R, or K; (b) HVR-H2 (SEQ ID NO: 224) comprising the amino acid sequence of SISX1X2SX3YIYYAX4SVX5G, wherein X1 is S or T, X2 is R or Q, X3 is G or H, X4 is D, E or R, and X5 is K or R; and (c) HVR-H3 (SEQ ID NO: 225) comprising the amino acid sequence of YGX1REDMLWVFDY, wherein X1 is K or A.

12. The antibody of claim 11, further comprising: (a) HVR-L1 (SEQ ID NO: 226) comprising the amino acid sequence of X1GX2STX3VGDYX4X5VX6, wherein X1 is T, D, Q, or E, X2 is T or P, X3 is D or G, X4 is N or T, X5 is Y or W, and X6 is S or H; (b) HVR-L2 (SEQ ID NO: 227) comprising the amino acid sequence of X1TX2X3KPX4, wherein X1 is E, F, or Y, X2 is S or I, X3 is K or S, and X4 is S, E, or K; and (c) HVR-L3 (SEQ ID NO: 228) comprising the amino acid sequence of X1TYAAPLGPX2, wherein X1 is S or Q and X2 is M or T.

13. The antibody of claim 10, comprising: (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 83 to 86, 98, and 135 to 141; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 88 to 95, 97, 99, 134, and 144 to 149; or (c) a VH sequence having the amino acid sequence of any one of SEQ ID NOs: 83 to 86, 98, and 135 to 141 and a VL sequence having the amino acid sequence of any one of SEQ ID NOs: 88 to 95, 97, 99, 134, and 144 to 149.

14. A pharmaceutical formulation comprising the antibody of any one of claims 10 to 13 and a pharmaceutically acceptable carrier.

15. The pharmaceutical preparation according to claim 14, wherein the pharmaceutical preparation is used to treat tumors.

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