Anti-CTLA-4 antibodies and use thereof

Abstract

To provide anti-CTLA-4 antibodies, and to provide methods for producing and using the same.SOLUTION: Provided is an anti-CTLA-4 antibody having CTLA-4 binding activity dependent on the concentration of an adenosine-containing compound, the antibody having at least one of the following: a. binding activity in the presence of a 100 μM adenosine-containing compound of more than 2 times higher than in its absence; b. KD value in the presence of a 100 μM adenosine-containing compound of 5×10-7M or less; c. KD value in the absence of adenosine-containing compounds of 1×10-6M or more; d. forming a tripartite complex with an adenosine-containing compound and CTLA-4; e. binding to the region from amino acid at position 97 to amino acid at position 106 of human CTLA-4; f. competing with a particular antibody for binding to CTLA-4; g. binding to the same epitope as the particular antibody; h. exhibiting cytotoxic activity against CTLA-4 expressing cells; and i. binding to human and mouse derived CTLA-4.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to anti-CTLA-4 antibodies and methods of use thereof. [Background technology]

[0002] Mutated cells in the body due to genetic mutations and other factors are monitored and eliminated by the immune surveillance system. However, sustained excessive immune responses can be harmful to the body itself, such as by causing autoimmune damage to normal tissues. Therefore, the immune system is equipped with a negative feedback mechanism (immune checkpoint) to suppress immune responses once activated (see, for example, Non-Patent Document 1). Immune checkpoints are thought to play an important role in maintaining homeostasis in the immune system. On the other hand, it has become clear that some tumors utilize immune checkpoints to evade the immune system. Currently, extensive research is being conducted on the immunosuppressive functions mediated by major immune checkpoint molecules such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1), and programmed cell death ligand 1 (PD-L1). CTLA-4 is a glycoprotein belonging to the immunoglobulin superfamily whose gene was cloned from a cDNA library of a mouse-derived killer T cell clone in 1987 (see, for example, Non-Patent Document 2). It is known that T cell immune responses are suppressed via CTLA-4. Based on the idea that suppressing CTLA-4 function and promoting T cell activation could lead to cancer regression, it was reported in 1996 that administration of an anti-CTLA-4 antibody to tumor-bearing mice resulted in tumor regression (see, for example, Non-Patent Document 3). Since 2000, evaluation of the efficacy of anti-CTLA-4 antibodies in humans has been ongoing, and in 2011, an anti-human CTLA-4 monoclonal antibody (ipilimumab) was approved by the U.S. Food and Drug Administration (FDA) as the world's first immunostimulatory antibody drug. In addition to ipilimumab, numerous other anti-CTLA-4 monoclonal antibodies have been produced (see, for example, Patent Documents 1, 2, 3, and 4), and attempts are being made to develop them as pharmaceuticals. These drugs, which inhibit immune checkpoints to release the immune suppression mechanism and thereby enhance immune activity, are called immune checkpoint inhibitors. On the other hand, it has long been known that some T cells have immunosuppressive functions. In 1995, these cells were identified as CD25+CD4+ T cells and named regulatory T cells (see, for example, Non-Patent Document 4). In 2003, the Foxp3 gene was identified as a master gene that is specifically expressed in regulatory T cells and controls their development and function. Foxp3 acts as a transcription factor and controls the expression of various immune response-related genes. Foxp3 is particularly involved in the constitutive expression of CTLA-4 in regulatory T cells, which is thought to play an important role in the immunosuppressive function of regulatory T cells (see, for example, Non-Patent Document 5). It is believed that the infiltration of regulatory T cells into tumor tissue results in the weakening or inhibition of immune surveillance against tumors. In fact, it has been revealed that regulatory T cells are increased in many human carcinomas (see, for example, Non-Patent Document 6), and it has been reported that the infiltration of regulatory T cells into the local tumor can be a factor in poor prognosis for cancer patients. Conversely, it is expected that the removal or reduction of regulatory T cells from tumor tissue will lead to the enhancement of anti-tumor immunity. Currently, the development of cancer immunotherapy targeting regulatory T cells is being actively pursued. Administration of the anti-CTLA-4 antibody ipilimumab enhances antitumor immunity, but it has also been reported that its systemic enhancement of immune activity can lead to the development of autoimmune diseases. In one clinical trial, 60% of patients treated with ipilimumab experienced adverse events, most of which were autoimmune diseases related to the skin or gastrointestinal tract. In other clinical trials, approximately half of patients treated with ipilimumab also developed similar autoimmune diseases. To prevent these side effects, patients treated with ipilimumab are sometimes administered immunosuppressants. The development of new drugs that can maintain antitumor immune responses while minimizing the side effects of immune checkpoint inhibitors is desirable. 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 tools for achieving antitumor effects using antibodies (see, for example, Non-Patent Documents 7 and 8). These effector functions are induced by the binding of the Fc region of IgG antibodies to antibody receptors (FcγR) or various complement components present on the surface of effector cells such as natural killer cells and macrophages. Numerous studies have been conducted on Fc region mutants, and mutants with various properties, such as higher FcγR binding activity than the wild-type, have been identified (see, for example, Patent Documents 5 and 6, Non-Patent Documents 9 and 10). It has also been reported that antibody Fc regions bind to FcγR in a 1:1 ratio and asymmetrically recognize FcγR at the lower hinge and CH2 regions (see, for example, Non-Patent Document 11). Based on this, methods have been reported in which different modifications are made to the two polypeptide chains that make up the Fc region of an antibody to create asymmetric Fc region mutants, thereby optimizing the interaction with FcγR (see, for example, Patent Documents 7, 8, 9, and 10). When a therapeutic antibody is administered in vivo, it is desirable that the target antigen be specifically expressed only at the lesion site. However, in many cases, the same antigen is also expressed in non-lesioned normal tissues, which can cause undesirable side effects from a therapeutic perspective. For example, while an antibody against a tumor antigen can exert cytotoxic activity against tumor cells through ADCC or other mechanisms, if the same antigen is also expressed in normal tissues, it may also cytotoxicize normal cells. To solve the above problems, a technology has been developed that focuses on the phenomenon in which specific compounds are present in large amounts in target tissues (e.g., tumor tissues) to create antigen-binding molecules whose antigen-binding activity changes depending on the concentration of such compounds (see, for example, Patent Document 11). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO 2000 / 037504 [Patent Document 2] WO 2001 / 014424 [Patent Document 3] WO 2012 / 120125 [Patent Document 4] WO 2016 / 196237 [Patent Document 5] WO 2000 / 042072 [Patent Document 6] WO 2006 / 019447 [Patent Document 7] WO 2012 / 058768 [Patent Document 8] WO 2012 / 125850 [Patent Document 9] WO 2013 / 002362 [Patent Document 10] WO 2014 / 104165 [Patent Document 11] WO 2013 / 180200 [Non-patent literature]

[0004] [Non-Patent Document 1] Pardoll, Nat Rev Cancer (2012) 12: 252-264 [Non-patent document 2] Brunet et al., Nature (1987) 328: 267-270 [Non-patent document 3] Leach et al., Science (1996) 271: 1734-1736 [Non-patent document 4] Sakaguchi et al., J Immunol (1995) 155: 1151-1164 [Non-Patent Document 5] Takahashi et al., J Exp Med (2000) 192: 303-310 [Non-patent document 6] Nishikawa & Sakaguchi, Int J Cancer (2010) 127: 759-767 [Non-Patent Document 7] Clynes et al., Proc Natl Acad Sci USA (1998) 95: 652-656 [Non-patent document 8] Clynes et al., Nat Med (2000) 6: 443-446 [Non-Patent Document 9] Lazar et al., Proc Natl Acad Sci USA (2006) 103: 4005-4010 [Non-Patent Document 10] Chu et al., Mol Immunol (2008) 45: 3926-3933 [Non-Patent Document 11] Radaev et al., J Biol Chem (2001) 276: 16469-16477 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides anti-CTLA-4 antibodies and methods of using them. The present invention also provides polypeptides comprising variant Fc regions and methods of producing them. [Means for solving the problem]

[0006] More specifically, the present invention provides the following [1] to

[47] . [1] An anti-CTLA-4 antibody having CTLA-4 binding activity dependent on the concentration of an adenosine-containing compound, the antibody having at least one characteristic selected from the following (a) to (i): (a) the binding activity in the presence of 100 μM of an adenosine-containing compound is at least two-fold higher than the binding activity in the absence of the adenosine-containing compound; (b) KD value of 5 × 10 in the presence of 100 μM adenosine-containing compound.-7 M or less, (c) KD value of 1 × 10 in the absence of adenosine-containing compounds -6 M or more, (d) forming a ternary complex with an adenosine-containing compound and CTLA-4; (e) binds to the region of amino acids 97 to 106 of human CTLA-4 (extracellular domain, SEQ ID NO: 28); (f) competes with ABAM004 (VH, SEQ ID NO:10; and VL, SEQ ID NO:11) for binding to CTLA-4; (g) binds to the same epitope as that bound by ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11); (h) exhibiting cytotoxic activity against CTLA-4 expressing cells; and (i) It binds to human and mouse CTLA-4. [2] The antibody according to [1], which is a monoclonal antibody. [3] The antibody according to [1] or [2], which is a human antibody, a humanized antibody, or a chimeric antibody. [4] The antibody according to any one of [1] to [3], which is an antibody fragment that binds to CTLA-4. [5] The antibody of any one of [1] to [4], comprising: (a) an HVR-H1 (SEQ ID NO: 223) comprising the amino acid sequence SX1TMN, wherein X1 is H, A, R, or K; (b) an HVR-H2 (SEQ ID NO: 224) comprising the amino acid sequence SISX1X2SX3YIYYAX4SVX5G, wherein X1 is S or T, X2 is R or Q, X3 is G or H, X4 is D, E, or R, and X5 is K or R; and (c) an HVR-H3 (SEQ ID NO: 225) comprising the amino acid sequence YGX1REDMLWVFDY, wherein X1 is K or A. [6] The antibody of [5], further comprising: (a) an HVR-L1 (SEQ ID NO: 226) comprising the amino acid sequence X1GX2STX3VGDYX4X5VX6, where X1 is T, D, Q, or E, X2 is T or P, X3 is D or G, X4 is N or T, X5 is Y or W, and X6 is S or H; (b) an HVR-L2 (SEQ ID NO: 227) comprising the amino acid sequence X1TX2X3KPX4, where X1 is E, F, or Y, X2 is S or I, X3 is K or S, and X4 is S, E, or K; and (c) an HVR-L3 (SEQ ID NO: 228) comprising the amino acid sequence X1TYAAPLGPX2, where X1 is S or Q, and X2 is M or T. [7] The antibody of [5], further comprising a heavy chain variable domain FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 229 to 232, FR2 comprising the amino acid sequence of SEQ ID NO: 233, FR3 comprising the amino acid sequence of SEQ ID NO: 234, and FR4 comprising the amino acid sequence of SEQ ID NO: 235. [8] The antibody of [6], further comprising a light chain variable domain FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 236 to 238, FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 240 to 241, FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 242 to 244, and FR4 comprising the amino acid sequence of any one of SEQ ID NOs: 245 to 246. [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.

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

[11] The antibody described in

[10] , wherein the Fc region is a mutant Fc region containing amino acid modifications, and the mutant Fc region has enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to a native Fc region.

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

[11] .

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

[12] .

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

[13] so that the antibody is produced.

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

[11] and a pharmaceutically acceptable carrier.

[16] The pharmaceutical formulation of

[15] , wherein the antibody is an immunoconjugate.

[17] The pharmaceutical formulation of

[15] or

[16] , which is used in combination with at least one selected from the group consisting of an immune checkpoint inhibitor, an EGFR inhibitor, a HER2 inhibitor, and a chemotherapeutic agent.

[18] The pharmaceutical preparation of any one of

[15] to

[17] for use in treating a tumor.

[19] The pharmaceutical preparation according to

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

[20] The pharmaceutical preparation of any one of

[15] to

[17] for use in damaging cells.

[21] The pharmaceutical preparation of any one of

[15] to

[17] for use in damaging Treg cells.

[22] The pharmaceutical preparation according to

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

[23] The pharmaceutical preparation according to

[20] or

[21] , wherein the immune system is activated by damaging Treg cells.

[24] The pharmaceutical preparation of any one of

[15] to

[17] for use in immune activation.

[25] The pharmaceutical preparation according to

[24] , wherein the immune activation is T cell activation.

[26] The pharmaceutical preparation of

[24] or

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

[27] The pharmaceutical preparation of any one of

[24] to

[26] , which induces a lower level of immune activation in non-tumor tissue compared to a pharmaceutical preparation containing a control anti-CTLA-4 antibody.

[28] The pharmaceutical formulation of any one of

[24] to

[27] , which has a lower level of side effects compared to a pharmaceutical formulation containing a control anti-CTLA-4 antibody.

[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 that depends on the concentration of an adenosine-containing compound.

[30] The pharmaceutical preparation according to

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

[31] The pharmaceutical preparation of any one of

[18] ,

[19] ,

[26] to

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

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

[33] The polypeptide described in

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

[34] The polypeptide of

[32] or

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

[35] The polypeptide of any one of

[32] to

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

[36] A polypeptide described in any one of

[32] to

[35] , wherein the mutant Fc region further comprises an amino acid modification at position 356 (EU numbering) in the first polypeptide of the parent Fc region.

[37] The polypeptide of any one of

[32] to

[36] , wherein the mutant Fc region further comprises an amino acid modification at position 366 (EU numbering) in the first polypeptide of the parent Fc region.

[38] The polypeptide of any one of

[32] to

[37] , wherein the mutant Fc region further comprises an amino acid modification at position 439 (EU numbering) in a second polypeptide of the parent Fc region.

[39] The polypeptide of any one of

[32] to

[38] , wherein the mutant Fc region further comprises amino acid modifications at positions 366, 368, and 407, as represented by EU numbering, in a second polypeptide of the parent Fc region.

[40] The polypeptide of any one of

[32] to

[39] , comprising at least one amino acid modification selected from the amino acid modifications described below: (i) in a first polypeptide of a parent Fc region, Tyr or Phe at position 234, Gln 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, as indicated by EU numbering; and (ii) in the second polypeptide of the parent Fc region, Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met, 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, as indicated by EU numbering.

[41] The polypeptide of any one of

[32] to

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

[42] A polypeptide described in any one of

[32] to

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

[43] The polypeptide of

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

[44] A polypeptide described in any one of

[32] to

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

[45] the polypeptide of

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

[46] The polypeptide of any one of

[32] to

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

[47] A method for producing a polypeptide containing a mutant Fc region, comprising the step of introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 250, 270, 298, 307, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows the CTLA-4 binding activity of the anti-CTLA-4 antibody ABAM004 depending on the ATP, ADP, or AMP concentration, as described in Examples 1-9. [Figure 2] FIG. 2 shows the binding activity of the anti-CTLA-4 antibody ABAM004 to CTLA-4 expressing cells depending on the AMP concentration, as described in Examples 1-10. [Figure 3] FIG. 3 shows the ADCC activity of the anti-CTLA-4 antibody ABAM004 against CTLA-4 expressing cells in the presence and absence of AMP, as described in Examples 1-11. [Figure 4]Figure 4 shows the binding mode between the ABAM004 Fab fragment and AMP, as described in Examples 2-13. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and AMP as a ball-and-stick model. Amino acid residues that interact with AMP are shown as stick models. The dashed lines and their associated numbers indicate the distance (Å) between each amino acid residue and AMP. [Figure 5] Figure 5 shows the binding mode of the ABAM004 Fab fragment to AMP and human CTLA4 (hCTLA4), as described in Examples 2-14. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, hCTLA4 in white, and AMP in a ball-and-stick model. The epitope is defined as an amino acid residue of hCTLA4 containing one or more non-hydrogen atoms located within 4.2 Å of either the antibody or AMP, and is shown in a stick model. [Figure 6] Figure 6 shows the mapping of the epitope of the ABAM004 Fab fragment to the amino acid sequence of hCTLA4, as described in Examples 2-14. In the figure, black amino acid residues indicate hCTLA4 amino acid residues that contain one or more non-hydrogen atoms located within 4.2 Å of either ABAM004 or AMP in the crystal structure. Gray amino acid residues indicate residues that were disordered in the crystal structure and therefore could not be modeled. [Figure 7] Figure 7 shows the superposition of the crystal structures of the ABAM004 Fab fragment alone in complex with AMP, and the ternary complex with AMP and CTLA4, with the structure of the antibody and AMP extracted from the crystal structures, as described in Example 2-15. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and AMP as a ball-and-stick model. The structure of the ABAM004 Fab fragment alone is shown in thin lines, the structure of the binary complex with AMP in medium-thick lines, and the structure of the ternary complex in thick lines. [Figure 8]8 shows the CTLA-4 binding activity of the anti-CTLA-4 antibody ABAM004 and its variants 04H0150 / 04L0072, depending on the ATP, ADP, or AMP concentration, as described in Example 3-2. In the figure, WT represents ABAM004, and H150L072 represents 04H0150 / 04L0072. [Figure 9] FIG. 9 shows the ATP concentration-dependent neutralizing activity of anti-CTLA-4 antibody SW1077 against CTLA-4, as described in Example 3-6. [Figure 10] Figure 10 shows the antitumor effect of the anti-CTLA-4 antibody mNS-mFa55 (control antibody) in a mouse model transplanted with the FM3A cell line, as described in Example 3-7-4. The antibody was administered via the tail vein at 0.01 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 1 mg / kg, 10 mg / kg, 30 mg / kg, or 100 mg / kg. Each point represents the mean tumor volume for n=4 mice per group. [Figure 11] Figure 11 shows the antitumor effect of anti-CTLA-4 antibody SW1208-mFa55 (switch antibody) in a mouse model transplanted with the FM3A cell line, as described in Example 3-7-4. The antibody was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, or 500 mg / kg. Each point represents the mean tumor volume for n=4 per group. [Figure 12]Figure 12 shows the change in the proportion of effector Treg cells in tumors following administration of the anti-CTLA-4 antibodies mNS-mFa55 (control antibody) and SW1208-mFa55 (switch antibody) in a mouse model transplanted with the FM3A cell line, as described in Example 3-7-7. mNS-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 100 mg / kg, and SW1208-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, or 500 mg / kg. Tumors were harvested 6 days after administration, and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents the proportion of effector Tregs (CD4+ FoxP3+ KLRG1+) relative to CD45+ cells. The average value is shown for n = 3. [Figure 13] Figure 13 shows the change in the percentage of activated helper T cells in the spleen following administration of the anti-CTLA-4 antibodies mNS-mFa55 (control antibody) and SW1208-mFa55 (switch antibody) in a mouse model transplanted with the FM3A cell line, as described in Example 3-7-8. mNS-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 100 mg / kg, and SW1208-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, or 500 mg / kg. Six days after administration, spleens were collected and the increase or decrease in activated helper T cells was assessed by FACS analysis. The vertical axis represents the percentage of activated helper T cells (CD4+ Foxp3- ICOS+) relative to CD45+ cells. The average value for n = 3 is shown. [Figure 14] Figure 14 shows the antitumor effect of anti-CTLA-4 antibody SW1389-mFa55 (switch antibody) in a mouse model implanted with the Hepa1-6 / hGPC3 cell line, as described in Example 4-3-5. The antibody was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 100 mg / kg. Each point represents the mean tumor volume for n=4 groups. [Figure 15] Figure 15 shows the antitumor effect of the anti-CTLA-4 antibody hNS-mFa55 (control antibody) in a mouse model implanted with the Hepa1-6 / hGPC3 cell line, as described in Example 4-3-5. The antibody was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 30 mg / kg. Each point represents the mean tumor volume for n=4 groups. [Figure 16] Figure 16 shows the change in the proportion of effector Treg cells in tumors following administration of the anti-CTLA-4 antibodies hNS-mFa55 (control antibody) and SW1389-mFa55 (switch antibody) in a mouse model implanted with the Hepa1-6 / hGPC3 cell line, as described in Example 4-3-8. hNS-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 30 mg / kg, and SW1389-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, or 500 mg / kg. Tumors were harvested 6 days after administration, and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents the proportion of effector Tregs (CD4+ FoxP3+ CCR7lowKLRG1+) relative to CD45+ cells. The average value is shown for n = 3. [Figure 17] Figure 17 shows the change in the percentage of activated helper T cells in the spleen following administration of the anti-CTLA-4 antibodies hNS-mFa55 (control antibody) and SW1389-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 4-3-9. hNS-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 30 mg / kg, and SW1389-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, or 500 mg / kg via the tail vein. Six days after administration, spleens were collected and the increase or decrease in activated helper T cells was assessed by FACS analysis. The vertical axis represents the percentage of activated helper T cells (CD4+ Foxp3- ICOS+) relative to CD45+ cells. The average value for n = 3 is shown. [Figure 18] Figure 18 shows the antitumor effect of anti-CTLA-4 antibody SW1610-mFa55 (switch antibody) in a mouse model implanted with the Hepa1-6 / hGPC3 cell line, as described in Example 5-4-5. The antibody was administered via the tail vein at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg. Each point represents the mean tumor volume for a group of 5 (n = 5). [Figure 19] Figure 19 shows the antitumor effect of anti-CTLA-4 antibody SW1612-mFa55 (switch antibody) in a mouse model implanted with the Hepa1-6 / hGPC3 cell line, as described in Example 5-4-5. The antibody was administered via the tail vein at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg. Each point represents the mean tumor volume for a group of 5 (n = 5). [Figure 20] Figure 20 shows the antitumor effect of anti-CTLA-4 antibody SW1615-mFa55 (switch antibody) in a mouse model implanted 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, or 3 mg / kg. Each point represents the mean tumor volume for a group of 5 (n = 5). [Figure 21]21 shows the change in the proportion of effector Treg cells in tumors following administration of the anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, and SW1615-mFa55 (all switch antibodies) in a mouse model implanted with the Hepa1-6 / hGPC3 cell line, as described in Example 5-4-8. SW1610-mFa55 was administered at 50 mg / kg, 100 mg / kg, or 200 mg / kg; SW1612-mFa55 was administered at 50 mg / kg, 100 mg / kg, or 200 mg / kg; SW1615-mFa55 was administered at 50 mg / kg, 100 mg / kg, 200 mg / kg, or 400 mg / kg; and the negative control antibody KLH-mFa55 was administered at 400 mg / kg via the tail vein. Tumors were harvested 6 days after administration and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents the ratio of effector Tregs (CD4+ FoxP3+ CCR7lowKLRG1+) to CD45+ cells. The mean value of n=3 is shown. [Figure 22] 22 shows the change in the percentage of activated helper T cells in the spleen following administration of the anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, and SW1615-mFa55 (all switch antibodies) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Example 5-4-9. SW1610-mFa55 was administered at 50 mg / kg, 100 mg / kg, or 200 mg / kg; SW1612-mFa55 was administered at 50 mg / kg, 100 mg / kg, or 200 mg / kg; SW1615-mFa55 was administered at 50 mg / kg, 100 mg / kg, 200 mg / kg, or 400 mg / kg; and the negative control antibody KLH-mFa55 was administered at 400 mg / kg via the tail vein. Six days after administration, spleens were collected and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis represents the ratio of activated helper T cells (CD4+ Foxp3- ICOS+) to CD45+ cells. The mean value of n=3 is shown. [Figure 23]Figure 23 shows a comparison of the in vitro ADCC activity of antibodies with various altered constant regions that have enhanced FcγR binding, as described in Example 6-2. In the figure, IgG1 represents MDX10D1H-G1m / MDX10D1L-k0MT, GASDALIE represents MDX10D1H-GASDALIE / MDX10D1L-k0MT, ART6 represents MDX10D1H-Kn462 / MDX10D1H-H1445 / MDX10D1L-k0MT, and ART8 represents MDX10D1H-Kn461 / MDX10D1H-H1443 / MDX10D1L-k0MT. Here, IgG1 represents an antibody with a control constant region, GASDALIE represents an antibody with a constant region described in a prior literature, and ART6 and ART8 represent antibodies with altered constant regions prepared in Example 6-1. [Figure 24] Figure 24 shows a comparison of the in vitro ADCP activity of antibodies with various altered constant regions that exhibit enhanced FcγR binding, as described in Example 6-3. In the figure, IgG1 represents MDX10D1H-G1m / MDX10D1L-k0MT, GASDIE represents MDX10D1H-GASDIE / MDX10D1L-k0MT, ART6 represents MDX10D1H-Kn462 / MDX10D1H-H1445 / MDX10D1L-k0MT, and ART8 represents MDX10D1H-Kn461 / MDX10D1H-H1443 / MDX10D1L-k0MT. Here, IgG1 represents an antibody with a control constant region, GASDIE represents an antibody with a constant region described in a prior publication, and ART6 and ART8 represent antibodies with altered constant regions prepared in Example 6-1. [Figure 25] Figure 25 shows the in vitro ADCC activity of anti-CTLA4 switched antibody SW1389-ART6, which has an altered constant region with enhanced binding to FcγR, as described in Example 6-4. [Figure 26] Figure 26 shows the in vitro ADCC activity of anti-CTLA4 switched antibody SW1610-ART6, which has a modified constant region with enhanced binding to FcγR, as described in Example 6-4. [Figure 27] Figure 27 shows the in vitro ADCC activity of anti-CTLA4 switched antibody SW1612-ART6, which has an altered constant region with enhanced binding to FcγR, as described in Example 6-4. [Figure 28] Figure 28 shows the neutralizing activity of anti-CTLA4 switch antibody SW1389 against CTLA4 (the activity of canceling the CTLA4 signal that acts suppressively on the activation of effector cells), as described in Example 6-5. [Figure 29] Figure 29 shows the neutralizing activity of anti-CTLA4 switch antibody SW1610 against CTLA4 (the activity of canceling the CTLA4 signal that acts suppressively on the activation of effector cells), as described in Example 6-5. [Figure 30] Figure 30 shows the neutralizing activity of anti-CTLA4 switch antibody SW1612 against CTLA4 (the activity of canceling the CTLA4 signal that acts suppressively on the activation of effector cells), as described in Example 6-5. [Figure 31] Figure 31 shows the neutralizing activity of anti-CTLA4 switch antibody SW1615 against CTLA4 (the activity of canceling the CTLA4 signal that acts suppressively on the activation of effector cells), as described in Example 6-5. [Figure 32] Figure 32 shows the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1389-ART5+ACT1 against CTLA4-positive regulatory T cells, as described in Example 6-6. [Figure 33] Figure 33 shows the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1389-ART6+ACT1 against CTLA4-positive regulatory T cells, as described in Example 6-6. [Figure 34] Figure 34 shows the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1610-ART5+ACT1 against CTLA4-positive regulatory T cells, as described in Example 6-6. [Figure 35] Figure 35 shows the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1610-ART6+ACT1 against CTLA4-positive regulatory T cells, as described in Example 6-6. DETAILED DESCRIPTION OF THE INVENTION

[0008] The techniques and procedures described or cited herein are generally well understood and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and C.C.Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993), using conventional techniques commonly used by those skilled in the art.

[0009] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide those skilled in the art with general guidance for many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0010] For purposes of interpreting this specification, the following definitions will apply, and wherever applicable, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. In the event that any of the definitions below conflict with any document incorporated herein by reference, the definition below shall control.

[0011] An "acceptor human framework," for purposes of this specification, is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise those same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0012] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" (antibody-dependent cell-mediated cytotoxicity) refers to a form of cytotoxicity in which secreted immunoglobulins bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxins. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), can be performed. Useful effector cells for such assays include PBMCs and NK cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be assessed in vivo in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0013] Examples of "cytotoxic activity" include the above-mentioned antibody-dependent cell-mediated cytotoxicity (ADCC) activity, the below-mentioned complement-dependent cytotoxicity (CDC) activity, and T-cell cytotoxic activity. CDC activity refers to cytotoxic activity mediated by the complement system. On the other hand, ADCC activity refers to the activity of an antibody binding to an antigen present on the surface of a target cell, and then effector cells binding to the antibody, causing the effector cells to injure the target cell. Whether an antibody of interest has ADCC activity or CDC activity can be determined by known methods (e.g., Current Protocols in Immunology, Chapter 7. Immunologic studies in humans, edited by Coligan et al. (1993)).

[0014] "Neutralizing activity" refers to the activity of an antibody that inhibits a biological activity by binding to a molecule involved in that biological activity. In some embodiments, the biological activity is brought about by the binding of a ligand to a receptor. In certain embodiments, the antibody inhibits the binding of the ligand to the receptor by binding to the ligand or receptor. An antibody having such neutralizing activity is called a neutralizing antibody. The neutralizing activity of a test substance can be measured by comparing the biological activity in the presence of a ligand with that in the presence or absence of the test substance.

[0015] The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to the process by which antibody-coated cells, either in whole or in part, are internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the immunoglobulin Fc region.

[0016] The term "binding activity" refers to the strength of the total noncovalent interactions between one or more binding sites of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Here, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). For example, when members of a binding pair reflect a monovalent 1:1 interaction, binding activity refers to the intrinsic binding affinity ("affinity"). When members of a binding pair are capable of both monovalent and multivalent binding, binding activity is the sum of these avidities. The binding activity of a molecule X to its partner Y can generally be expressed as a dissociation constant (KD) or "amount of analyte bound per unit amount of ligand." Binding activity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding activity are described below.

[0017] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0018] The term "anti-CTLA-4 antibody" or "antibody that binds to CTLA-4" refers to an antibody that can bind to CTLA-4 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeted to CTLA-4. In one embodiment, the extent of binding of the anti-CTLA-4 antibody to an unrelated, non-CTLA-4 protein is less than about 10% of the binding of the antibody to CTLA-4, as measured (e.g., by radioimmunoassay (RIA)). In certain embodiments, an antibody that binds to CTLA-4 has a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10-8 M or less, e.g. 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 In certain embodiments, the anti-CTLA-4 antibody binds to an epitope of CTLA-4 that is conserved among CTLA-4 from different species.

[0019] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0020] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0021] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its own antigen in a competition assay, e.g., by 50% or more, and / or the reference antibody blocks the binding of the antibody to its own antigen in a competition assay, e.g., by 50% or more. Exemplary competition assays are provided herein.

[0022] "Autoimmune disease" refers to a non-malignant disease or disorder arising from and directed against an individual's own tissues. As used herein, autoimmune disease specifically excludes malignant or cancerous diseases or conditions, and specifically excludes B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia.Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic sclerosis and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, such as eczema and asthma and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; and systemic lupus erythematosus (SLE). (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, typically seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases associated with leukocyte leakage; and the central nervous system (CNS). Inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs' positive anemia); myasthenia gravis; antigen-antibody complex-mediated disease; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; Stiffman syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0023] 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.

[0024] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism for inducing cell death in which the Fc effector domain of a target-bound antibody activates a series of enzymatic reactions that result in the formation of holes in the target cell's membrane. Typically, antigen-antibody complexes formed on the target cell bind to and activate complement component C1q, which then activates the complement cascade, leading to target cell death. Complement activation can also result in the deposition of complement components on the surface of target cells, which promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0025] "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylolmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including the synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®)), acetylcamptothecin, and scopolectin. (including scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide (chlorophosphamide), estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, fenesterine, prednimustine, trofosfamide, nitrogen mustards, such as uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Enediyne antibiotics {e.g., calicheamicin, particularly calicheamicin gamma 11 and calicheamicin omega 11 (e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)) See also); CDP323, an oral alpha-4 integrin inhibitor; dynemicins, including dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposomal for injection (DOXIL®), liposomal doxorubicin TCL D-99 (including MYOCET®), pegylated liposomal doxorubicin (including CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, and lodorubicin antibiotics, such as rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calucelone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane. (anti-adrenals); folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydrochloride; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verraculin A) A), Roridin A, and Anguidine; urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as paclitaxel (TAXOL®), albumin-modified nanoparticle formulations of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin;Vinces, which interfere with the formation of microtubules by tubulin polymerization, include vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine; DMFO; retinoids, such as retinoic acids, including bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGFR). EGF-R; vaccines such as THERATOPE® vaccine and gene therapy vaccines (e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine); topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341);Bcl-2 inhibitors such as bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); sorafenib, ABT510; oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is an abbreviation for a treatment regimen with oxaliplatin (ELOXANTIN™) in combination with 5-FU and leucovorin;

[0026] 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.

[0027] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0028] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 radioactive isotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and the various chemotherapeutic agents disclosed above.

[0029] "Effector cell" refers to a leukocyte that expresses one or more FcRs and exerts effector function. In certain embodiments, the cell expresses at least FcγRIII and exerts ADCC effector function. Examples of leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, e.g., from blood. In certain embodiments, effector cells can be human effector cells.

[0030] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0031] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets that antigen and includes specific amino acids that directly contact the antibody. Epitopic determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on that target antigen in a complex mixture of proteins and / or macromolecules.

[0032] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-492 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126: 330-341 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

[0033] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for regulating maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol. Today 18(12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7): 637-640 (1997); Hinton et al., J. Biol. Chem. 279(8): 6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0034] The in vivo binding to human FcRn and serum half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides with mutated Fc regions are administered. WO2000 / 42072 (Presta) describes antibody mutants with improved or reduced binding to FcR. See, for example, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001).

[0035] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0036] The term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of a nucleic acid encoding the antibody. Thus, a composition containing an antibody with an Fc region according to the present invention can contain an antibody with G446-K447, an antibody with G446 but without K447, an antibody with G446-K447 completely removed, or a mixture of the above three types of antibodies.

[0037] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0038] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0039] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0040] A "functional Fc region" comprises an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors (BCRs)); and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays, for example, as disclosed within the definitions herein.

[0041] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0042] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0043] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0044] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0045] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0046] An "isolated" antibody is one that has been separated from a component of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0047] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0048] An "isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including nucleic acid molecules carried on a single vector or separate vectors, and including nucleic acid molecules present in one or more locations in a host cell.

[0049] 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 as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0050] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0051] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0052] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0053] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0054] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes); native-sequence human IgG2 Fc regions; native-sequence human IgG3 Fc regions; and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0055] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, for example, about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide, compared to the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region and / or the Fc region of the parent polypeptide, more preferably at least about 90% homology thereto, and most preferably at least about 95% homology thereto.

[0056] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0057] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0058] The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0059] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0060] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0061] An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount, at dosages and for periods of time necessary, effective to achieve a desired therapeutic or prophylactic result.

[0062] The term "package insert" is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, method of administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0063] As used herein, the term "CTLA-4," unless otherwise indicated, refers to any native form of CTLA-4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CTLA-4 as well as any form of CTLA-4 resulting from processing in cells. The term also encompasses naturally occurring variants of CTLA-4, such as splice variants and allelic variants. An exemplary amino acid sequence of human CTLA-4 is set forth in SEQ ID NO:214, mouse CTLA-4 in SEQ ID NO:247, monkey CTLA-4 in SEQ ID NO:248, and the amino acid sequence of the extracellular domain of human CTLA-4 in SEQ ID NO:28. CTLA-4 may also be referred to herein as CTLA4.

[0064] The term "regulatory T (Treg) cells" refers to a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. These cells generally suppress or downregulate the induction and proliferation of effector T cells. The best understood Treg cells are those that express CD4, CD25, and Foxp3 (CD4 + CD25 + These Tregs are distinct from helper T cells. Several different methods are used to identify and monitor Treg cells. These Tregs are defined by CD4 and CD25 expression (CD4 + CD25 +cells), Treg cells are mature CD4 + They comprise approximately 5-10% of the T cell subpopulation, while approximately 1-2% of Tregs can be measured in whole blood. Expression of Foxp3 may also be measured (CD4 + CD25 + Foxp3 + Alternatively, the absence or low expression of CD127, in combination with the presence of CD4 and CD25, may be used as another marker. Treg cells also express high levels of CTLA-4 and GITR. Tregs can also be identified by the methods described in the Examples below.

[0065] As used herein, the terms "substantially similar," "substantially equal," or "substantially the same" refer to a similarity between two numerical values ​​(e.g., between one relating to an antibody of the invention and one relating to a reference / comparator antibody) that is sufficiently high that one of skill in the art would consider the difference between the two numerical values ​​to have little or no biological and / or statistical significance in terms of the biological characteristic measured by the numerical values ​​(e.g., KD value).

[0066] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0067] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

[0068] The term "tumor tissue" refers to tissue containing at least one tumor cell. Tumor tissue usually consists of a group of tumor cells (parenchyma), which form the main body of the tumor, and the connective tissue and blood vessels (stroma) that exist between them and support the tumor. In some cases, the distinction between the two is clear, while in other cases, the two are mixed. Immune cells and other substances may infiltrate tumor tissue. On the other hand, "non-tumor tissue" refers to tissue other than tumor tissue in the body. Healthy tissue / normal tissue that is not in a diseased state is a typical example of non-tumor tissue.

[0069] II. Compositions and Methods In one aspect, the present invention is based in part on anti-CTLA-4 antibodies and their uses. In certain embodiments, antibodies that bind to CTLA-4 are provided. The antibodies of the present invention are useful, for example, for the diagnosis or treatment of cancer.

[0070] A. Exemplary anti-CTLA-4 antibodies In one aspect, the present invention provides isolated antibodies that bind to CTLA-4. In certain embodiments, the anti-CTLA-4 antibodies of the present invention have CTLA-4 binding activity that is dependent on the concentration of an adenosine-containing compound. In some embodiments, the binding activity to CTLA-4 is higher in the presence of an adenosine-containing compound than in the absence of the adenosine-containing compound. Alternatively, in other embodiments, the binding activity to CTLA-4 is higher in the presence of a high concentration of an adenosine-containing compound than in the presence of a low concentration of the adenosine-containing compound. In further embodiments, the difference in binding activity to CTLA-4 is, for example, 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 500-fold or more, 1×10 3 More than twice, 2×10 3More than double, 3×10 3 More than twice, 5×10 3 More than twice, 1×10 4 More than twice, 2×10 4 More than double, 3×10 4 More than twice, 5×10 4 More than double, or 1×10 5 It is more than double.

[0071] In some embodiments, the binding activity of an anti-CTLA-4 antibody can be expressed as a KD (Dissociation constant) value. In a further embodiment, the KD value of the anti-CTLA-4 antibody is smaller in the presence of an adenosine-containing compound than in the absence of the adenosine-containing compound. Alternatively, in another embodiment, the KD value of the anti-CTLA-4 antibody is smaller in the presence of a high concentration of an adenosine-containing compound than in the presence of a low concentration of the adenosine-containing compound. In a further embodiment, the difference in the KD values ​​of the anti-CTLA-4 antibody is, for example, 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 500-fold or more, 1×10 3 More than twice, 2×10 3 More than double, 3×10 3 More than twice, 5×10 3 More than twice, 1×10 4 More than twice, 2×10 4 More than double, 3×10 4 More than twice, 5×10 4 More than double, or 1×10 5 The KD value of an anti-CTLA-4 antibody in the presence of an adenosine-containing compound or in the presence of a high concentration of an adenosine-containing compound is, for example, 9 × 10 -7 M or less, 8×10 -7 M or less, 7×10 -7 M or less, 6×10 -7 M or less, 5×10 -7 M or less, 4×10 -7 M or less, 3×10 -7 M or less, 2×10 -7 M or less, 1×10 -7 M or less, 9×10 -8 M or less, 8×10 -8 M or less, 7×10-8 M or less, 6×10 -8 M or less, 5×10 -8 M or less, 4×10 -8 M or less, 3×10 -8 M or less, 2×10 -8 M or less, 1×10 -8 M or less, 9×10 -9 M or less, 8×10 -9 M or less, 7×10 -9 M or less, 6×10 -9 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, 1×10 -9 M or less, 9×10 -10 M or less, 8×10 -10 M or less, 7×10 -10 M or less, 6×10 -10 M or less, 5×10 -10 M or less, 4×10 -10 M or less, 3×10 -10 M or less, 2×10 -10 M or less, or 1 x 10 -10 The KD value of an anti-CTLA-4 antibody in the absence of an adenosine-containing compound or in the presence of a low concentration of an adenosine-containing compound can be, for example, 1 × 10 -8 M or more, 2×10 -8 M or more, 3×10 -8 M or more, 4×10 -8 M or more, 5×10 -8 M or more, 6×10 -8 M or more, 7×10 -8 M or more, 8×10 -8 M or more, 9×10 -8 M or more, 1×10 -7 M or more, 2×10 -7 M or more, 3×10 -7 M or more, 4×10 -7 M or more, 5×10 -7 M or more, 6×10 -7 M or more, 7×10 -7 M or more, 8×10 -7 M or more, 9×10 -7 M or more, 1×10 -6 M or more, 2×10-6 M or more, 3×10 -6 M or more, 4×10 -6 M or more, 5×10 -6 M or more, 6×10 -6 M or more, 7×10 -6 M or more, 8×10 -6 M or larger, or 9 x 10 -6 It can be M or more.

[0072] In another embodiment, the binding activity of an anti-CTLA-4 antibody may be expressed as a kd (Dissociation rate constant) value instead of a KD value.

[0073] In another embodiment, the binding activity of an anti-CTLA-4 antibody may be expressed as the amount of CTLA-4 bound per unit amount of antibody. For example, in a surface plasmon resonance assay, the amount of antibody bound to a sensor chip and the amount of antigen bound thereto are each measured as a response unit (RU). The value obtained by dividing the amount of antigen bound thereto by the amount of antibody bound thereto can be defined as the amount of antigen bound per unit amount of antibody. Specific methods for measuring and calculating such binding amounts are described in the Examples below. In some embodiments, the amount of CTLA-4 bound in the presence of an adenosine-containing compound is greater than that in the absence of an adenosine-containing compound. Alternatively, in another embodiment, the amount of CTLA-4 bound in the presence of a high concentration of an adenosine-containing compound is greater than that in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in the amount of CTLA-4 binding is, for example, 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 500-fold or more, 1×10 3 More than twice, 2×10 3 More than double, 3×10 3 More than twice, 5×10 3 More than twice, 1×10 4 More than twice, 2×10 4 More than double, 3×10 4 More than twice, 5×10 4 More than double, or 1×10 5The value of the amount of CTLA-4 binding in the presence of an adenosine-containing compound, or in the presence of a high concentration of an adenosine-containing compound, can be, for example, 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, or 1 or more. The value of the amount of CTLA-4 binding in the absence of an adenosine-containing compound or in the presence of a low concentration of an adenosine-containing compound can be, for example, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less, 0.006 or less, 0.005 or less, 0.004 or less, 0.003 or less, 0.002 or less, or 0.001 or less.

[0074] In some embodiments, the KD values, kd values, binding amounts, etc., described herein are measured or calculated by performing a surface plasmon resonance assay at 25°C or 37°C (see, e.g., Example 3 herein).

[0075] The concentration of the adenosine-containing compound can be selected at any concentration as long as it detects a difference in the binding activity of anti-CTLA-4 antibodies. In certain embodiments, high concentrations can include, for example, 1 nM or higher, 3 nM or higher, 10 nM or higher, 30 nM or higher, 100 nM or higher, 300 nM or higher, 1 μM or higher, 3 μM or higher, 10 μM or higher, 30 μM or higher, 100 μM or higher, 300 μM or higher, 1 mM or higher, 3 mM or higher, 10 mM or higher, 30 mM or higher, 100 mM or higher, 300 mM or higher, and 1 M or higher. Alternatively, the high concentration herein can be a sufficient amount such that each anti-CTLA-4 antibody exhibits maximum binding activity. In one embodiment, the high concentration herein can be selected to be 1 μM, 10 μM, 100 μM, 1 mM, or a sufficient amount such that each anti-CTLA-4 antibody exhibits maximum binding activity. In a specific embodiment, the low concentration can include, for example, 1 mM or lower, 300 μM or lower, 100 μM or lower, 30 μM or lower, 10 μM or lower, 3 μM or lower, 1 μM or lower, 300 nM or lower, 100 nM or lower, 30 nM or lower, 10 nM or lower, 3 nM or lower, 1 nM or lower, 300 pM or lower, 100 pM or lower, 30 pM or lower, 10 pM or lower, 3 pM or lower, 1 pM or lower, etc. Alternatively, the low concentration here can be the concentration at which each anti-CTLA-4 antibody exhibits minimal binding activity.A substantially zero concentration (in the absence of an adenosine-containing compound) can also be selected as an embodiment of a low concentration. In one embodiment, 1 mM, 100 μM, 10 μM, or 1 μM, the concentration at which each anti-CTLA-4 antibody exhibits minimal binding activity, or the absence of an adenosine compound, can be selected as a low concentration. In another embodiment, the ratio of the high concentration to the low concentration can be, for example, 3-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300-fold or more, or 1×10. 3 Double or more, 3 x 10 3 Double or more, 1 x 10 4 Double or more, 3 x 10 4 Double or more, 1 x 10 5 Double or more, 3 x 10 5 Double or more, 1 x 10 6 Double or more, 3 x 10 6 Double or more, 1 x 10 7 Double or more, 3 x 10 7 Double or more, 1 x 10 8 Double or more, 3 x 10 8 Double or more, 1 x 10 9 Double or more, 3 x 10 9 Double or more, 1 x 10 10 Double or more, 3 x 10 10 Double or more, 1 x 10 11 Double or more, 3 x 10 11 Double or more, 1 x 10 12 A value of double or more can be selected.

[0076] In another embodiment, the anti-CTLA-4 antibody of the present invention also has binding activity to adenosine-containing compounds. Using the above-mentioned method, the amount of adenosine-containing compounds bound per unit antibody amount can be calculated, and this can be used as the binding activity of the anti-CTLA-4 antibody to adenosine-containing compounds. Specific methods for measuring and calculating such binding amounts are described in the Examples below. The amount of adenosine-containing compound bound per unit antibody amount of an anti-CTLA-4 antibody of the present invention can be, for example, 0.0001 or more, 0.0002 or more, 0.0003 or more, 0.0004 or more, 0.0005 or more, 0.0006 or more, 0.0007 or more, 0.0008 or more, 0.0009 or more, 0.001 or more, 0.002 or more, 0.003 or more, 0.004 or more, 0.005 or more, 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more, or 0.01 or more.

[0077] In another embodiment, the anti-CTLA-4 antibody of the present invention forms a ternary complex with an adenosine-containing compound and CTLA-4. In one embodiment, the anti-CTLA-4 antibody binds to an adenosine-containing compound via heavy chain CDR1, CDR2, and CDR3. In one embodiment, the anti-CTLA-4 antibody has a binding motif for an adenosine-containing compound. The binding motif for an adenosine-containing compound may consist of at least one amino acid at positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c, for example, according to the Kabat numbering system. In a further embodiment, the anti-CTLA-4 antibody binds to an adenosine-containing compound via at least one amino acid selected from the group consisting of positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c, for example, according to the Kabat numbering system. In certain embodiments, the anti-CTLA-4 antibody has at least one amino acid selected from the group consisting of Thr at position 33, Ser at position 52, Ser at position 52a, Arg at position 53, Tyr at position 56, Tyr at position 58, Tyr at position 95, Gly at position 96, Met at position 100a, Leu at position 100b, and Trp at position 100c, as determined by the Kabat numbering system. CTLA-4 may also bind to the complex formed by binding of the anti-CTLA-4 antibody and the adenosine-containing compound. Alternatively, the adenosine-containing compound may be present at the interface where the anti-CTLA-4 antibody and CTLA-4 interact, binding to both of them. The formation of a ternary complex between the anti-CTLA-4 antibody, the adenosine-containing compound, and CTLA-4 can be confirmed, for example, by techniques such as crystal structure analysis described below (see Examples).

[0078] In another embodiment, the anti-CTLA-4 antibodies of the present invention bind to at least one amino acid selected from the group consisting of amino acid 3 (Met), amino acid 33 (Glu), amino acid 35 (Arg), amino acid 53 (Thr), amino acid 97 (Glu), amino acid 99 (Met), amino acid 100 (Tyr), amino acid 101 (Pro), amino acid 102 (Pro), amino acid 103 (Pro), amino acid 104 (Tyr), amino acid 105 (Tyr), and amino acid 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). These amino acids may constitute the epitope of the anti-CTLA-4 antibodies of the present invention. In another embodiment, the anti-CTLA-4 antibodies of the present invention bind to the region from amino acid 97 (Glu) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). In another embodiment, the anti-CTLA-4 antibodies of the invention bind to the region from amino acid 99 (Met) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO:28).

[0079] In another embodiment, an anti-CTLA-4 antibody of the invention competes with ABAM004 (VH, SEQ ID NO:10; VL, SEQ ID NO:11; HVR-H1, SEQ ID NO:100; HVR-H2, SEQ ID NO:101; HVR-H3, SEQ ID NO:102; HVR-L1, SEQ ID NO:113; HVR-L2, SEQ ID NO:114; HVR-L3, SEQ ID NO:115) for binding to CTLA-4. In another embodiment, an anti-CTLA-4 antibody of the invention binds to the same epitope as ABAM004. When an anti-CTLA-4 antibody is present in excess, the binding of ABAM004 to CTLA-4 can be reduced by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Exemplary competition assays are provided herein.

[0080] In another embodiment, the anti-CTLA-4 antibodies of the present invention exhibit cytotoxic activity against CTLA-4-expressing cells. When CTLA-4 is expressed on the surface of a target cell and an anti-CTLA-4 antibody binds to the cell, the cell can be cytotoxic. Cell cytotoxicity may be induced by effector cells bound to the antibody, such as through antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), or by complement bound to the antibody, such as through complement-dependent cytotoxicity (CDC). Alternatively, the cytotoxicity may be induced by a cytotoxic agent (e.g., a radioisotope or a chemotherapeutic agent) bound to the antibody, such as in an immunoconjugate. Here, cytotoxicity may include effects such as inducing cell death, inhibiting cell proliferation, or impairing cell function. When present in sufficient amounts, an anti-CTLA-4 antibody can cause cytotoxicity in, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of CTLA-4-expressing cells. Such cytotoxicity measurements can be compared to measurements in the absence of the antibody or in the presence of a negative control antibody. Exemplary cytotoxicity assays are provided herein.

[0081] In another embodiment, the anti-CTLA-4 antibody of the present invention exhibits neutralizing activity against CTLA-4. CTLA-4 is known to function by interacting with its ligands, CD80 (B7-1) or CD86 (B7-2). In certain embodiments, the anti-CTLA-4 antibody inhibits the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2). When present in a sufficient amount, the anti-CTLA-4 antibody can inhibit the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2), for example, by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Such inhibitory activity measurements can be made relative to measurements in the absence of the antibody or in the presence of a negative control antibody. Specific methods for measuring neutralizing activity are provided herein.

[0082] In another embodiment, the anti-CTLA-4 antibodies of the present invention bind to CTLA-4 derived from multiple animal species. Exemplary animal species include mammals, such as humans, monkeys, mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, goats, horses, sheep, camels, dogs, and cats. In a specific embodiment, the anti-CTLA-4 antibodies bind to CTLA-4 derived from humans and non-humans (e.g., monkeys, mice, rats, etc.). The amino acid sequence of human CTLA-4 is set forth in SEQ ID NO: 214, the amino acid sequence of monkey CTLA-4 is set forth in SEQ ID NO: 247, and the amino acid sequence of mouse CTLA-4 is set forth in SEQ ID NO: 248. The amino acid sequences of CTLA-4 derived from other animal species can also be determined appropriately by methods known to those skilled in the art.

[0083] In certain embodiments, examples of adenosine-containing compounds of the present invention include adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP), deoxyadenosine (dADO), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), deoxyadenosine monophosphate (dAMP), adenosine gamma thiotriphosphate (ATPγS), and the like.

[0084] In one aspect, the invention provides antibodies comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225.

[0085] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 228.

[0086] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 228.

[0087] 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.

[0088] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0089] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0090] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0091] 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.

[0092] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0093] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0094] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0095] 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.

[0096] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0097] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0098] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0099] 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.

[0100] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0101] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153.

[0102] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153.

[0103] 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.

[0104] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0105] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0106] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0107] 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.

[0108] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0109] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0110] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0111] 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.

[0112] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152.

[0113] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0114] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0115] 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.

[0116] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0117] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0118] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0119] 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.

[0120] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152.

[0121] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0122] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0123] 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.

[0124] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0125] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0126] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0127] In another aspect, the present invention provides an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133.

[0128] In certain embodiments, any one or more amino acids of the above-described anti-CTLA-4 antibodies are substituted at the following HVR positions: - in HVR-H1 (SEQ ID NO: 223): position 2 - in HVR-H2 (SEQ ID NO: 224): positions 4, 5, 7, 13, and 16 - in HVR-H3 (SEQ ID NO: 225): position 3 - in HVR-L1 (SEQ ID NO: 226): positions 1, 3, 6, 11, 12, and 14 - in HVR-L2 (SEQ ID NO: 227): positions 1, 3, 4, and 7 - in HVR-L3 (SEQ ID NO: 228): positions 1 and 10

[0129] In certain embodiments, the substitutions provided herein are conservative substitutions. In certain embodiments, any one or more of the following substitutions may be made in any combination: - in HVR-H1 (SEQ ID NO: 100): H2A, R or K - in HVR-H2 (SEQ ID NO: 101): S4T; R5Q; G7H; D13E or R; K16R - in HVR-H3 (SEQ ID NO: 102): K3A - in HVR-L1 (SEQ ID NO: 113): T1D, Q or E; T3P; D6G; N11T; Y12W; S14H - in HVR-L2 (SEQ ID NO: 114): E1F or Y; S3I; K4S; S7E or K - in HVR-L3 (SEQ ID NO: 115): S1Q;M10T

[0130] All possible combinations of the above substitutions are encompassed in the consensus sequences of SEQ ID NOs: 223, 224, 225, 226, 227, and 228 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, respectively.

[0131] In any of the above-described embodiments, the anti-CTLA-4 antibody is humanized. In one embodiment, the anti-CTLA-4 antibody comprises the HVR of any of the above-described embodiments and further comprises an acceptor human framework (e.g., a human immunoglobulin framework or a human consensus framework). In another embodiment, the anti-CTLA-4 antibody comprises the HVR of any of the above-described embodiments and further comprises a VH or VL comprising FR sequences. In a further embodiment, the anti-CTLA-4 antibody comprises the following heavy and / or light chain variable domain FR sequences: for the heavy chain variable domain, FR1 comprises the amino acid sequence of any one of SEQ ID NOs:229-232, FR2 comprises the amino acid sequence of SEQ ID NO:233, FR3 comprises the amino acid sequence of SEQ ID NO:234, and FR4 comprises the amino acid sequence of SEQ ID NO:235. For the light chain variable domain, FR1 comprises any one of the amino acid sequences of SEQ ID NOs: 236 to 238, FR2 comprises any one of the amino acid sequences of SEQ ID NOs: 240 to 241, FR3 comprises any one of the amino acid sequences of SEQ ID NOs: 242 to 244, and FR4 comprises any one of the amino acid sequences of SEQ ID NOs: 245 to 246.

[0132] In another aspect, an anti-CTLA-4 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CTLA-4 antibody comprising such a sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 10. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-CTLA-4 antibody comprises the VH sequence in SEQ ID NO: 10, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0133] In another aspect, an anti-CTLA-4 antibody is provided that comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CTLA-4 antibody comprising such a sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 11. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-CTLA-4 antibody comprises the VL sequence in SEQ ID NO: 11, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0134] In another aspect, an anti-CTLA-4 antibody is provided that comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 149. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CTLA-4 antibody comprising such a sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 149. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-CTLA-4 antibody comprises the VL sequence in SEQ ID NO: 149, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0135] In another aspect, an anti-CTLA-4 antibody is provided, comprising the VH of any of the above embodiments and the VL of any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:10 and SEQ ID NO:11, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:98 and SEQ ID NO:99, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:83 and SEQ ID NO:97, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:86 and SEQ ID NO:134, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:136 and SEQ ID NO:95, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 146, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 147, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 136 and SEQ ID NO: 149, respectively, including post-translational modifications of said sequences. In a further aspect, a heterologous anti-CTLA-4 antibody is provided, comprising at least two different variable regions selected from among the variable regions comprising the VH and VL sequences described above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 146, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of said sequences.In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 147, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of such sequences, including, but not limited to, modification of the heavy or light chain N-terminus glutamine or glutamic acid to pyroglutamic acid by pyroglutamylation.

[0136] 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.

[0137] In a further aspect, the present invention provides antibodies that bind to the same epitope as the anti-CTLA-4 antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind to the same epitope as the antibodies listed in Tables 4, 9, 14, and 19. In certain embodiments, antibodies are provided that bind to an epitope in a fragment of CTLA-4 that contains at least one amino acid selected from the group consisting of amino acid 3 (Met), amino acid 33 (Glu), amino acid 35 (Arg), amino acid 53 (Thr), amino acid 97 (Glu), amino acid 99 (Met), amino acid 100 (Tyr), amino acid 101 (Pro), amino acid 102 (Pro), amino acid 103 (Pro), amino acid 104 (Tyr), amino acid 105 (Tyr), and amino acid 106 (Leu) of SEQ ID NO:28. In certain embodiments, antibodies are provided that bind to an epitope in a fragment of CTLA-4 consisting of amino acid 97 (Glu) to amino acid 106 (Leu) of SEQ ID NO: 28. In certain embodiments, antibodies are provided that bind to an epitope in a fragment of CTLA-4 consisting of amino acid 99 (Met) to amino acid 106 (Leu) of SEQ ID NO: 28.

[0138] In further aspects of the invention, the anti-CTLA-4 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-CTLA-4 antibody is an antibody fragment, such as, for example, an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as, for example, a full IgG1 antibody or a full IgG4 antibody, or other antibody classes or isotypes defined herein.

[0139] In a further aspect, the anti-CTLA-4 antibody of the present invention comprises an Fc region. In a further aspect, the anti-CTLA-4 antibody of the present invention comprises a constant region. The constant region may be a heavy chain constant region (including an Fc region), a light chain constant region, or both. In some embodiments, the Fc region is a native-sequence Fc region. Exemplary heavy chain constant regions derived from native antibodies include heavy chain constant regions 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). Other exemplary heavy chain constant regions include heavy chain constant regions such as those of SEQ ID NO: 82 and SEQ ID NO: 158. Exemplary light chain constant regions derived from native antibodies include light chain constant regions such as human κ chain (SEQ ID NO: 33, SEQ ID NO: 63, SEQ ID NO: 159) and human λ chain (SEQ ID NO: 53, SEQ ID NO: 87).

[0140] In another embodiment, the Fc region is a mutant Fc region created by amino acid alterations to a native-sequence Fc region. In a specific embodiment, the mutant Fc region has enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa, compared to a native-sequence Fc region. In a further embodiment, the mutant Fc region has enhanced binding activity to FcγRIIa and FcγRIIIa, compared to a native-sequence Fc region. Examples of heavy chain constant regions comprising such mutant Fc regions include the heavy chain constant regions listed in Tables 26 to 30, and the heavy chain constant regions listed in SEQ ID NOs: 31, 32, 41 to 46, 65, 66, 81, 207, 239, 253 to 271, 276, 277, and 278.

[0141] Native sequence Fc regions are usually structured as homodimers consisting of two identical polypeptide chains. In certain embodiments, mutant Fc regions may be homodimers consisting of polypeptide chains with the same sequence, or may be heterodimers consisting of polypeptide chains with different sequences. Similarly, heavy chain constant regions comprising Fc regions may be homodimers consisting of polypeptide chains with the same sequence, or may be heterodimers consisting of polypeptide chains with different sequences. Examples of heterologous heavy chain constant regions include a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 31 and a polypeptide chain of SEQ ID NO: 32, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 43 and a polypeptide chain of SEQ ID NO: 44, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 45 and a polypeptide chain of SEQ ID NO: 46, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 254 and a polypeptide chain of SEQ ID NO: 256, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 257 and a polypeptide chain of SEQ ID NO: 258, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 259 and a polypeptide chain of SEQ ID NO: 260, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 261 and a polypeptide chain of SEQ ID NO: 263, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 262 and a polypeptide chain of SEQ ID NO: 264, Examples of such a heavy chain constant region include a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 265 and the polypeptide chain of SEQ ID NO: 267, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 266 and the polypeptide chain of SEQ ID NO: 268, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 269 and the polypeptide chain of SEQ ID NO: 270, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 271 and the polypeptide chain of SEQ ID NO: 81, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 65 and the polypeptide chain of SEQ ID NO: 66, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 239 and the polypeptide chain of SEQ ID NO: 207, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 259 and the polypeptide chain of SEQ ID NO: 276, and a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 65 and the polypeptide chain of SEQ ID NO: 278.

[0142] In further aspects, anti-CTLA-4 antibodies according to any of the above embodiments may incorporate, alone or in combination, any of the features described in items 1-7 below.

[0143] 1. Antibody binding activity In certain embodiments, the binding activity of the antibodies provided herein is ≦10 μM, ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 is the dissociation constant (KD) of the ATPase (M).

[0144] In one embodiment, antibody binding activity is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by measuring the binding affinity of the Fab to the antigen at a minimum concentration ( 125 I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57: 4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0145] In one embodiment, antibody binding activity is measured using a ligand capture method based on surface plasmon resonance analysis, for example, using a BIACORE™ T200 or BIACORE™ 4000 (GE Healthcare, Uppsala, Sweden). BIACORE™ Control Software is used to operate the instrument. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions to immobilize a ligand capture molecule, such as an anti-tag antibody, anti-IgG antibody, or protein A, on a carboxymethyldextran-coated sensor chip (GE Healthcare, Uppsala, Sweden). The ligand capture molecule is diluted with 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate and injection time. Binding activity measurements are performed using a buffer containing 0.05% polysorbate 20 (also known as Tween®-20) as the measurement buffer, at a flow rate of 10-30 μL / min, and at a temperature of preferably 25°C or 37°C. When measurements are performed by capturing an antibody as a ligand on a ligand capture molecule, the antibody is injected and captured in a desired amount, and then a serial dilution (analyte) of an antigen or Fc receptor prepared using the measurement buffer is injected. When measurements are performed by capturing an antigen or Fc receptor as a ligand on a ligand capture molecule, the antigen or Fc receptor is injected and captured in a desired amount, and then a serial dilution (analyte) of an antibody prepared using the measurement buffer is injected.

[0146] In one embodiment, the measurement results are analyzed using BIACORE® Evaluation Software. Kinetic parameters are calculated by simultaneously fitting the binding and dissociation sensorgrams using a 1:1 binding model, and the binding rate (k or ka), dissociation rate (k or k), and equilibrium dissociation constant (KD) can be calculated. When the binding activity is weak, particularly when dissociation is rapid and calculation of kinetic parameters is difficult, the equilibrium dissociation constant (KD) may be calculated using a steady state model. Another parameter of binding activity may be calculated by dividing the amount of analyte bound (RU) at a specific concentration by the amount of ligand captured (RU), i.e., the "amount of analyte bound per unit amount of ligand."

[0147] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased half-lives in vivo.

[0148] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0149] A single-domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0150] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0151] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0152] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0153] Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and also see, 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 a "guide selection" approach for FR shuffling).

[0154] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see Sims et al. J. Immunol. 151: 2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992) and Presta et al. J. Immunol., 151: 2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening of FR libraries (see Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0155] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0156] Human antibodies may be prepared by administering an immunogen to transgenic animals that have been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of human immunoglobulin loci, which either replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0157] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4): 265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-191 (2005).

[0158] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0159] 5. Library-derived Antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further see, for example, McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0160] In a specific phage display method, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers encoding the hypervariable CDR3 regions and containing random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent literature describing human antibody phage libraries includes, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0161] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0162] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites. In certain embodiments, one of the binding specificities is for CTLA-4 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of CTLA-4. Bispecific antibodies may be used to localize cytotoxic agents to cells expressing CTLA-4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0163] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole technology (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152: 6444-6448 (1993)). 5368 (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0164] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576 A1).

[0165] As used herein, antibody or fragment also includes a "dual-acting Fab" or "DAF" that contains one antigen-binding site that binds to CTLA-4 and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0166] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0167] a) Substitution, insertion, and deletion mutants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the products may be screened for a desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0168] [Table 1]

[0169] Amino acids can be divided into groups according to common side chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the exchange of a member of one of these classes for one from another class.

[0170] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study will have a modified (e.g., improved) specific biological property compared to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain the specific biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0171] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigen, and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0172] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, as long as such modifications do not substantially reduce the antibody's ability to bind to antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such modifications may, for example, be outside the antigen-contacting residues of the HVRs. In certain embodiments of the above-described mutant VH and VL sequences, each HVR is unaltered or contains only one, two, or three amino acid substitutions.

[0173] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted as substitution candidates or can be excluded from the list. Mutants can be screened to determine whether they contain desired properties.

[0174] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as internal insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the plasma half-life of the antibody.

[0175] b) Glycosylation variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0176] If the antibody contains an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, e.g., Wright et al. TIBTECH 15: 26-32 (1997). Oligosaccharides include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention may be performed to create antibody variants with specific improved properties.

[0177] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 represents an asparagine residue located approximately at position 297 in the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application Publication No. US2003 / 0157108 A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003 / 085107).

[0178] Further provided are antibody variants having bisected oligosaccharides, for example, bisected oligosaccharides attached to the Fc region of the antibody by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0179] c) Fc region mutants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0180] In certain embodiments, antibody variants that retain some, but not all, effector functions are also contemplated by the present invention, making them desirable candidates for applications where in vivo half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo in an animal model, e.g., as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). Furthermore, determination of FcRn binding and in vivo clearance / half-life may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0181] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0182] Certain antibody variants with increased or decreased binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0183] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (EU numbering) of the Fc region).

[0184] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0185] Antibodies with increased half-lives and increased binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976); Kim et al., J. Immunol. 24: 249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0186] For other examples of Fc region variants, see also Duncan & Winter, Nature 322: 738-740 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0187] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody have been substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to generate immunoconjugates, as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0188] e) Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0189] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, wavelengths that heat the non-protein moiety to temperatures that are not harmful to normal cells but that kill cells in close proximity to the antibody-non-protein moiety.

[0190] B. Recombinant Methods and Constructs Antibodies can be produced using recombinant methods or constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CTLA-4 antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, or Sp2 / 0 cell)). In one aspect, a method for producing an anti-CTLA-4 antibody is provided, comprising culturing a host cell containing nucleic acid encoding the antibody as described above under conditions suitable for expression of the anti-CTLA-4 antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0191] For recombinant production of an anti-CTLA-4 antibody, nucleic acid encoding the antibody (e.g., such as those described above) is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0192] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (also see Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste and further purified.

[0193] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0194] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

[0195] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0196] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36: 59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)). (described in

[1999] ); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0197] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. The relevant antigen is conjugated to a protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, with a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R. 1 N=C=NR (where R and R 1 are different alkyl groups).

[0198] Animals (usually non-human mammals) are immunized against an antigen, immunogenic conjugate, or derivative by combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animal is boosted with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animal is bled and the serum is assayed for antibody titer. The animal is boosted until the titer plateaus. Preferably, the animal is boosted with a conjugate of the same antigen but conjugated to a different protein and / or via a different cross-linking reagent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Aggregating agents, such as alum, are also suitably used to enhance the immune response.

[0199] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0200] For example, monoclonal antibodies can be produced using the hybridoma method first described in Kohler et al., Nature 256(5517): 495-497 (1975). In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described hereinabove to induce lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0201] The immunizing agent typically includes an antigen protein or a fusion variant thereof. Generally, peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0202] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells thus generated are seeded and grown in an appropriate culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0203] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center in San Diego, California, USA, and SP-2 cells (and their derivatives, such as X63-Ag8-653) available from the American Type Culture Collection in Manassas, Virginia, USA, are preferred. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor et al., J. Immunol. 133(6):3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0204] The culture medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson, Anal Biochem. 107(1): 220-239 (1980).

[0205] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also be grown in vivo as tumors in mammals.

[0206] 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.

[0207] Antibodies may be produced by immunizing a suitable host animal against an antigen. In one embodiment, the antigen is a polypeptide comprising full-length CTLA-4. In one embodiment, the antigen is a polypeptide comprising soluble CTLA-4. In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acid 97 (Glu) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO:28). In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acid 99 (Met) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO:28). The present invention also encompasses antibodies produced by immunizing an animal against an antigen. The antibody may incorporate any of the features described above for the exemplary anti-CTLA-4 antibodies, alone or in combination.

[0208] C. Assay The anti-CTLA-4 antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0209] 1. Binding and other assays In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, Western blot, surface plasmon resonance assay, and the like.

[0210] In another aspect, a competition assay can be used to identify antibodies that compete with the anti-CTLA-4 antibodies described herein (e.g., the anti-CTLA-4 antibodies described in Table 4, Table 9, Table 14, and Table 19) for binding to CTLA-4. In certain embodiments, when such a competing antibody is present in excess, it prevents (e.g., reduces) binding of the reference antibody to CTLA-4 by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some examples, binding is inhibited by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as that bound by an anti-CTLA-4 antibody described herein (e.g., an anti-CTLA-4 antibody described in Table 4, Table 9, Table 14, and Table 19). Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ).

[0211] In an exemplary competitive assay, immobilized CTLA-4 is incubated in a solution containing a first, labeled antibody that binds to CTLA-4 and a second, unlabeled antibody to be tested for its ability to compete with the first antibody for binding to CTLA-4. The second antibody may be present in hybridoma supernatant. As a control, immobilized CTLA-4 is incubated in a solution containing the first, labeled antibody but not the second, unlabeled antibody. After incubation under conditions that allow binding of the first antibody to CTLA-4, excess unbound antibody is removed and the amount of label bound to immobilized CTLA-4 is measured. A substantial decrease in the amount of label bound to immobilized CTLA-4 in the test sample compared to the control sample indicates that the second antibody competes with the first antibody for binding to CTLA-4. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0212] 2.Activity measurement method In one aspect, a method for identifying an anti-CTLA-4 antibody having biological activity is provided. Biological activity may include, for example, cell growth inhibitory activity, cytotoxic activity (e.g., ADCC / CDC activity, ADCP activity), immunostimulatory activity, and CTLA-4 inhibitory activity. Also provided are antibodies having such biological activity in vivo and / or in vitro.

[0213] In certain embodiments, antibodies of the invention are tested for such biological activities.

[0214] In certain embodiments, antibodies of the present invention are tested for their ability to inhibit cell growth or proliferation in vitro. Assays for cell growth or proliferation inhibition are well known in the art. Certain cell proliferation assays, exemplified by the "cell killing" assay described herein, measure cell viability. One such assay is the CellTiter-Glo™ Luminescent Cell Viability Assay, commercially available from Promega (Madison, WI). This assay determines the number of viable cells in culture based on the presence of ATP, an indicator of metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160: 81-88; U.S. Patent No. 6,602,677. Assays may also be performed in 96- or 384-well formats, making them amenable to automated high-throughput screening (HTS). See Cree et al. (1995) Anticancer Drugs 6: 398-404. The assay procedure involves adding a single reagent (CellTiter-Glo® Reagent) directly to cultured cells, which lyses the cells and generates a luminescent signal via the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in the culture. Data can be recorded by a luminometer or a CCD camera imager. Luminescence values ​​are expressed in relative light units (RLU).

[0215] Another cell proliferation assay is the "MTT" assay, a colorimetric assay that measures the oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by mitochondrial reductase. Similar to the CellTiter-Glo™ assay, this assay indicates the number of metabolically active cells present in cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65: 55-63 and Zhang et al. (2005) Cancer Res. 65: 3877-3882.

[0216] Cells for use in any of the above in vitro assays include cells or cell lines that naturally express CTLA-4 or that have been engineered to express CTLA-4. Such cells also include cell lines that express CTLA-4 and cell lines that do not normally express CTLA-4 but that have been transfected with a nucleic acid encoding CTLA-4.

[0217] In one aspect, anti-CTLA-4 antibodies are tested for their ability to inhibit cell growth or proliferation in vivo. In certain embodiments, anti-CTLA-4 antibodies are tested for their ability to inhibit tumor growth in vivo. An in vivo model system, such as a xenograft model, can be used for such testing. In an exemplary xenograft system, human tumor cells are introduced into an appropriately immunocompromised non-human animal, such as an athymic "nude" mouse. An antibody of the present invention is administered to the animal. The ability of the antibody to inhibit or reduce tumor growth is measured. In certain embodiments of the above xenograft system, the human tumor cells are tumor cells derived from a human patient. Such xenograft models are commercially available from Oncotest GmbH (Frieberg, Germany). In certain embodiments, human tumor cells are introduced into an appropriately immunocompromised non-human animal by subcutaneous injection or by implantation into a suitable site, such as the mammary fat pad.

[0218] It will be understood that any of the above assays can be performed using an immunoconjugate of the invention in place of or in addition to an anti-CTLA-4 antibody.

[0219] A representative assay for measuring the ADCC activity of a therapeutic antibody is 51 It is based on a Cr release assay and includes the following steps: 51 The method involves labeling the cells with [Cr]Na2CrO4; opsonizing target cells expressing the antigen on their surface with the antibody; combining the opsonized radiolabeled target cells with effector cells at a suitable ratio in a microtiter plate in the presence or absence of the test antibody; incubating the cell mixture for 16-18 hours, preferably at 37°C; collecting the supernatant; and analyzing the radioactivity in the supernatant sample. The cytotoxicity of the test antibody is then determined, for example, using the following formula: specific cytotoxicity (%) = (radioactivity in the presence of antibody - radioactivity in the absence of antibody) / (maximum radioactivity - radioactivity in the absence of antibody) x 100. Graphs can be generated by varying the target cell:effector cell ratio or antibody concentration.

[0220] To assess complement activation, a complement-dependent cytotoxicity (CDC) assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Briefly, various concentrations of the polypeptide variant and human complement are diluted in buffer. Approximately 1 x 10 cells expressing the antigen to which the polypeptide variant binds are cultured. 6The mixture is diluted to a density of 1000 cells / ml. The mixture of polypeptide variant, diluted human complement, and antigen-expressing cells is added to a flat-bottom 96-well tissue culture plate and incubated at 37°C and 5% CO2 for 2 hours to promote complement-mediated cell lysis. 50 μl of Alamar Blue (Accumed International) is then added to each well and incubated overnight at 37°C. Absorbance is measured using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm. Results are expressed in relative fluorescence units (RFU). Sample concentrations can be calculated from the standard curve, and percent activity compared to the non-mutant polypeptide is reported for the polypeptide variant of interest.

[0221] An exemplary assay for ADCP activity may include coating a target bioparticle, such as E. coli-labeled FITC (Molecular Probes) or Staphylococcus aureus-FITC, with a test antibody to form an opsonized particle; adding the opsonized particle to THP-1 effector cells (a monocytic cell line available from the American College of Cancer Research) at a ratio of 1:1, 10:1, 30:1, 60:1, 75:1, or 100:1 to allow FcγR-mediated phagocytosis to occur; preferably incubating the cells with E. coli-FITC / antibody at 37°C for 1.5 hours; after incubation, adding trypan blue to the cells (preferably for 2-3 minutes at room temperature) to quench the fluorescence of bacteria that are not internalized but adhere to the outer cell surface; transferring the cells to FACS buffer (e.g., 0.1% BSA, 0.1% sodium azide in PBS) and assaying the fluorescence of the THP-1 cells using FACS (e.g., BD FACS Calibur). To assay the degree of ADCP, a gate is preferably set on THP-1 cells and the median fluorescence intensity is measured. In the most preferred embodiment, the ADCP assay is performed using E. coli-FITC in culture medium (control), E. coli-FITC and THP-1 cells (used as FcγR-independent ADCP activity), or E. coli-FITC, THP-1 cells, and the test antibody (used as FcγR-dependent ADCP activity).

[0222] The cytotoxic activity of an antibody is usually accompanied by binding of the antibody to the cell surface. Whether or not an antigen is expressed on the surface of a target cell can be appropriately confirmed by techniques known to those skilled in the art, such as FACS.

[0223] Immune activation can be detected using cellular or humoral immune responses as indicators. Specifically, this includes increased expression of cytokines (e.g., IL-6, G-CSF, IL-12, TNFα, and IFNγ) or their receptors, and enhanced proliferation, activation, function, or cytotoxic activity of immune cells (e.g., B cells, T cells, NK cells, macrophages, monocytes). In particular, T cell activation can be detected by measuring increased expression of activation markers such as CD25, CD69, and ICOS. For example, patients administered the anti-CTLA-4 antibody ipilimumab showed significantly increased ICOS levels in peripheral blood after administration. + CD4 + It is known that T cells increase, which is thought to be the result of activating the systemic immune state by administering anti-CTLA-4 antibodies (Cancer Immunol. Res. (2013) 1(4): 229-234).

[0224] T cell activation requires not only stimulation via the antigen receptor (TCR) but also costimulation via CD28. When CD28 on the T cell surface binds to B7-1 (CD80) or B7-2 (CD86) present on the surface of an antigen-presenting cell, a costimulatory signal is transmitted within the T cell, resulting in T cell activation. CTLA-4 is also expressed on the surface of activated T cells. CTLA-4 binds to CD80 and CD86 with stronger affinity than CD28, so it interacts with CD80 and CD86 preferentially over CD28, ultimately suppressing T cell activation.

[0225] Based on this mechanism of action, inhibitory activity against CTLA-4 can be measured as the activity of inhibiting the binding of CTLA-4 to CD80 or CD86. In one embodiment, an assay for measuring inhibitory activity against CTLA-4 comprises the following steps: binding purified CTLA-4 protein to a support such as a microtiter plate or magnetic beads, adding a test antibody and labeled soluble CD80 or CD86, washing away unbound components, and quantifying the bound labeled CD80 or CD86. Whether a test antibody cross-reacts with CD28 can be confirmed by performing a similar assay in which CTLA-4 is replaced with CD28. In another embodiment, inhibitory activity against CTLA-4 can also be measured using a functional assay for detecting T cell activation, as described above. For example, in a system in which a T cell population is stimulated with cells expressing CD80 or CD86 to measure T cell activation, the addition of a test antibody with CTLA-4 inhibitory activity results in further enhancement of T cell activation.

[0226] D. Immunoconjugates The present invention also provides immunoconjugates comprising the anti-CTLA-4 antibodies herein conjugated to one or more cytotoxic agents (e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant, or animal origin, an enzymatically active toxin, or fragment thereof), or a radioactive isotope).

[0227] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to: maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235 B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53: 3336-3342). (1993); and see Lode et al., Cancer Res. 58: 2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chem. 16: 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al., J. Med. Chem. 45: 4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0228] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0229] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include: 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Radioactive isotopes include Pb and Lu. When radioactive conjugates are used for detection, they are used in combination with radioactive atoms for scintigraphic examinations (e.g., Tc-99m or 123 I), or spin labels (again, e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0230] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein linking agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0231] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0232] E. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-CTLA-4 antibodies provided herein are useful for detecting the presence of CTLA-4 in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, milk, colostrum, mammary gland secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, peritoneal fluid, ocular fluid, or mucus.

[0233] In one embodiment, an anti-CTLA-4 antibody is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of CTLA-4 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-CTLA-4 antibody described herein under conditions that allow binding of the anti-CTLA-4 antibody to CTLA-4, and detecting whether a complex is formed between the anti-CTLA-4 antibody and CTLA-4. Such a method can be an in vitro method or an in vivo method. In one embodiment, the anti-CTLA-4 antibody is used to select subjects suitable for treatment with an anti-CTLA-4 antibody, for example, when CTLA-4 is a biomarker for patient selection.

[0234] The antibodies of the present invention can be used, for example, to check the status of the immune response or to diagnose immune system dysfunction.

[0235] In certain embodiments, labeled anti-CTLA-4 antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and moieties that are indirectly detected, for example, through enzymatic reactions or molecular interactions (e.g., enzymes or ligands). Exemplary labels include, but are not limited to, radioisotopes 32 P, 14 C. 125 I, 3 H and 131 Fluorophores such as I, rare earth chelates or those linked to fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases such as uricase and xanthine oxidase, enzymes that oxidize dye precursors using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0236] F. Pharmaceutical Formulations Pharmaceutical formulations of the anti-CTLA-4 antibodies described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the antibody having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.; small (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral activated hyaluronidase glycoproteins (sHASEGPs) (e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Certain exemplary sHASEGPs and methods of use thereof (including rHuPH20) are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0237] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0238] The formulations herein may contain more than one active ingredient as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. Such active ingredients are present in suitable combinations and in amounts that are effective for the purpose intended.

[0239] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by droplet formation (coacervation) techniques or by interfacial polymerization, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0240] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0241] Preparations to be used for in vivo administration are generally sterile, and sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0242] G. Therapeutic Methods and Compositions Any of the anti-CTLA-4 antibodies provided herein may be used in therapeutic methods. In one aspect, an anti-CTLA-4 antibody is provided for use as a pharmaceutical. In a further aspect, an anti-CTLA-4 antibody is provided for use in treating tumors. In a particular embodiment, an anti-CTLA-4 antibody is provided for use in a therapeutic method. In a particular embodiment, the present invention provides an anti-CTLA-4 antibody for use in a method of treating an individual having a tumor, the method comprising administering to the individual an effective amount of an anti-CTLA-4 antibody. In 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 a further embodiment, the present invention provides an anti-CTLA-4 antibody for use in cell damage. In a particular embodiment, the present invention provides an anti-CTLA-4 antibody for use in a method of cell damage in an individual, the method comprising administering to the individual an effective amount of an anti-CTLA-4 antibody to damage the cells. In a further embodiment, 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 immune activation in an individual, the method comprising administering to the individual an effective amount of the anti-CTLA-4 antibody to activate the immune system. The "individual" according to any of the above embodiments is preferably a human.

[0243] In some embodiments, the tumor is a solid tumor. In solid tumors, tumor cells typically proliferate and form clusters, which then form the tumor tissue. Furthermore, immune cells such as lymphocytes are often infiltrated into tumor tissue in vivo, and these also constitute part of the tumor tissue. In some embodiments, immune cells, particularly regulatory T (Treg) cells, are infiltrated into tumor tissue. In one embodiment, cell damage is induced by ADCC activity, CDC activity, or ADCP activity. In one embodiment, cells expressing CTLA-4 on their surface are damaged. In a further embodiment, the damaged cells are Treg cells. In a specific embodiment, Treg cells infiltrating tumor tissue are damaged. In one embodiment, damage to Treg cells results in immune activation (immunosuppression by Treg cells is relieved). In a further embodiment, immunity (particularly anti-tumor immunity) is activated in tumor tissue. In some embodiments, immune activation is T cell activation.

[0244] In a further aspect, the extent of the pharmaceutical effect provided by the anti-CTLA-4 antibody of the present invention varies depending on the tissue within an individual. In certain embodiments, the extent varies depending on the concentration of adenosine-containing compounds in the tissue. In further embodiments, the effect is enhanced in tissues with high concentrations of adenosine-containing compounds compared to tissues with low concentrations of adenosine-containing compounds. Tissues with high concentrations of adenosine-containing compounds include, for example, tumor tissue. Tissues with low concentrations of adenosine-containing compounds include, for example, non-tumor tissues, such as normal tissue. In some embodiments, stronger immune activation is observed in tumor tissue compared to non-tumor tissue. Such a difference in response need not be observed at all doses of the anti-CTLA-4 antibody, but may be observed within a certain range of doses. In another embodiment, immune activation is observed at a lower dose in tumor tissue compared to non-tumor tissue. In another embodiment, a therapeutic effect is observed at a dose lower than that at which side effects are observed. In a specific embodiment, the therapeutic effect is the manifestation of an anti-tumor effect (e.g., tumor regression, induction of cell death or inhibition of proliferation of tumor cells, etc.), and the side effect is the onset of an autoimmune disease (including damage to normal tissues due to an excessive immune response).

[0245] In a further aspect, the degree of pharmaceutical effect provided by the anti-CTLA-4 antibodies of the present invention depends on whether they have adenosine-containing compound-dependent (i.e., adenosine-containing compound concentration-dependent) binding activity to CTLA-4. In some embodiments, the anti-CTLA-4 antibodies of the present invention are antibodies whose CTLA-4 binding activity increases as the concentration of the adenosine-containing compound increases. In some embodiments, the control anti-CTLA-4 antibody is an antibody that does not have adenosine-containing compound concentration-dependent CTLA-4 binding activity. In certain embodiments, an antibody that does not have adenosine-containing compound concentration-dependent CTLA-4 binding activity refers to an antibody whose CTLA-4 binding activity in the presence and absence of the compound differs, for example, by 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 CTLA-4 binding activity of the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody be substantially equivalent to each other in the presence of a sufficient amount of an adenosine-containing compound.

[0246] In certain aspects, the pharmaceutical effects of the anti-CTLA-4 antibodies of the present invention differ from those of a control anti-CTLA-4 antibody. In certain embodiments, the pharmaceutical effects differ in tissues with low concentrations of adenosine-containing compounds. Tissues with low concentrations of adenosine-containing compounds include non-tumor tissues such as normal tissues. The anti-CTLA-4 antibodies can also be provided as pharmaceutical formulations containing them. In some embodiments, the anti-CTLA-4 antibodies of the present invention induce a lower level of immune activation in tissues with low concentrations of adenosine-containing compounds compared to a control anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibodies of the present invention require a higher dose to induce immune activation in tissues with low concentrations of adenosine-containing compounds compared to a control anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibodies of the present invention induce a lower level of side effects in tissues with low concentrations of adenosine-containing compounds compared to a control anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody of the present invention exhibits a higher dose at which adverse effects are observed in tissues with low concentrations of adenosine-containing compounds compared to a control anti-CTLA-4 antibody. Such differential responses need not be observed in all tissues (e.g., all tissues with low concentrations of adenosine-containing compounds), but may be observed in some tissues. In certain embodiments, the adverse effects are autoimmune diseases (including damage to normal tissues due to an excessive immune response).

[0247] In certain aspects, the pharmaceutical effects of the anti-CTLA-4 antibodies of the present invention and the control anti-CTLA-4 antibodies are substantially equivalent. In certain embodiments, the pharmaceutical effects are substantially equivalent in tissues with high concentrations of adenosine-containing compounds. Tissues with high concentrations of adenosine-containing compounds include, for example, tumor tissue. The anti-CTLA-4 antibodies can be provided as pharmaceutical formulations containing them. In some embodiments, the levels of immune activation are substantially equivalent between the anti-CTLA-4 antibodies of the present invention and the control anti-CTLA-4 antibodies in tissues with high concentrations of adenosine-containing compounds. In some embodiments, the doses required for immune activation are substantially equivalent between the anti-CTLA-4 antibodies of the present invention and the control anti-CTLA-4 antibodies in tissues with high concentrations of adenosine-containing compounds. In some embodiments, the levels of therapeutic effects are substantially equivalent between the anti-CTLA-4 antibodies of the present invention and the control anti-CTLA-4 antibodies in tissues with high concentrations of adenosine-containing compounds. In some embodiments, in tissues with high concentrations of adenosine-containing compounds, the doses at which therapeutic effects are observed are substantially equivalent between the anti-CTLA-4 antibodies of the present invention and the control anti-CTLA-4 antibodies. In certain embodiments, the therapeutic effect is the manifestation of an anti-tumor effect (e.g., tumor regression, induction of tumor cell death or inhibition of tumor cell proliferation, etc.).

[0248] In certain embodiments, the tumor is selected from the group consisting of breast cancer and liver cancer.

[0249] In a further aspect, the present invention provides use of an anti-CTLA-4 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a tumor. In a further embodiment, the medicament is for use in a method of treating a tumor, the method comprising administering an effective amount of the medicament to an individual having a tumor. In 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 a further embodiment, the medicament is for cell damage. In a further embodiment, the medicament is for use in a method of cell damage in an individual, the method comprising administering to the individual an effective amount of the medicament to damage the cells. In a further embodiment, the medicament is for immune activation. In a further embodiment, the medicament is for use in a method of immune activation in an individual, the method comprising administering to the individual an effective amount of the medicament to activate the immune system. An "individual" according to any of the above embodiments may be a human.

[0250] In a further aspect, the present invention provides methods of treating tumors. 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 may be a human.

[0251] In a further aspect, the present invention provides a method for damaging cells in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-CTLA-4 antibody to damage the cells. In a further aspect, 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.

[0252] In a further aspect, the present invention provides pharmaceutical formulations comprising any of the anti-CTLA-4 antibodies provided herein (e.g., for use in any of the therapeutic methods described above). In one embodiment, the pharmaceutical formulation 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 use in treating tumors. In one embodiment, the present invention provides pharmaceutical formulations for use in cellular damage. In one embodiment, the present invention provides pharmaceutical formulations for use in immune activation. 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).

[0253] In a further aspect, the present invention provides a method for preparing a medicament or pharmaceutical formulation (e.g., for use in any of the therapeutic methods described above) comprising combining 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.

[0254] The antibodies of the present invention can be used in therapy either alone or in combination with other agents. For example, the antibodies of the present invention may 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. Examples of immune checkpoint inhibitors include 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. Examples of anti-CTLA-4 inhibitors include ipilimumab and tremelimumab. Examples of anti-PD-1 inhibitors include nivolumab and pembrolizumab. Examples of anti-PD-L1 inhibitors include atezolizumab, durvalumab, and avelumab. Examples of anti-PD-L2 inhibitors include anti-PD-L2 inhibitory antibodies. Examples of anti-TIM-3 inhibitors include anti-TIM-3 inhibitory antibodies. Examples of anti-LAG-3 inhibitors include anti-LAG-3 inhibitory antibodies. Examples of anti-TIGIT inhibitors include anti-TIGIT inhibitory antibodies. Examples of anti-BTLA inhibitors include anti-BTLA inhibitory antibodies. Examples of anti-VISTA inhibitors include anti-VISTA inhibitory antibodies. Examples of EGFR inhibitors include cetuximab, panitumumab, nimotuzumab, necitumumab, zalutumumab, etc. Examples of HER2 inhibitors include trastuzumab, trastuzumab emtansine, pertuzumab, etc.

[0255] Combination therapy as described above encompasses combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, in which the antibody of the invention can be administered prior to, simultaneously with, and / or subsequent to the administration of the additional therapeutic agent. In one embodiment, the administration of the anti-CTLA-4 antibody and the additional therapeutic agent is performed within about 1 month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other. The antibodies of the invention can also be used in combination with radiation therapy.

[0256] The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and, if desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injection, e.g., intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or repeated doses over various time periods, bolus administration, and pulse infusion.

[0257] The antibodies of the present invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of agent delivery, the method of administration, the administration schedule, and other factors known to medical professionals. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will typically be used in the same dosages and by any route of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0258] The appropriate dose of an antibody of the invention (whether used alone or in combination with one or more other additional therapeutic agents) for disease prevention or treatment will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered prophylactically or therapeutically, previous medical history, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of antibody may be an initial candidate dose for administration to a patient, whether by one or more separate administrations or by continuous infusion. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors discussed above. For repeated administrations over several days or longer, depending on the situation, treatment is usually maintained until a desired suppression of disease symptoms occurs. An exemplary dose of antibody is in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., so that the patient receives about 2 to about 20, or, for example, about 6 doses of antibody). A high initial loading dose may be followed by one or more lower doses. The progress of this therapy is easily monitored by conventional techniques and measurements.

[0259] It will be understood that any of the above-described formulations or therapeutic methods may be practiced using an immunoconjugate of the invention in place of, or in addition to, an anti-CTLA-4 antibody.

[0260] H.Product In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders is provided. The article of manufacture includes a container and a label on the container or a package insert associated with the container. Preferred containers include, for example, bottles, vials, syringes, and intravenous (IV) solution bags. The containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active ingredient in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise: (a) a first container with a composition comprising an antibody of the invention contained therein; and (b) a second container with a composition comprising an additional cytotoxic or otherwise therapeutic agent contained therein. The article of manufacture of this aspect of the invention may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other equipment desirable from a commercial or user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0261] It will be understood that any of the above-described products may include an immunoconjugate of the present invention in place of or in addition to an anti-CTLA-4 antibody. <Polypeptides containing mutant Fc regions>

[0262] In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the mutant Fc region comprises at least one amino acid residue modification (e.g., substitution) compared to the corresponding sequence in the Fc region of a native sequence or a reference mutant sequence (sometimes collectively referred to herein as a "parent" Fc region). Native sequence Fc regions are typically organized as homodimers consisting of two identical polypeptide chains. The amino acid modification in the mutant Fc region of the present invention may be introduced into either one of the two polypeptide chains of the parent Fc region, or into both of the two polypeptide chains.

[0263] In some aspects, the present invention provides mutant Fc regions with altered function compared to their parent Fc regions. In certain aspects, the mutant Fc regions of the present invention have enhanced binding activity to Fcγ receptors compared to their parent Fc regions. In certain embodiments, the mutant Fc regions of the present invention have enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to their parent Fc regions. In some embodiments, the mutant Fc regions of the present invention have enhanced binding activity to FcγRIIa. In some embodiments, the mutant Fc regions of the present invention have enhanced binding activity to FcγRIIIa. In a further embodiment, the mutant Fc regions of the present invention have enhanced binding activity to FcγRIIa and FcγRIIIa.

[0264] In some embodiments, the mutant Fc regions of the present invention comprise at least one amino acid modification at at least one position selected from the group consisting of positions 234, 235, 236, 298, 330, 332, and 334 (EU numbering). Alternatively, amino acid modifications such as those described in International Publication WO2013 / 002362 and WO2014 / 104165 may also be used in the present invention.

[0265] In a specific embodiment, the binding activity of the parent Fc region and the mutant Fc region can be expressed as KD (Dissociation constant) values. In one embodiment, the ratio of [KD value of the parent Fc region for FcγRIIa] / [KD value of the mutant Fc region for FcγRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In a further embodiment, FcγRIIa may be FcγRIIa R or FcγRIIa H, or both. In one embodiment, the ratio of [binding activity of parent Fc region to FcγRIIIa] / [binding activity of mutant Fc region to FcγRIIIa] is, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1 × 10 3 That's it, 2 x 10 3 That's it, 3 x 10 3 or more, or 5 x 10 3 In a further embodiment, FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both.

[0266] In one embodiment, the KD value of the mutant Fc region for FcγRIIa is, for example, 1.0×10 -6 M or less, 5.0×10 -7 M or less, 3.0×10 -7 M or less, 2.0×10 -7 M or less, 1.0×10 -7 M or less, 5.0×10 -8 M or less, 3.0×10 -8 M or less, 2.0×10 -8 M or less, 1.0×10 -8 M or less, 5.0×10 -9 M or less, 3.0×10 -9 M or less, 2.0×10 -9 M or less, or 1.0 x 10 -9In a further embodiment, FcγRIIa may be FcγRIIa R or FcγRIIa H, or both. In one embodiment, the KD value of the mutant Fc region for FcγRIIIa is, for example, 1.0 × 10 -6 M or less, 5.0×10 -7 M or less, 3.0×10 -7 M or less, 2.0×10 -7 M or less, 1.0×10 -7 M or less, 5.0×10 -8 M or less, 3.0×10 -8 M or less, 2.0×10 -8 M or less, 1.0×10 -8 M or less, 5.0×10 -9 M or less, 3.0×10 -9 M or less, 2.0×10 -9 M, 1.0 × 10 -9 M or less, 5.0×10 -10 M or less, 3.0×10 -10 M or less, 2.0×10 -10 M, or 1.0 x 10 -10 In a further embodiment, FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both.

[0267] In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as kd (Dissociation rate constant) values ​​instead of KD values.

[0268] In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as the amount of Fcγ receptor binding per unit amount. For example, in a surface plasmon resonance assay, the amount of Fc region bound to a sensor chip and the amount of Fcγ receptor binding thereto are each measured in resonance units (RU). The amount of Fcγ receptor binding thereto divided by the amount of Fc region binding can be defined as the amount of Fc region binding to Fcγ receptor per unit amount. Specific methods for measuring and calculating such binding amounts are described in the Examples below. In some embodiments, the ratio of [amount of mutant Fc region binding to FcγRIIa] / [amount of parent Fc region binding to FcγRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In some embodiments, the ratio of [amount of binding of mutant Fc region to FcγRIIIa] / [amount of binding of parent Fc region to FcγRIIIa] is, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1 × 10 3 That's it, 2 x 10 3 That's it, 3 x 10 3 or more, or 5 x 10 3 That's all.

[0269] In certain embodiments, the KD values, kd values, binding amounts, etc., presented herein are measured or calculated by surface plasmon resonance assays performed at 25°C or 37°C (see, e.g., Example 6 herein).

[0270] In certain aspects, the mutant Fc regions of the present invention have improved selectivity between activating Fcγ receptors and inhibitory Fcγ receptors compared to the parent Fc region. In other words, the mutant Fc regions of the present invention have a greater enhanced binding activity to activating Fcγ receptors than to inhibitory Fcγ receptors compared to the parent Fc region. In certain embodiments, the activating Fcγ receptor is at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, and FcγRIIIa V, and the inhibitory Fcγ receptor is FcγRIIb. In some embodiments, the mutant Fc regions of the present invention have improved selectivity between FcγRIIa and FcγRIIb. In some embodiments, the mutant Fc regions of the present invention have improved selectivity between FcγRIIIa and FcγRIIb. In a further aspect, the mutant Fc regions of the present invention have improved selectivity between FcγRIIa and FcγRIIb and between FcγRIIIa and FcγRIIb.

[0271] In some embodiments, the mutant Fc regions of the present invention comprise at least one amino acid modification at at least one position selected from the group consisting of positions 236, 239, 268, 270, and 326 (EU numbering). Alternatively, the amino acid modifications described in International Publication Nos. WO2013 / 002362 and WO2014 / 104165 can also be used in the present invention.

[0272] In certain embodiments, the binding activity of the parent Fc region and the mutant Fc region can be expressed as KD (Dissociation constant) values. The binding activity for FcγRIIa and FcγRIIIa is as described above. In one embodiment, the ratio of [KD value of the parent Fc region for FcγRIIb] / [KD value of the mutant Fc region for FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as kd (Dissociation rate constant) values ​​instead of KD values.

[0273] In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as the amount of binding of the Fc region to an Fcγ receptor per unit amount as described above. In some embodiments, the ratio of [amount of binding of the mutant Fc region to FcγRIIb] / [amount of binding of the parent Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In some embodiments, the amount of binding of the mutant Fc region to FcγRIIb is, for example, 0.5 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.005 or less, 0.003 or less, 0.002 or less, or 0.001 or less.

[0274] In certain aspects, the mutant Fc regions of the present invention have improved stability compared to the parent Fc region. In certain embodiments, the stability is thermodynamic stability. The thermodynamic stability of a polypeptide can be determined, for example, using the Tm value as an indicator. The Tm value can be measured using techniques known to those skilled in the art, such as CD (circular dichroism), DSC (differential scanning calorimetry), and DSF (differential scanning fluorometry). In one embodiment, the Tm value of the CH2 region of the mutant Fc regions of the present invention is increased by 0.1° or more, 0.2° or more, 0.3° or more, 0.4° or more, 0.5° or more, 1° or more, 2° or more, 3° or more, 4° or more, 5° or more, or 10° or more compared to the parent Fc region.

[0275] In some embodiments, the mutant Fc region of the present invention comprises at least one amino acid modification at at least one position selected from the group consisting of positions 250 and 307 (EU numbering). Alternatively, the amino acid modifications described in International Publication WO2013 / 118858 may also be used in the present invention.

[0276] In a specific aspect, the mutant Fc region of the present invention is composed of two polypeptide chains with different sequences. In a further aspect, the mutant Fc region of the present invention exhibits enhanced heterodimerization between a first polypeptide and a second polypeptide. When producing heterodimeric proteins using recombinant techniques, it is preferable that different peptide chains preferentially associate to form heterodimers, rather than identical polypeptide chains associating to form homodimers. Whether or not heterodimerization of the mutant Fc region has been enhanced can be determined, for example, by separating homodimers and heterodimers from the produced mutant Fc region using techniques such as chromatography and determining the ratio of each component.

[0277] In some embodiments, the mutant Fc regions of the present invention comprise at least one amino acid modification at at least one position selected from the group consisting of positions 349, 356, 366, 368, 407, and 439 (EU numbering). Alternatively, the amino acid modifications described in International Publication WO2006 / 106905 and WO1996 / 027011 can also be used in the present invention.

[0278] In certain aspects, the mutant Fc regions of the present invention have enhanced FcRn-binding activity at acidic pH. In some embodiments, acidic pH refers to pH 4.0 to 6.5. In further embodiments, acidic pH is at least one selected from the group consisting of pH 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In certain embodiments, the acidic pH is pH 5.8.

[0279] In some embodiments, the mutant Fc region of the present invention comprises at least one amino acid modification at at least one position selected from the group consisting of positions 428, 434, 436, 438, and 440 (EU numbering). Alternatively, the amino acid modifications described in International Publication WO2016 / 125495 may also be used in the present invention.

[0280] In one aspect, the mutant Fc region of the present invention comprises at least one amino acid modification at at least one position selected from the group consisting of positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, 407, 428, 434, 436, 438, 439, and 440, as expressed in EU numbering.

[0281] In certain aspects, the variant Fc regions of the invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 334, as designated by EU numbering. In further aspects, the variant Fc regions comprise amino acid modifications at positions (i) 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, as designated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) 236, 250, 270, 298, 307, 326, and 334, as designated by EU numbering, in a second polypeptide of the parent Fc region.

[0282] In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 332 (EU numbering). In a further aspect, the variant Fc region further comprises an amino acid modification at position 332 (EU numbering) in the first polypeptide of the parent Fc region.

[0283] In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 356 (EU numbering). In a further aspect, the variant Fc region further comprises an amino acid modification at position 356 (EU numbering) in the first polypeptide of the parent Fc region.

[0284] In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 366 (EU numbering). In a further aspect, the first polypeptide of the parent Fc region comprises an amino acid modification at position 366 (EU numbering).

[0285] In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 349 (EU numbering). In a further aspect, the variant Fc region further comprises an amino acid modification at position 349 (EU numbering) in the first polypeptide of the parent Fc region.

[0286] In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 332 (EU numbering). In a further aspect, the parent Fc region comprises an amino acid modification at position 332 (EU numbering) in a second polypeptide.

[0287] In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 330 (EU numbering). In a further aspect, the parent Fc region comprises an amino acid modification at position 330 (EU numbering) in a second polypeptide.

[0288] In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 439 (EU numbering). In a further aspect, the parent Fc region comprises an amino acid modification at position 439 (EU numbering) in a second polypeptide.

[0289] In another aspect, the variant Fc region of the invention further comprises amino acid modifications at positions 366, 368, and 407 (EU numbering). In a further aspect, the parent Fc region comprises amino acid modifications at positions 366, 368, and 407 (EU numbering) in a second polypeptide.

[0290] In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 356 (EU numbering). In a further aspect, the parent Fc region comprises an amino acid modification at position 356 (EU numbering) in a second polypeptide.

[0291] In some aspects, the variant Fc regions of the invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 334, 349, 356, 366, 368, and 407, as designated by EU numbering. In further aspects, the variant Fc regions comprise amino acid modifications at positions (i) 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366, as designated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) 236, 250, 270, 298, 307, 326, 334, 356, 366, 368, and 407, as designated by EU numbering, in a second polypeptide of the parent Fc region.

[0292] In some aspects, the variant Fc regions of the invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 334, 356, and 439, as designated by EU numbering. In further aspects, the variant Fc regions comprise amino acid modifications at positions (i) 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 356, as designated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) 236, 250, 270, 298, 307, 326, 334, and 439, as designated by EU numbering, in a second polypeptide of the parent Fc region.

[0293] In some aspects, the mutant Fc regions of the present invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, and 407, as expressed in EU numbering. In a further aspect, the parent Fc region comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366, as indicated by EU numbering, in a first polypeptide, and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 356, 366, 368, and 407, as indicated by EU numbering, in a second polypeptide.

[0294] In some aspects, the variant Fc regions of the invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 356, and 439, as designated by EU numbering. In further aspects, the variant Fc regions comprise amino acid modifications at positions (i) 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 356, as designated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) 236, 250, 270, 298, 307, 326, 330, 332, 334, and 439, as designated by EU numbering, in a second polypeptide of the parent Fc region.

[0295] In some aspects, the variant Fc regions of the invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 349, 356, 366, 368, and 407, as designated by EU numbering. In further aspects, the variant Fc regions comprise amino acid modifications at positions (i) 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 349, and 366, as designated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) 236, 250, 270, 298, 307, 326, 332, 334, 356, 366, 368, and 407, as designated by EU numbering, in a second polypeptide of the parent Fc region.

[0296] In some aspects, the variant Fc regions of the invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 356, and 439, as designated by EU numbering. In further aspects, the variant Fc regions comprise amino acid modifications at positions (i) 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 356, as designated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) 236, 250, 270, 298, 307, 326, 332, 334, and 439, as designated by EU numbering, in a second polypeptide of the parent Fc region.

[0297] In some aspects, the variant Fc regions of the invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 366, 368, and 407, as designated by EU numbering. In further aspects, the variant Fc regions comprise amino acid modifications at positions (i) 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 366, as designated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) 236, 250, 270, 298, 307, 326, 330, 332, 334, 366, 368, and 407, as designated by EU numbering, in a second polypeptide of the parent Fc region.

[0298] In some aspects, the variant Fc regions of the invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 366, 368, and 407, as designated by EU numbering. In further aspects, the variant Fc regions comprise amino acid modifications at positions (i) 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 366, as designated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) 236, 250, 270, 298, 307, 326, 332, 334, 366, 368, and 407, as designated by EU numbering, in a second polypeptide of the parent Fc region.

[0299] In some aspects, the mutant Fc regions of the present invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, and 407, as expressed in EU numbering. In a further aspect, the parent Fc region comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366, as indicated by EU numbering, in a first polypeptide, and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 356, 366, 368, and 407, as indicated by EU numbering, in a second polypeptide.

[0300] In some aspects, the variant Fc regions of the invention comprise amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 349, 356, 366, 368, and 407, as designated by EU numbering. In further aspects, the variant Fc regions comprise amino acid modifications at positions (i) 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 349, and 366, as designated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) 236, 250, 270, 298, 307, 326, 332, 334, 356, 366, 368, and 407, as designated by EU numbering, in a second polypeptide of the parent Fc region.

[0301] In a further embodiment, the variant Fc region of the invention comprises (i) a first polypeptide of a parent Fc region, comprising: Tyr, 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, as indicated by EU numbering; and (ii) The parent Fc region comprises at least one amino acid modification in the second polypeptide selected from the group consisting of Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met, Lys at position 330, Asp, 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, as indicated by EU numbering.

[0302] In a further aspect, the mutant Fc region of the present invention further comprises any of the following amino acid modifications (a) to (d): (a) Ala at position 434, as expressed in EU numbering; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, as indicated by EU numbering; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, Glu at position 440, as indicated by EU numbering; (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, as indicated by EU numbering.

[0303] In a further aspect, 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.

[0304] The term "Fcγ receptor" (herein referred to as Fcγ receptor, FcγR, or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and effectively refers to any member of a family of proteins encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγR, FcγR isoform, or allotype. FcγRIIb1 and FcγRIIb2 have been reported as splice variants of human FcγRIIb. Furthermore, a splice variant designated FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splice variants, human FcγRIIb includes all splice variants registered with NCBI: NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. Furthermore, human FcγRIIb includes, in addition to FcγRIIb, all genetic polymorphisms that have been reported in the past (Arthritis Rheum. 48: 3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14: 2881-2892 (2005); and Kyogoju et al., Arthritis Rheum. 46: 1242-1254 (2002)), as well as all genetic polymorphisms that will be reported in the future.

[0305] There are two allotypes of FcγRIIa: one in which the amino acid at position 131 of FcγRIIa is histidine (H type), and the other in which the amino acid at position 131 is substituted with arginine (R type) (Warrmerdam, J. Exp. Med. 172: 19-25(1990)).

[0306] FcγRs may be derived from any organism, including, but not limited to, FcγRs from humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isoform.

[0307] The amino acid sequence of human FcγRI is set forth in SEQ ID NO: 131 (NP_000557.1); the amino acid sequence of human FcγRIIa is set forth in SEQ ID NO: 132 (AAH20823.1), SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 150; the amino acid sequence of human FcγRIIb is set forth in SEQ ID NO: 151 (AAI46679.1), SEQ ID NO: 169, or SEQ ID NO: 172; the amino acid sequence of human FcγRIIIa is set forth in SEQ ID NO: 174 (AAH33678.1), SEQ ID NO: 175, SEQ ID NO: 176, or SEQ ID NO: 177; and the amino acid sequence of human FcγRIIIb is set forth in SEQ ID NO: 178 (AAI28563.1).

[0308] Unlike Fcγ receptors, which belong to the immunoglobulin superfamily, human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides, sharing 22–29% sequence identity with class I MHC molecules (Ghetie et al., Immunol. Today (1997) 18(12), 592–598). FcRn is expressed as a heterodimer consisting of a transmembrane α or heavy chain complexed with a soluble β or light chain (β2-microglobulin). Like MHC, the α chain of FcRn consists of three extracellular domains (α1, α2, and α3), and a short cytoplasmic domain anchors the protein to the cell surface. The α1 and α2 domains interact with the FcRn-binding domain in the Fc region of an antibody (Raghavan et al. (Immunity (1994) 1, 303-315). The amino acid sequence of human FcRn is set forth in SEQ ID NO: 179 (NP_004098.1), and the amino acid sequence of β2-microglobulin is set forth in SEQ ID NO: 180.

[0309] As used herein, the term "parent Fc region" refers to an Fc region prior to the introduction of the amino acid modifications described herein. In some embodiments, the parent Fc region is a native sequence Fc region (or an Fc region of a native antibody). Examples of antibodies include IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM. Antibodies may be derived from humans or monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons). Native antibodies may contain naturally occurring mutations. Multiple allotype sequences of IgG due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and any of these sequences may be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356-358 (EU numbering) may be either 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 a further embodiment, the parent Fc region may be an Fc region created by adding amino acid modifications other than those described herein to a native-sequence Fc region (reference variant sequence Fc region). Native-sequence Fc regions are typically organized as homodimers consisting of two identical polypeptide chains.

[0310] Additionally, amino acid modifications made for other purposes can be combined in the variant Fc regions described herein. For example, amino acid substitutions that enhance FcRn-binding activity (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); WO2006 / 019447; WO2006 / 053301; and WO2009 / 086320) and amino acid substitutions that improve antibody heterogeneity or stability (WO2009 / 041613) may be made. Alternatively, the mutant Fc regions described herein can be combined with polypeptides having the property of promoting antigen clearance as described in WO2011 / 122011, WO2012 / 132067, WO2013 / 046704, or WO2013 / 180201, polypeptides having the property of specifically binding to target tissues as described in WO2013 / 180200, or polypeptides having the property of repeatedly binding to multiple antigen molecules as described in WO2009 / 125825, WO2012 / 073992, or WO2013 / 047752. Alternatively, the amino acid modifications disclosed in EP1752471 and EP1772465 can be combined in the CH3 of the mutant Fc regions described herein to confer binding ability to other antigens. Alternatively, the mutant Fc region described herein may be combined with amino acid modifications that decrease the pI of the constant region (WO2012 / 016227) to increase plasma retention, or with amino acid modifications that increase the pI of the constant region (WO2014 / 145159) to promote cellular uptake.Alternatively, amino acid modifications that increase the pI of the constant region (WO2016 / 125495) may be combined in the mutant Fc region described herein to promote plasma clearance of the target molecule. In one embodiment, such modifications may include, for example, substitutions at at least one position selected from the group consisting of positions 311, 343, 384, 399, 400, and 413, as represented by EU numbering. In a further embodiment, such substitutions may be substitutions of the amino acid at each position with Lys or Arg.

[0311] In addition, the heterodimerized antibody production technique described in WO2011 / 028952, which utilizes the combination of antibody CH1 and CL and VH and VL, can also be used.

[0312] Similar to the methods described in WO2008 / 119353 and WO2011 / 131746, it is also possible to use a heterodimeric antibody production technique in which two types of homodimeric antibodies are prepared in advance, the antibodies are incubate under reducing conditions to dissociate them, and then the antibodies are re-bound.

[0313] Furthermore, it is also possible to use techniques for producing heterodimerized antibodies by modifying the CH2 and CH3 domains, similar to the method described in WO2012 / 058768.

[0314] When two polypeptides containing mutant Fc regions with different amino acid sequences are simultaneously expressed to produce a polypeptide containing a heterologous mutant Fc region, a polypeptide containing a homologous mutant Fc region is usually also produced as an impurity. In such cases, the polypeptide containing the heterologous mutant Fc region can be efficiently obtained by separating and purifying it from the polypeptide containing the homologous mutant Fc region using known techniques. A method for efficiently separating and purifying a heterodimerized antibody from a homodimerized antibody using ion exchange chromatography has been reported (WO 2007 / 114325) by introducing amino acid modifications into the variable regions of the two antibody heavy chains that result in a difference in the isoelectric point between the homodimerized antibody and the heterodimerized antibody. Another method for purifying a heterodimerized antibody using protein A chromatography has been reported (WO 1998 / 050431 and WO 1995 / 033844) by constructing a heterodimerized antibody containing two heavy chains derived from mouse IgG2a, which binds to protein A, and rat IgG2b, which does not bind to protein A.

[0315] Furthermore, by substituting amino acids such as Tyr or His for the amino acid residues at positions 435 and 436 (EU numbering) located in the protein A binding site of the antibody heavy chain to confer different protein A binding affinities, heterodimerized antibodies can be efficiently purified using protein A chromatography.

[0316] In the present invention, amino acid modification refers to any substitution, deletion, addition, insertion, and modification, or a combination thereof. In the present invention, amino acid modification can be rephrased as amino acid mutation.

[0317] The number of amino acid modifications introduced into the Fc region is not limited, and in certain embodiments, may 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.

[0318] In one aspect, the present invention provides a method for producing a polypeptide comprising a mutant Fc region. In a further aspect, the present invention provides a method for producing a polypeptide comprising a mutant Fc region with altered function. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the methods comprise introducing at least one amino acid modification into a parent Fc region. In certain embodiments, the methods comprise (i) providing a polypeptide comprising the parent Fc region, and (ii) introducing at least one amino acid modification into the parent Fc region. In certain embodiments, the methods may further comprise (iii) measuring the function of the polypeptide comprising the mutant Fc region. A native Fc region is usually composed of two identical polypeptide chains. The amino acid modification to the parent Fc region may be introduced into either or both of the two polypeptide chains of the parent Fc region.

[0319] In another embodiment, a method for producing a polypeptide comprising a mutant Fc region comprises the steps of: (i) providing one or more nucleic acids encoding a polypeptide comprising a parent Fc region; (ii) introducing at least one mutation into the region of the nucleic acid encoding the parent Fc region; (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 mutant Fc region. In a specific embodiment, the method may further comprise the step of (v) recovering the polypeptide comprising the mutant Fc region from the host cell culture described in (iv).

[0320] In certain embodiments, the nucleic acids produced in (ii) may be contained in one or more vectors (eg, expression vectors).

[0321] In some embodiments, the amino acid modifications used in this production method are selected from any single modification, combination of single modifications, or combination modifications listed in Tables 26 to 30, selected from the amino acid modifications that can be included in the mutant Fc region described above.

[0322] The Fc region may be obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. with a protease such as pepsin, followed by re-elution of the fraction adsorbed to the Protein A column. The protease is not particularly limited, as long as it is capable of digesting full-length antibodies, thereby producing Fab and F(ab')2 in a limiting manner by appropriately setting the enzyme reaction conditions, such as pH, and examples include pepsin and papain.

[0323] In addition to the production methods described above, polypeptides comprising mutant Fc regions of the present invention may also be produced by other methods known in the art. Polypeptides comprising mutant Fc regions produced by the production methods described herein are also included in the present invention.

[0324] Various assays described herein or known in the art may be used to identify or screen the mutant Fc regions provided herein, or to characterize their physical or chemical properties or biological activities.

[0325] Assays for determining the binding activity of polypeptides comprising variant Fc regions to one or more FcR family members are described herein or known in the art, including, but not limited to, surface plasmon resonance assays, amplified luminescence 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).

[0326] In one embodiment, the binding activity of a polypeptide comprising a mutant Fc region to an FcR family member can be measured using a surface plasmon resonance assay. For example, various FcRs are allowed to interact as analytes with a polypeptide comprising a mutant Fc region 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 antibody, anti-κ chain antibody, antigen peptide, antigen protein, etc.). Alternatively, FcRs may be immobilized or captured on a sensor chip, and the polypeptide comprising a mutant Fc region may be used as the analyte. As a result of such interactions, binding sensorgrams are obtained, and by analyzing these, the dissociation constant (KD) of the binding can be calculated. 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 also be used as an indicator of the binding activity of a polypeptide comprising a mutant Fc region to the FcR. Furthermore, the corrected value (i.e., the amount of FcR binding per unit amount of polypeptide containing a mutant Fc region) obtained by dividing the amount of FcR binding by the difference in RU values ​​in the sensorgram before and after the polypeptide containing a mutant Fc region is immobilized or captured on a sensor chip (i.e., the amount of polypeptide containing a mutant Fc region bound) can be used as an indicator of binding activity.

[0327] In a further aspect, the present invention provides a pharmaceutical formulation comprising a polypeptide comprising a mutant Fc region provided herein. In one embodiment, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier. [Example]

[0328] The following are examples of the methods and compositions of the present invention. In light of the above general description, it will be understood that various other embodiments may be practiced.

[0329] [Example 0] Concept of switch antibodies that exert antibody-dependent cytotoxic activity against cell surface markers of regulatory T cells only in the cancer microenvironment Ipilimumab was thought to exert its antitumor effects by inhibiting the suppression of effector T cell activation by CTLA4 expressed on the surface of effector T cells. However, it has recently been reported that antibody-dependent cellular cytotoxicity (ADCC activity) against CTLA4-expressing T cells is also important, and it has been found that the elimination of regulatory T cells in tumors and ADCC activity are important mechanisms of action for the antitumor effect of anti-CTLA4 antibodies.

[0330] It is also known that the ADCC activity of IgG1 antibodies is induced by the binding of the antibody's constant region to FcγR on NK cells or macrophages, resulting in cytotoxic activity, and that antibodies with constant regions modified to enhance this binding induce stronger cytotoxic activity and exhibit antitumor effects.

[0331] On the other hand, it has also been reported that depletion of regulatory T cells throughout the body can cause systemic reactions similar to autoimmune diseases, and it is thought that the balance between cytotoxic activity and the control of systemic reactions is extremely important for exerting antitumor effects.

[0332] In other words, if an antibody could exert a strong antitumor effect by binding strongly to regulatory T cells or exhausted T cells in the tumor microenvironment and eliminating them through cytotoxic activity, and if the response could be restricted to the tumor microenvironment, it would be expected to exert even stronger cytotoxic activity and suppress systemic reactions. No antibody with this mechanism of action has been reported to date. Therefore, we have actually generated and tested an antibody (CTLA4 switch antibody) that acts only locally on tumors and has a constant region that enhances binding to FcγR expressed on NK cells and macrophages.

[0333] [Example 1] Isolation of antibodies that bind to antigens in the presence of ATP or its metabolites from a naive library and a rationally designed antibody library using phage display technology (1-1) Antigen preparation for obtaining antibodies that bind to antigens in the presence of small molecules Biotinylated mouse CTLA4 extracellular domain (mCTLA4), human CTLA4 extracellular domain (hCTLA4), and abatacept were prepared as antigens. Specifically, the hCTLA4 extracellular domain was fused to a His tag and a BAP tag at the C-terminus, resulting in gene synthesis of hCTLA4-His-BAP (SEQ ID NO: 1), which was then inserted into an animal expression plasmid. The antigen protein was expressed and purified using the following method. 1.33 × 10 cells were added to FreeStyle 293 Expression Medium (Invitrogen) at 100 μg / ml. 6 The prepared plasmid was introduced by lipofection into human embryonic kidney cell-derived FreeStyle 293-F strain (Invitrogen) suspended at a cell density of 100 μM / mL and seeded in a flask. Three hours after plasmid introduction, biotin was added to a final concentration of 100 μM. The cells were cultured in a CO2 incubator (37°C, 8% CO2, 125 rpm) for 4 days. The antigen was purified from the culture supernatant using a method 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 measurement value 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 a His tag is fused to the extracellular domain of mCTLA4, and abatacept (Alfresa Corporation), in which the human IgG1 constant domain is fused to hCTLA4, were biotinylated by the amine coupling method (PIERCE Cat. No. 21329).

[0334] (1-2) Identification of antibodies that bind to mouse CTLA4 in the presence of small molecules from a naive human antibody library by bead panning A human antibody phage display library consisting of multiple phages displaying Fab domains of different human antibody sequences was constructed according to methods known to those skilled in the art using polyA RNA prepared from human PBMCs or commercially available human polyA RNA as a template.

[0335] The constructed naive human antibody phage display library was screened for antibodies whose binding activity to the murine CTLA4 extracellular domain (mCTLA4) changed in the presence and absence of small molecules. Phages displaying antibodies that exhibited binding activity to mCTLA4 captured on beads in the presence of small molecules were collected. Phages were recovered from the phage eluate eluted from the beads in the absence of small molecules. Biotin-labeled mCTLA4 (mCTLA4-His-Biotin) was used as the antigen in this method.

[0336] Phages produced from E. coli harboring the constructed phage display phagemid were purified using standard methods. The phage library solution was then dialyzed against TBS. 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.

[0337] To efficiently obtain small molecule-dependent small molecule switch antibodies that can act as switches in cancer tissues, panning was performed to enrich for antibodies that bind to antigens in the presence of adenosine 5'-triphosphate (ATP) and ATP metabolites but do not bind to antigens in the absence of ATP, using a method described in the prior patent WO2013 / 180200 as a reference.

[0338] (1-3) Evaluation of binding activity in the presence and absence of small molecules by phage ELISA Phage-containing culture supernatants were recovered from the single colonies of E. coli obtained in (1-2) according to standard methods (Methods Mol. Biol. (2002) 178, 133-145). The recovered culture supernatants were ultrafiltered using a NucleoFast 96 (MACHERY-NAGEL). 100 μL of culture supernatant was applied to each well of the NucleoFast 96 and centrifuged at 4,500 g for 45 minutes. The flow-through was removed. 100 μL of H2O was added, and the cells were washed again by centrifugation at 4,500 g for 30 minutes. Then, 100 μL of TBS was added, and the cells were left to stand at room temperature for 5 minutes. The phage solution contained in the supernatant was then recovered.

[0339] The purified phage, supplemented with TBS, was subjected to ELISA as follows: StreptaWell 96 microtiter plates (Roche) were coated overnight with 100 μL of TBS containing mCTLA4-His-Biotin. After washing each well of the plate with TBST to remove unbound mCTLA4-His-Biotin, the wells were blocked with 250 μL of 2% skim milk-TBS for at least 1 hour. After removing the 2% skim milk-TBS, the purified phage were added to each well and the plate was left to stand at room temperature for 1 hour. The phage displaying the antibody were allowed 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 plate was incubated for 1 hour. After washing with TBST or ATP / TBST, the color reaction of the solution in each well to which TMB single solution (ZYMED) had been added was stopped by adding sulfuric acid, and the color development was measured by absorbance at 450 nm. As a result, several antibodies that bound to mCTLA4 only in the presence of ATP were confirmed. The results of the phage ELISA are shown in Table 2. Here, clones with an absorbance of greater than 0.2 in the presence of ATP were determined to be positive, and clones with an absorbance ratio in the presence / absence of ATP of greater than 2 were determined to have ATP-dependent antigen-binding ability (switch clones). In this example, SM is sometimes used as an abbreviation for small molecules such as ATP.

[0340] [Table 2]

[0341] (1-4) Identification of antibodies that bind to antigens in the presence of small molecules from a rationally designed library using ATP or its metabolites Antibodies that exhibit antigen-binding activity in the presence of ATP or ATP metabolites (e.g., ADP, AMP, adenosine (ADO), etc.) were obtained from the rationally designed antibody phage display library constructed in the prior patent WO2015 / 083764. To obtain the antibodies, phages displaying antibodies that exhibit binding activity to the antigen captured on beads were collected in the presence of ATP or ATP metabolites, and then the phages were collected from the eluate eluted from the beads in the absence of ATP or ATP metabolites.

[0342] Phages were produced from E. coli harboring the constructed phage display phagemid using standard methods. The phage-producing E. coli culture medium was supplemented with 2.5 M NaCl / 10% PEG to precipitate the phage population, which was then diluted with TBS to obtain a phage library solution. 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.

[0343] To efficiently obtain small molecule-dependent small molecule switch antibodies that can act as switches in cancer tissues, panning was performed to enrich for antibodies that bind to antigens in the presence of adenosine 5'-triphosphate (ATP) or ATP metabolites but do not bind to antigens in the absence of ATP or ATP metabolites, using a method described in the prior patent WO2015 / 083764 as a reference.

[0344] (1-5) Evaluation of binding activity in the presence and absence of ATP or its metabolites by phage ELISA Phage-containing culture supernatants were recovered from single colonies of E. coli obtained by the above method according to a standard method (Methods Mol. Biol. (2002) 178, 133-145). The recovered culture supernatants were ultrafiltered using a NucleoFast 96 (Macherey-Nagel). 100 μL of culture supernatant was applied to each well of the NucleoFast 96, and the NucleoFast 96 was 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 the NucleoFast 96 was again washed by centrifugation (4,500 g, 30 minutes). Finally, 100 μL of TBS was added, and the NucleoFast 96 was left to stand at room temperature for 5 minutes, and the phage solution contained in the supernatant from each well was recovered.

[0345] Purified phage supplemented with TBS or TBS containing ATP or its metabolites (SM / TBS) were subjected to ELISA as follows: StreptaWell 96 microtiter plates (Roche) were coated overnight with 100 μL of TBS containing the biotin-labeled antigen (Abatacept-Biotin) prepared in Example 1-1. Free Abatacept-Biotin was removed by washing each well of the plate with TBST, and then the wells were blocked with 250 μL of 2% Skim Milk-TBS for at least 1 hour. After removing the 2% Skim Milk-TBS, the plate containing the purified phage was incubated at 37°C for 1 hour, allowing the antibody-displaying phage to bind to Abatacept-Biotin present in each well in the absence and presence of ATP or its metabolites. After washing with TBST or TBST containing ATP or its metabolites (SM / TBST), each well was added with HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted in TBS or SM / TBS, and the plate was incubated for 1 hour. After washing with TBST or SM / TBST, TMB single solution (ZYMED) was added to each well. The color reaction was stopped by adding sulfuric acid, and the absorbance at 450 nm was measured. Several antibodies were identified whose binding activity to Abatacept changed 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 of greater than 2 in the presence of ATP or its metabolites were considered positive, and clones with an absorbance ratio of greater than 2 in the presence / absence of ATP or its metabolites were considered to have antigen-binding ability dependent on ATP or its metabolites (switch clones).

[0346] [Table 3]

[0347] (1-6) Sequence analysis of switch antibodies whose antigen-binding activity changes depending on the presence or absence of ATP and its metabolites The phage ELISA results showed that clones had antigen-binding activity in the presence of ATP and its metabolites. The nucleotide sequences of the genes amplified using specific primers lacPF (SEQ ID NO: 2) and G1seqR (SEQ ID NO: 3) were analyzed. Clones ABADh11-4_020, ABADh11-4_086, ABADh12-4_014, ABADh12-5_001, ABADh12-5_046, and ABADh5-5_041 were identified as having binding activity to biotin-labeled abatacept in the presence of ATP and its metabolites. These clones were renamed ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, respectively (Table 4).

[0348] [Table 4]

[0349] (1-7) Expression and purification of switch antibodies whose antigen-binding activity changes depending on the presence or absence of ATP and its metabolites The genes encoding the variable regions of ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006 obtained from the human rationally designed phage library were inserted into an animal expression plasmid for human IgG1 / Lambda. Antibodies were expressed using the following method: 1.33 × 10 cells / ml in FreeStyle 293 Expression Medium (Invitrogen). 6The prepared plasmid was introduced by lipofection into human embryonic kidney cell-derived FreeStyle 293-F (Invitrogen) cells suspended at a cell density of 1000 cells / mL and seeded in 3 mL volumes into each well of a 6-well plate. After 4 days of incubation in a CO2 incubator (37°C, 8% CO2, 90 rpm), antibodies were purified from the culture supernatant using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) according to methods known to those skilled in the art. 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 absorbance coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

[0350] (1-8) Evaluation of the binding activity of the obtained antibodies against hCTLA4 by IgG ELISA in the presence and absence of AMP. The six antibodies obtained, ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, were subjected to IgG ELISA. The buffers shown in Table 5 were prepared appropriately. Biotin-labeled human CTLA4 (hCTLA4-His-Biotin) was used as the antigen.

[0351] [Table 5]

[0352] First, a StreptaWell 96 microtiter plate (Roche) was coated with 100 μL of TBS containing hCTLA4-His-Biotin for at least 1 hour at room temperature. After washing each well with wash buffer to remove unbound hCTLA4-His-Biotin, the wells were blocked with 250 μL of blocking buffer for at least 1 hour. After removing the blocking buffer, 100 μL of purified IgG adjusted to 2.5 μg / mL in sample buffer containing AMP at a final concentration of 1 mM was added to each well. The plate was then left to stand at room temperature for 1 hour, allowing each IgG to bind to the hCTLA4-His-Biotin present in each well. After washing with wash buffer containing AMP at a final concentration of 1 mM, HRP-conjugated anti-human IgG antibody (BIOSOURCE) diluted in sample buffer was added to each well, and the plate was then incubated for 1 hour. After washing with wash buffer containing each small molecule, the color reaction of the solution in each well to which TMB single solution (ZYMED) had been added was stopped by adding sulfuric acid, and the color development was measured by absorbance at 450 nm. The buffer used had the composition shown in Table 5.

[0353] The measurement results are shown in Table 6. Note that the value for wells in which the value overflowed was provisionally set to 5.00. For all clones, ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, the absorbance in the absence of AMP was significantly lower than the absorbance in the presence of AMP. These results confirmed that all clones, ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, have the property that their antigen binding changes depending on the presence or absence of small molecules.

[0354] [Table 6]

[0355] (1-9) Effect of ATP and its metabolites on binding to human CTLA4 assessed by surface plasmon resonance Further evaluation of ABAM004 as a CTLA4 switch antibody was performed. The antigen-antibody interaction between ABAM004 and hCTLA4-His-BAP was analyzed using a Biacore T200 (GE Healthcare). ABAM004 was captured onto a Sensor Chip CM5 (GE Healthcare) to which an appropriate amount of protein A / G (Pierce) had been immobilized by amine coupling, and the antigen, hCTLA4-His-BAP prepared in Example 1-1, was allowed to interact with it. TBS was used as the running buffer, and 10 mM glycine-HCl (pH 1.5) was used as the regeneration solution.

[0356] After capturing 1 μg / mL ABAM004 suspended in TBS, a solution containing 500 nM hCTLA4-His-BAP, 10 concentrations of ATP, ADP, or AMP diluted 4:1 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 constituted the binding phase of hCTLA4-His-BAP. After the binding phase, the running buffer was replaced with the running buffer for 2 minutes, which constituted the dissociation phase of hCTLA4-His-BAP. After the dissociation phase, the regeneration solution was injected at a flow rate of 30 μL / min for 30 seconds. This completed the ABAM004 binding activity measurement cycle. The amount of hCTLA-4-His-BAP bound to ABAM004 during the binding phase was normalized for the amount of antibody captured. Biacore T200 Evaluation Software Version: 2.0 and Microsoft Excel 2013 (Microsoft) were used for data analysis and plotting.

[0357] The binding amounts of ABAM004 and hCTLA4-His-BAP in the presence of ATP and its metabolites obtained by this measurement are shown in Figure 1.

[0358] As shown in Figure 1, ABAM004 was confirmed to bind to hCTLA4 not only via ATP but also via ATP metabolites as a switch. Furthermore, it was shown that this antibody had the strongest binding activity in the presence of AMP.

[0359] (1-10) Evaluation of antibody binding to human CTLA4-expressing cells Using a flow cytometer, the antigen-antibody interaction between ABAM004 and human CTLA4 was evaluated in the presence and absence of AMP. CHO cells stably expressing human CTLA4 (hCTLA4-CHO cells) were prepared at appropriate concentrations and suspended in PBS containing 0.1% BSA (FACS Buffer). To 100 μL of the cell suspension, antibody was added to a final concentration of 10 mg / mL, followed by the addition of AMP to final concentrations of 0, 0.4, 4, 40, 200, or 1000 μM. The mixture was then incubated at 4°C for 30 minutes. The cell lines were then washed using a wash buffer containing AMP added to the FACS buffer to final concentrations of 0, 0.4, 4, 40, 200, or 1000 μM. FITC-labeled secondary antibody (Goat F(ab'2) Anti-Human IgG Mouse ads-FITC, Beckman 732598) was then added, and the cells were again incubated at 4°C for 30 minutes in the dark. After another wash, the cells were measured using a flow cytometer (FACS CyAn TM The results are shown in Figure 2.

[0360] These results demonstrate that ABAM004 exhibits AMP concentration-dependent binding activity to hCTLA4-expressing cells, and that it also exhibits AMP concentration-dependent binding activity to not only soluble antigens but also membrane antigens.

[0361] (1-11) ADCC activity of test antibodies using human peripheral blood mononuclear cells as effector cells Antibody concentration-dependent ADCC activity of antibodies that bind to antigens in an ATP-dependent manner was measured according to the following method, using human peripheral blood mononuclear cells (hereinafter referred to as human PBMCs) as effector cells.

[0362] First, a human PBMC solution was prepared. 50 mL of peripheral blood was collected from a healthy adult male volunteer using a syringe prefilled with 200 μL of 1,000 U / mL heparin solution (Novo Heparin Injection 5,000 U, Novo Nordisk). The peripheral blood was diluted 2-fold with PBS(-), divided into four equal portions, and added to a Leucosep lymphocyte separator tube (Greiner Bio-One) prefilled with 15 mL of Ficoll-Paque PLUS and centrifuged. The tube containing the peripheral blood was centrifuged at 2,150 rpm for 10 minutes at room temperature, and the mononuclear cell fraction was isolated. The cells contained in the fraction were washed once with RPMI-1640 (Nacalai Tesque) containing 10% FBS (hereinafter referred to as 10% FBS / RPMI), and then the cells were cultured in 10% FBS / RPMI at a cell density of 1 × 10 7 The cells were suspended at 1000 cells / mL, and the resulting cell suspension was used as a human PBMC solution in subsequent experiments.

[0363] Next, human CTLA4 extracellular domain-expressing CHO cells were used as target cells. hCTLA4-CHO cells were cultured at 2 × 10 5 The cells were suspended and prepared to a concentration of 1000 cells / mL. AMP (Sigma) was further diluted to 4 mM with RPMI and used as the AMP solution for the subsequent tests.

[0364] 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, and 40 μg / mL) was added to each well of a 96-well U-bottom plate, and 50 μL of target cells (1 × 10 ) were seeded. 4Then, 50 μL of AMP solution was added to each well, and the plate was left to stand at room temperature for 15 minutes. Each well was filled with 50 μL of human PBMC solution (5 × 10 5 The plate containing the LDH-released 100 μL of Catalyst (C) and Dye solution (D) was centrifuged and placed in a 5% CO2 incubator at 37°C for 4 hours. After the reaction was completed, 100 μL of the culture supernatant was collected and transferred to a 96-well plate for measurement. 100 μL of a 1:45 mixture of Catalyst (C) and Dye solution (D) included in the LDH detection kit (TaKaRa) was added. After 15 minutes at room temperature, the reaction was stopped by adding 50 μL of 1N HCl. The absorbance at 492 nm was measured to determine ADCC activity due to LDH release. ADCC activity was calculated using the following formula: ADCC activity (%)={(AD)-(CD)}×100 / {(BD)-(CD)}

[0365] In the above formula, A represents the average LDH activity (OD492nm) in wells to which each test antibody was added. Furthermore, B represents the average LDH activity (OD492nm) in wells to which 10 μL of 20% Triton-X aqueous solution was added after the reaction. Furthermore, C represents the average LDH activity (OD492nm) in 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 target cells. D represents the average LDH activity (OD492nm) in wells to which only 10% FBS / RPMI was added. The test was performed in duplicate, and the average ADCC activity (%) in the test, which reflects the ADCC activity of the test antibody, was calculated. The results are shown in Figure 3.

[0366] The above results demonstrated 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.

[0367] [Example 2] Crystal structure analysis of anti-CTLA4 antibody with ATP-dependent binding properties (2-1) X-ray crystal structure analysis of ABAM004, an anti-CTLA4 binding antibody with an AMP switch The crystal structures of the Fab fragment of the hCTLA4-binding antibody ABAM004, which uses AMP as a switch and was obtained from the library in Example 1, alone, a complex of the Fab fragment of ABAM004 with AMP, and a complex of the Fab fragment of ABAM004 with AMP and the extracellular domain of hCTLA4 were analyzed.

[0368] (2-2) Preparation of ABAM004 full-length antibody for crystallization Preparation and purification of the ABAM004 full-length antibody for crystallization was carried out by methods known to those skilled in the art.

[0369] (2-3) Preparation of ABAM004 Fab fragment for crystal structure analysis The Fab fragment of ABAM004 was prepared by conventional methods using restriction digestion with rLys-C (Promega, catalog no. V1671), followed by loading onto a Protein A column (MabSelect SuRe, GE Healthcare) to remove the Fc fragment, a cation exchange column (HiTrap SP HP, GE Healthcare), and a gel filtration column (Superdex200 16 / 60, GE Healthcare). Fractions containing the Fab fragment were pooled and stored at -80°C.

[0370] (2-4) Preparation of crystals of ABAM004 Fab fragment The ABAM004 Fab fragment for crystallization, purified as described in Method 2-3, was concentrated to approximately 13 mg / mL and crystallized by the sitting-drop vapor diffusion method at 20°C. The reservoir solution consisted of 0.1 M MES pH 6.5, 25% w / v polyethylene glycol 4000. The resulting crystals were immersed in a solution of 0.08 M MES pH 6.5, 20% w / v polyethylene glycol 4000, and 20% ethylene glycol.

[0371] (2-5) Collection of X-ray diffraction data from crystals of ABAM004 Fab fragment and structure determination X-ray diffraction data were collected at BL-17A of the Photon Factory, a synchrotron radiation facility of the High Energy Accelerator Research Organization (KEK). The crystal was kept frozen under a nitrogen stream at -178°C throughout the measurements. A total of 360 X-ray diffraction images were collected using a Quantum 270 CCD detector (ADSC) connected to the beamline, rotating the crystal 0.5° at a time. Cell parameters, diffraction spot indexing, and processing of the diffraction data obtained from the diffraction images were performed using the Xia2 program (J. Appl. Cryst. 43: 186-190 (2010)), the XDS package (Acta. Cryst. D66: 125-132 (2010)), and Scala (Acta. Cryst. D62: 72-82 (2006)). Finally, diffraction intensity data were obtained to a resolution of 1.70 Å. Crystallographic data statistics are shown in Table 7.

[0372] The structure was determined by molecular replacement using the program Phaser (J. Appl. Cryst. (2007) 40, 658-674). The search model for the Fab fragment was derived from the published crystal structure of Fab (PDB code: 4NKI). The model was constructed using the Coot program (Acta Cryst. D66: 486-501 (2010)) and refined using the Refmac5 program (Acta Cryst. D67: 355-467 (2011)). The crystallographic confidence factor (R) for the diffraction intensity data from 52.92-1.70 Å was 16.92%, and the free R value was 21.22%. The structure refinement statistics are shown in Table 7.

[0373] [Table 7]

[0374] (2-6) Preparation of ...

Claims

1. An anti-CTLA-4 antibody having CTLA-4 binding activity dependent on the concentration of an adenosine-containing compound, the antibody having at least one characteristic selected from the following (a) to (i): (a) the binding activity in the presence of 100 μM of an adenosine-containing compound is at least two-fold higher than the binding activity in the absence of an adenosine-containing compound; (b) KD value of 5 × 10 in the presence of 100 μM adenosine-containing compound. -7 M or less, (c) KD value of 1×10 in the absence of adenosine-containing compounds -6 M or more, (d) forming a ternary complex with an adenosine-containing compound and CTLA-4; (e) binds to the region of amino acids 97 to 106 of human CTLA-4 (extracellular domain, SEQ ID NO: 28); (f) competes with ABAM004 (VH, SEQ ID NO:10; and VL, SEQ ID NO:11) for binding to CTLA-4; (g) binds to the same epitope as ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11); (h) exhibiting cytotoxic activity against CTLA-4 expressing cells; and (i) It binds to CTLA-4 of human and mouse origin.

2. (a) Amino acid sequence SX 1 Contains TMN, X 1 is H, A, R, or K; (b) HVR-H1 (SEQ ID NO: 223), 1 X 2 SX 3 YIYYAX 4 SVX 5 Contains G, X 1 is S, T, X 2 is R, Q, or X 3 is G, H, X 4 is D, E, or R, X 5 is K or R; and (c) the amino acid sequence YGX 1 REDMLWVFDY, including X 1 The antibody of claim 1, comprising HVR-H3 (sequence number: 225), wherein HVR-H3 is K or A.

3. (a) Amino acid sequence X 1 GX 2 STX 3 VGDYX 4 X 5 VX 6 Contains X 1 is T, D, Q, or E, X 2 is T, P, X 3 is D, G, or X 4 is N, T, X 5 is Y or W, X 6 is S or H; (b) HVR-L1 (SEQ ID NO: 226), 1 TX 2 X 3 KPX 4 Contains X 1 is E, F, or Y, X 2 is S, I, X 3 is K, S, or X 4 (c) an amino acid sequence X 1 TYAAPLGPX 2 Contains X 1 is S, Q, or X 2 The antibody of claim 2, further comprising HVR-L3 (sequence number: 228) which is M or T.

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

5. A pharmaceutical formulation comprising an antibody according to any one of claims 1 to 4 and a pharma- ceutically acceptable carrier.

6. 6. The pharmaceutical formulation of claim 5 for use in the treatment of tumors.

7. The pharmaceutical formulation of claim 6 , which is immune-activating at a lower dose in tumor tissue compared to non-tumor tissue.

8. A polypeptide comprising a mutant Fc region comprising an amino acid modification in a parent Fc region, the parent Fc region being composed of two polypeptide chains, and the mutant Fc region comprising an amino acid modification at the following positions: (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, as represented by EU numbering, in a first polypeptide of a parent Fc region; and (ii) positions 236, 250, 270, 298, 307, 326, and 334, as expressed in EU numbering, in a second polypeptide of the parent Fc region.

9. The polypeptide described in claim 8, wherein the mutant Fc region has enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to the parent Fc region.

10. A polypeptide described in claim 8 or 9, in which the selectivity between activating Fcγ receptors and inhibitory Fcγ receptors is improved in the mutant Fc region compared to the parent Fc region.

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