EA045931B1 - ANTI-CTLA-4 ANTIBODY AND ITS APPLICATION - Google Patents

Description

Technical field

The present invention relates to anti-CTA-4 antibodies and methods of using these antibodies. Background of the Invention

Cells carrying gene mutations and similar disorders in living organisms are tracked and destroyed by the immune surveillance system. However, the persistence of excessive immune reactions can cause harm to the body itself, such as damage to normal tissues as a result of an autoimmune reaction. Thus, the immune system is equipped with a negative feedback mechanism (immune checkpoints) to suppress immune responses once activated (see, for example, NPL 1). Immune checkpoints are thought to play an important role in maintaining homeostasis in the immune system. On the other hand, it appears that some tumors use immune checkpoints for immunological escape. Extensive research is currently underway into immunosuppressive function through the essential immune checkpoint molecules, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1), and programmed cell death ligand 1 (PD-L1). ).

CTLA-4 is a glycoprotein belonging to the immunoglobulin superfamily, the gene of which was cloned from a library of killer T cell cDNA clones obtained from mice in 1987 (see, for example, NPL2). The T cell immune response is known to be suppressed by CTLA-4. In 1996, it was reported that a tumor regression effect was observed when anti-CTLA4 antibody was administered to cancer-bearing mice, consistent with the notion that promoting T cell activation by inhibiting CTLA-4 function leads to cancer regression (see, e.g., NPL 3). The effectiveness of anti-CTLA-4 antibodies in humans has been evaluated since 2000, and the human monoclonal anti-CTLA-4 antibody (ipilimumab) was approved by the US Food and Drug Administration (FDA) as the world's first immunostimulatory therapeutic. antibodies in 2011. Many anti-CTLA-4 monoclonal antibodies other than ipilimumab have also been produced (see, for example, PTL 1, 2, 3 and 4), and their development as drugs is currently underway. Such drugs that inhibit immune checkpoints to neutralize the immunosuppressive mechanism, thereby increasing immunoreactivity, are called immune checkpoint inhibitors.

On the other hand, it was previously known that among T cells there are cells that have an immunosuppressive function, and they were identified as CD25- and CD4-positive T cells in 1995 and called regulatory T cells (see, for example, NPL 4). In 2003, the Foxp3 gene was identified as a major gene specifically expressed in regulatory T cells, where it regulates their development and function. Foxp3 regulates the expression of various immune response-related genes as a transcription factor. In particular, Foxp3 is involved in the constitutive expression of CTLA-4 in regulatory T cells and has been suggested to play an important role in the immunosuppressive function of regulatory T cells (see, for example, NPL 5).

It is assumed that the infiltration of regulatory T cells into tumor tissues leads to weakening or inhibition of the immune control mechanism of the tumor. Indeed, it has been found that many human carcinomas exhibit an increase in the number of regulatory T cells (see, for example, NPL 6), and it has been reported that local infiltration of regulatory T cells into the tumor may be a poor prognostic factor for the patient's cancer. Conversely, if regulatory T cells can be removed from or reduced in tumor tissues, this would be expected to result in enhanced antitumor immunity. Cancer immunotherapy targeting regulatory T cells is currently being actively developed.

Administration of the anti-CTLA-4 antibody, ipilimumab, enhances antitumor immunity, but has been reported to cause autoimmune diseases because it systemically increases immunoreactivity. In one clinical trial, adverse events occurred in 60% of patients treated with ipilimumab, many of whom had autoimmune diseases related to the skin or gastrointestinal tract. Another clinical study also reported that approximately half of the patients treated with ipilimumab developed similar autoimmune diseases. To suppress such side effects, in some cases, an immunosuppressant drug is administered to the patient receiving ipilimumab. Development of a new drug that can support the antitumor immune response while suppressing such side effects of immune checkpoint inhibitors is desirable.

The cytotoxic effector functions of IgG antibodies, the activity of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP antibody-dependent cellular phagocytosis) have attracted attention as promising means for achieving antitumor effects. effects using antibodies (see, for example, NPL 7 and 8). These effector functions are induced by binding of the Fc region of IgG antibodies to antibody receptors (FcyRs) present on the surface of effector cells, such as natural killer cells and macrophages, or to various complement components. To date, many studies have been carried out

- 1 045931 variants of the Fc region have been produced, and variants have been obtained having different properties, such as FcYR binding activity, which is higher than that of the wild type (see, for example, PTL 5 and 6 and NPL 9 and 10). In addition, the Fc region of antibodies has been reported to bind to FcyR in a 1:1 ratio and to asymmetrically recognize FcyR in the lower hinge and CH2 regions (see, for example, NPL 11). On this basis, methods have also been reported for optimizing the interaction with FcyR by making various changes to the two polypeptide chains constituting the Fc region of an antibody and producing asymmetric variants of the Fc region (see, for example, PTL 7, 8, 9 and 10).

It is desirable that when a therapeutic antibody is introduced into a living organism, its target antigen is specifically expressed only at the affected sites. However, in many cases the same antigen is also expressed in unlesioned areas of normal tissue and can cause side effects that are undesirable from a therapeutic point of view. For example, although an antibody against a tumor antigen may exhibit cytotoxic activity against tumor cells through ADCC and the like, if the same antigen is also expressed in normal tissues, the antibody may also damage normal cells. To solve the above problem, a technology has been developed that focuses on the phenomenon that a large amount of a compound is present in a target tissue (for example, tumor tissue) and creates an antigen-binding molecule that changes antigen-binding activity depending on the concentration of the compound (see eg PTL 11).

Patent Literature Cited:

[PTL 1] WO 2000/037504

[PTL2] WO 2001/014424

[PTL3] WO 2012/120125

[PTL4] WO 2016/196237

[PTL 5] WO 2000/042072

[PTL6] WO 2006/019447

[PTL7] WO 2012/058768

[PTL8] WO 2012/125850

[PTL9] WO 2013/002362

[PTL10] WO 2014/104165

[PTL 11] WO 2013/180200

Cited scientific publications.

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

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

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

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

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

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

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

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

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

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

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

Brief description of the invention

Technical problem.

The present invention provides anti-CTLA-4 antibodies and methods of using these antibodies. The present invention also provides polypeptides comprising a variant Fc region and methods for producing the polypeptides.

Solution to the problem.

More precisely, the present invention provides the following paragraphs [1] to [47]:

[1] Ahtu-CTLA-4 antibody, which has CTLA-4 binding activity that is dependent on

- 2 045931 concentration of an adenosine-containing compound, wherein said antibody has at least one feature selected from the following (a)-(i):

(a) binding activity in the presence of 100 μM adenosine-containing compound is twofold or greater than in the absence of adenosine-containing compound;

(b) the KD value in the presence of 100 μM adenosine-containing compound is 5x10 ^-7 M or less;

(c) the KD value in the absence of the adenosine-containing compound is 1x 10 ^-6 M or more;

(d) formation of a ternary complex with an adenosine-containing compound and CTLA-4;

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

(f) competition with ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11) for binding to CTLA4;

(g) binding to the same epitope as ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11);

(h) manifestation of cytotoxic activity against CTLA-4-expressing cells; and/or binding to human or mouse CTLA-4.

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

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

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

[5] The antibody of any one of [1] to [4], wherein the antibody comprises: (a) HVR-H1 (SEQ ID NO: 223) containing the amino acid sequence SX ₁ TMN, where X1 is H, A, R, or K; (b) HVR-H2 (SEQ ID NO: 224) containing the amino acid sequence SISX ₁ X ₂ SX ₃ YIYYAX ₄ SVX ₅ Q where X1 is S or T, X ₂ is R or Q, X ₃ is G or H, X ₄ means D, E or R and X ₅ means K or R; and (c) HVR-H3 (SEQ ID NO: 225) containing the amino acid sequence YGX1REDMLWVFDY, where X1 is K or A.

[6] The antibody of item [5], which further includes: (a) HVR-L1 (SEQ ID NO: 226) containing the amino acid sequence X1GX ₂ STX ₃ VGDYX ₄ X ₅ VX ₆ , where X1 means T, D, Q or E, X ₂ is T or P, X ₃ is D or Q, X ₄ is N or T, X ₅ is Y or W, and X ₆ is S or H; (b) HVR-L2 (SEQ ID NO: 227) containing the amino acid sequence X ₁ TX ₂ X ₃ KRX ₄ where X1 is E, F or Y, X ₂ is S or I, X ₃ is K or S, and X ₄ means S, E or K; and (c) HVRL3 (SEQ ID NO: 228) containing the amino acid sequence X1TYAAPLGPX2, where X1 is S or Q and _X2 is M or T.

[7] The antibody of item [5], which further includes: a heavy chain variable domain FR1 containing any amino acid sequence from SEQ ID NO: 229-232; FR2 containing the amino acid sequence SEQ ID NO: 233; FR3 containing the amino acid sequence SEQ ID NO: 234; and FR4 containing the amino acid sequence of SEQ ID NO: 235.

[8] The antibody of paragraph [6], which further includes: a light chain variable domain FR1 containing any amino acid sequence from SEQ ID NO: 236-238; FR2 containing any amino acid sequence from SEQ ID NO: 240 and 241; FR3 containing any amino acid sequence from SEQ ID NO: 242-244; and FR4 containing any amino acid sequence from SEQ ID NOs: 245 and 246.

[9] An antibody according to any one of [1]-[4], which includes: (a) a VH that is at least 95% identical to any amino acid sequence of SEQ ID NOs: 83-86, 98 and 135-141; (b) VL that is at least 95% identical to any amino acid sequence of SEQ ID NO: 88-95, 97, 99, 134 and 144-149; or (c) VH having any of the amino acid sequences of SEQ ID NOs: 83-86, 98 and 135-141, and VL having any of the amino acid sequences of SEQ ID NOs: 88-95, 97, 99, 134 and 144-149 .

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

[11] The antibody of claim [10], wherein the Fc region is a variant Fc region containing amino acid change(s), and wherein the variant Fc region has increased binding activity to at least one Fcy receptor selected from the group, consisting of FcYRIa, FcYRIIa, FcYRIIb and FcYRIIIa, compared to the native Fc region.

[12] An isolated nucleic acid encoding an antibody according to any one of [1]-[11].

[13] A host cell containing the nucleic acid of [12].

[14] A method for producing an antibody, comprising culturing host cells as in [13] so that the antibody is produced.

[15] A pharmaceutical composition comprising an antibody according to any one of [1] to [11] and a pharmaceutically acceptable carrier.

[16] The pharmaceutical composition according to paragraph [15], in which the antibody is an immunoconjugate.

- 3 045931

[17] The pharmaceutical composition of claim [15] or [16], wherein the pharmaceutical composition is used in combination with at least one agent from the group consisting of immune checkpoint inhibitors, EGFR inhibitors, HER2 inhibitors and chemotherapeutic agents.

[18] The pharmaceutical composition according to any one of paragraphs [15]-[17], wherein the pharmaceutical composition is used for treating a tumor.

[19] The pharmaceutical composition according to [18], wherein the tumor is a solid tumor into which regulatory T cells (Treg) are infiltrated.

[20] The pharmaceutical composition according to any one of paragraphs [15]-[17], wherein the pharmaceutical composition is intended to damage cells.

[21] The pharmaceutical composition according to any one of paragraphs [15]-[17], wherein the pharmaceutical composition is intended to damage Treg cells.

[22] The pharmaceutical composition according to [20], wherein the cell damage is caused by ADCC activity, CDC activity or ADCP activity.

[23] The pharmaceutical composition according to paragraph [20] or [21], wherein immunity is activated by damage to Treg cells.

[24] The pharmaceutical composition according to any one of paragraphs [15]-[17], wherein the pharmaceutical composition is intended for use in activating the immune system.

[25] The pharmaceutical composition according to paragraph [24], where the activation of immunity is the activation of T cells.

[26] The pharmaceutical composition according to paragraph [24] or [25], and the immunity in the tumor tissue is activated.

[27] The pharmaceutical composition according to any one of paragraphs [24]-[26], wherein the level of immune activation in non-tumor tissues is lower than the level of immune activation under the influence of a pharmaceutical composition containing a control anti-CTLA-4 antibody.

[28] The pharmaceutical composition according to any one of [24]-[27], wherein the level of side effect is lower compared to the level of side effect under the influence of a pharmaceutical composition containing a control anti-CTLA-4 antibody.

[29] The pharmaceutical composition as claimed in any one of [24]-[28], wherein the control anti-CTLA-4 antibody is an anti-CTLA-4 antibody that does not have binding activity for CTLA-4, which is dependent on the concentration of adenosine-containing compound.

[30] The pharmaceutical composition according to item [29], and the side effect is an autoimmune disease.

[31] The pharmaceutical composition according to any one of paragraphs [18], [19] and [26]-[30], wherein the tumor is breast cancer or liver cancer.

[32] A polypeptide that contains a variant Fc region containing amino acid changes in the original Fc region, wherein the original Fc region consists of two polypeptide chains, and the variant Fc region contains amino acid changes at the following positions:

(i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307 and 326 according to EU numbering in the first polypeptide of the parent Fc region; and (ii) positions 236, 250, 270, 298, 307, 326 and 334 according to EU numbering in the second polypeptide of the original Fc region.

[33] The polypeptide of [32], wherein the Fc region variant further comprises an amino acid change at position 332 consistent with EU numbering in the first polypeptide of the original Fc region.

[34] The polypeptide of [32] or [33], wherein the Fc region variant further comprises an amino acid change at position 332 consistent with EU numbering in the second polypeptide of the original Fc region.

[35] The polypeptide of any one of [32]-[34], wherein the Fc region variant further comprises an amino acid change at position 330 consistent with EU numbering in the second polypeptide of the original Fc region.

[36] The polypeptide of any one of [32]-[35], wherein the Fc region variant further comprises an amino acid change at position 356 consistent with EU numbering in the second polypeptide of the original Fc region.

[37] The polypeptide of any one of [32]-[36], wherein the Fc region variant further comprises an amino acid change at position 366 consistent with EU numbering in the first polypeptide of the original Fc region.

[38] The polypeptide of any one of [32]-[37], wherein the Fc region variant further comprises an amino acid change at position 439 consistent with EU numbering in the second polypeptide of the original Fc region.

[39] The polypeptide of any one of [32]-[38], wherein the Fc region variant further comprises an amino acid change at positions 366, 368 and 407 consistent with EU numbering in the second polypeptide of the original Fc region.

- 4 045931

[40] A polypeptide according to any one of paragraphs [32]-[39], comprising at least one amino acid change selected from the following amino acid changes:

(i) Tyr or Phe at position 234, Gln at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Glu at position 332, Cys at position 349, Lys at position 356, and Trp at position 366 according to EU numbering in the first polypeptide of the parent Fc region; and (ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met or Lys at position 330, Asp or Glu at position 332, Glu at position 334, Cys at position 356, Ser at position 366, Ala at position 368, Val at position 407, and Glu at position 439 according to the EU numbering in the second polypeptide of the parent Fc region.

[41] The polypeptide of any one of [32]-[40], wherein the variant Fc region further comprises any of the amino acid changes from (a) to (d) below in the first polypeptide and/or second polypeptide of the original Fc- areas:

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

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

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

[42] A polypeptide according to any one of [32]-[41], wherein the binding activity to at least one Fcy receptor selected from the group consisting of FcYRIa, FcYRIIa, FcYRIIb and FcYRIIIa is increased in the Fc region variant compared to that in the original Fc region.

[43] The polypeptide according to paragraph [42], wherein the binding activity to FcYRIIa and FcYRIIIa is increased in the Fc region variant compared to that in the original Fc region.

[44] A polypeptide according to any one of [32]-[43], wherein the selectivity between the activating Fcy receptor and the inhibitory Fcy receptor is improved in the Fc region variant compared to the original Fc region.

[45] The polypeptide of claim [44], wherein the activating Fcy receptor is at least one Fcy receptor selected from the group consisting of FcYRIa, FcYRIIa and FcYRIIIa, wherein the inhibitory Fcy receptor is FcYRIIb.

[46] The polypeptide of any one of [32]-[45], wherein the polypeptides comprising the F region variant are an antibody.

[47] A method of producing a polypeptide containing a variant Fc region, comprising introducing amino acid changes into the original Fc region, wherein the original Fc region consists of two polypeptide chains, and the amino acid changes are introduced into the original Fc region at the following positions:

(i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307 and 326 according to EU numbering in the first polypeptide of the original Fc region; and (ii) positions 236, 250, 270, 298, 307, 326 and 334 according to EU numbering in the second polypeptide of the original Fc region.

Brief description of the figures

Fig. 1. Binding activity of anti-CTLA-4 antibodies ABAM004 CTLA-4, and the binding activity depends on the concentration of ATP, ADP or AMP, as described in examples 1-9.

Fig. 2. Binding activity of the anti-CTLA-4 antibody ABAM004 with cells expressing CTLA-4, and the binding activity depends on the concentration of AMP, as described in examples 1-10.

Fig. 3. ADCC activity of the anti-CTLA-4 antibody ABAM004 against cells expressing CTLA-4 in the presence and absence of AMP, as described in Examples 1-11.

Fig. 4. Method of binding the ABAM004 Fab fragment and AMP, as described in examples 2-13. In the figure, the antibody heavy chain is shown in black, the light chain in gray, and AMP in a ball-and-stick model. Amino acid residues forming an interaction with AMP are indicated in the rod model. The dotted lines and their values indicate the distance (A) between each amino acid residue and AMP.

Fig. 5. The binding pattern of the ABAM004 Fab fragment, AMP and human CTLA4 (hCTLA4), as described in examples 2-14. In the figure, the antibody heavy chain is shown in black, the light chain in gray, hCTLA4 in white, and AMP in a ball-and-stick model. Amino acid residues of hCTLA4 containing one or more non-hydrogen atoms located at a distance of 4.2 A from any part of the antibody or AMP are taken as the epitope and are indicated in the rod model.

Fig. 6. Epitope mapping of the ABAM004 Fab fragment to the amino acid sequence of hCTLA4, as described in examples 2-14. In the figure, black indicates amino acid residues of hCTLA4 that contain one or more atoms that are not hydrogen atoms, located

- 5 045931 at a distance of 4.2 A from any part of ABAM004 or AMP in the crystal structure. Amino acid residues highlighted in gray indicate residues that were not modeled due to the disorder of their crystal structure.

Fig. 7. Overlay drawing of the antibody and AMP structures extracted from the crystal structures of ABAM004 Fab alone, the ABAM004 Fab-AMP complex, and the ternary complex of ABAM004 Fab, AMP, and CTLA4, as described in Example 2-15. In the figure, the heavy chain of the antibody is shown in black, the light chain is shown in gray, and AMP is shown as a ball-and-stick model. The structure of the ABAM004 Fab fragment is shown by a thin line, the structure of the binary complex with AMP by a medium-thick line, and the structure of the ternary complex by a thick line.

Fig. 8. Binding activity of the anti-CTLA-4 antibody ABAM004 and its variant 04H0150/04L0072 with CTLA-4, and the binding activity depends on the concentration of ATP, ADP or AMP, as described in Example 3-2. In the notation in the figure, WT and H150L072 are designated ABAM004 and 04H0150/04L0072, respectively.

Fig. 9. Neutralizing activity of the anti-CTLA-4 antibody SW1077 against CTLA-4, and the neutralizing activity depends on the ATP concentration, as described in examples 3-6.

Fig. 10. Antitumor effect of 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 is administered at doses of 0.01, 0.1, 0.25, 1, 10, 30 and 100 mg/kg via the tail vein. Each dot represents the mean tumor volume per group, n=4.

Fig. 11. Antitumor effect of anti-CTLA-4 antibody SW1208-mFa55 (switching antibody) in a mouse model transplanted with the FM3A cell line as described in Example 3-7-4. The antibody is administered in doses of 0.1, 1, 10, 100 and 500 mg/kg via the tail vein. Each dot represents the mean tumor volume per group, n=4.

Fig. 12. Changes in the proportion of effector Treg cells in the tumor upon administration of the anti-CTLA4 antibody mNS-mFa55 (control antibody) or SW1208-mFa55 (switch antibody) in a mouse model engrafted with the FM3A cell line as described in Example 3-7-7 . The mNS-mFa55 antibody was administered at doses of 0.1, 1, 10, and 100 mg/kg via the tail vein, and the SW1208-mFa55 antibody was administered at doses of 0.1, 1, 10, 100, and 500 mg/kg via the tail vein. The tumor is examined six days after injection and the increase or decrease of effector Tregs is assessed using FACS analysis. The longitudinal axis represents the ratio of Treg effector cells (CD4 ⁺ FoxP3 ⁺ KLRG1') to CD45+ cells. Mean n=3 shown.

Fig. 13. Changes in the proportion of activated helper T cells in the spleen upon administration of the anti-CTLA-4 antibody mNS-mFa55 (control antibody) or SW1208-mFa55 (switch antibody) in a mouse model engrafted with the FM3A cell line as described in Example 3 -7-8. The mNS-mFa55 antibody was administered at doses of 0.1, 1, 10, and 100 mg/kg via the tail vein, and the SW1208mFa55 antibody was administered at doses of 0.1, 1, 10, 100, and 500 mg/kg via the tail vein. The spleen is removed six days after injection and the increase or decrease in the number of activated helper T cells is assessed using FACS analysis. The longitudinal axis represents the ratio of activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS+) to CD45+ cells. Means shown, n=3.

Fig. 14. Antitumor effect of anti-CTLA-4 antibody SW1389-mFa55 (switching antibody) in a mouse model transplanted with the Hepa1-6/hGPC3 cell line as described in Example 4-3-5. The antibody is administered in doses of 0.1, 1, 10 and 100 mg/kg via the tail vein. Each dot represents the mean tumor volume per group, n=4.

Fig. 15. Antitumor effect of anti-CTLA-4 antibody hNS-mFa55 (control antibody) in a mouse model transplanted with the Hepa1-6/hGPC3 cell line as described in Example 4-3-5. The antibody is administered in doses of 0.1, 1, 10 and 30 mg/kg via the tail vein. Each dot represents the mean tumor volume per group, n=4.

Fig. 16. Change in the ratio of effector Treg cells in the tumor upon administration of the anti-CTLA-4 antibody hNS-mFa55 (control antibody) or SW1389-mFa55 (switching antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cells, as described in example 4- 3-8. The hNS-mFa55 antibody was administered at doses of 0.1, 1, 10 and 30 mg/kg, and SW1389-mFa55 was administered at doses of 0.1, 1, 10, 100 and 500 mg/kg via the tail vein. The tumor is examined six days after injection and the increase or decrease of effector Treg cells is assessed using FACS analysis. The longitudinal axis represents the ratio of effector Tregs (CD4 ⁺ FoxP3 ⁺ CCR7 ^low KLRG1+) to CD45+ cells. Means shown, n=3.

Fig. 17. Change in the ratio of activated helper T cells in the spleen by administration of the anti-CTLA-4 antibody hNS-mFa55 (control antibody) or SW1389-mFa55 (switch antibody) in a mouse model engrafted with the Hepa1-6/hGPC3 cell line as described. in example 4-3-9. The hNS-mFa55 antibody was administered at doses of 0.1, 1, 10, and 30 mg/kg, and the SW1389-mFa55 antibody was administered at doses of 0.1, 1, 10, 100, and 500 mg/kg via the tail vein. The spleen is removed six days after injection and the increase or decrease in the number of activated helper T cells is assessed using FACS analysis. Longitudinal axis represents the ratio of activated helper T cells

- 6 045931 (CD4 ⁺ Foxp3-ICOS ⁺ ) to CD45 ⁺ cells. Means shown, n=3.

Fig. 18. Antitumor effect of anti-CTk-LN antibody SW1610 ^- mFa55 (switching antibody) in a mouse model transplanted with the Hepa1-6/hGPC3 cell line, as described in Example 5-4-5. The antibody is administered in doses of 0.3, 1 and 3 mg/kg through the tail vein. Each dot represents the mean tumor volume per group, n=5.

Fig. 19. Antitumor effect of anti-CTCL-4 antibody SW1612-mFa55 (switching antibody) in a mouse model transplanted with the Hepa1-6/hGPC3 cell line as described in Example 5-4-5. The antibody is administered in doses of 0.3, 1 and 3 mg/kg through the tail vein. Each dot represents the mean tumor volume per group, n=5.

Fig. 20. Antitumor effect of anti-CTCL-4 antibody SW1615-mFa55 (switching antibody) in a mouse model transplanted with the Hepa1-6/hGPC3 cell line as described in Example 5-4-5. The antibody is administered in doses of 0.3, 1 and 3 mg/kg through the tail vein. Each dot represents the mean tumor volume per group, n=5.

Fig. 21. Changes in the ratio of effector Treg cells in the tumor when administered anti-CTCL-4 antibodies SW1610 ^- mFa55, SW1612-mFa55 or SW1615-mFa55 (all of which are switching antibodies) in a mouse model transplanted with the Hepa1-6/hGPC3 cell line, as described in example 5-4-8. Antibody SW1610 ^- mFa55 is administered at doses of 50, 100 and 200 mg/kg, SW1612-mFa55 is administered at doses of 50, 100 and 200 mg/kg, SW1615-mFa55 is administered at doses of 50, 100, 200 and 400 mg/kg and negative control the KLH-mFa55 antibody is administered at a dose of 400 mg/kg through the tail vein. The tumor is examined six days after injection and the increase or decrease of effector Treg cells is assessed using FACS analysis. The longitudinal axis represents the ratio of Treg effector cells (CD4 ⁺ FoxP3 ⁺ CCR7 ^low KLRG1') to CD45+ cells. Means shown, n=3.

Fig. 22. Changes in the proportion of activated helper T cells in the spleen upon administration of the anti-CTCL-4 antibody SW1610 ^- mFa55, SW1612-mFa55 or SW1615-mFa55 (all of which are switch antibodies) in a mouse model transplanted with the Hepa1-6 cell line/ hGPC3 as described in Example 5-4-9. Antibody SW1610 ^- mFa55 is administered at doses of 50, 100 and 200 mg/kg, SW1612-mFa55 is administered at doses of 50, 100 and 200 mg/kg, SW1615-mFa55 is administered at doses of 50, 100, 200 and 400 mg/kg, and negative control antibody KLH-mFa55 is administered at a dose of 400 mg/kg via the tail vein. The spleen is examined six days after administration and the increase or decrease in the number of activated helper T cells is assessed using FACS analysis. The longitudinal axis represents the ratio of activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS+) to CD45+ cells. Means shown, n=3.

Fig. 23. Comparison of in vitro ADCC activity of antibodies having various altered constant regions with increased binding to FcyR, as described in Example 6-2. As shown in the figure, IgG1 is MDX10D1H-G1m/MDX10D1L-k0MT, GASDALIE is MDX10D1H-GASDALIE/MDX10D1L-k0MT, ART6 is MDX10D1H-Kn462/MDX10D1HH1445/MDX10D1L-k0MT, ART8 is MDX10D1H-K n461/MDX10D1HH1443/MDX10D1L -k0MT. Here, IgG1 is an antibody having a control constant region, GASDALIE is an antibody having a constant region described previously, ART6 and ART8 are antibodies having a modified constant region obtained in Example 6-1.

Fig. 24. Comparison of in vitro activity of ADCP antibodies having various altered constant regions with increased binding to FcyR, as described in Example 6-3. In the figure, IgG1 is MDX10D1H-G1m/MDX10D1L-k0MT, GASDIE is MDX10D1HGASDIE/MDX10D1L-k0MT, ART6 is MDX10D1H-Kn462/MDX10D1HH1445/MDX10D1L-k0MT and ART8 is MDX10D1H-Kn461/MDX 10D1HH1443/MDX10D1L-k0MT. Here, IgG1 is an antibody having a control constant region, GASDIE is an antibody having a previously described constant region, and ART6 and ART8 are antibodies having a modified constant region obtained in Example 6-1.

Fig. 25. In vitro ADCC activity of the anti-CTLA-4 switch antibody SW1389-ART6 having an altered constant region with increased binding to FcyR, as described in Example 6-4.

Fig. 26. In vitro ADCC activity of the anti-CTLA-4 switch antibody SW1610-ART6 having an altered constant region with increased binding to FcyR, as described in Example 6-4.

Fig. 27. In vitro ADCC activity of the anti-CTLA-4 switch antibody SW1612-ART6 having an altered constant region with increased binding to FcyR, as described in Example 6-4.

Fig. 28. Neutralizing activity of the anti-CTLA4 switch antibody SW1389 (activity to cancel the CTLA4 signal that acts in an inhibitory manner against effector cell activation) as described in Example 6-5.

Fig. 29. Neutralizing activity of the anti-CTLA4 switching antibody SW1610 (activity to cancel the CTLA4 signal, which acts in an inhibitory manner against the activation of effectors

- 7 045931 cells), as described in example 6-5.

Fig. 30. Neutralizing activity of the anti-CTE-A4 switching antibody SW1612 (activity to cancel the CTLA4 signal, which acts in an inhibitory manner against effector cell activation), as described in Example 6-5.

Fig. 31. Neutralizing activity of the anti-CTLA4 switching antibody SW1615 (activity to cancel the CTLA4 signal that acts in an inhibitory manner against effector cell activation) as described in Example 6-5.

Fig. 32. In vitro cytotoxic activity of anti-CTLA4 switching antibody SW1389ART5+ACT1 against CTLA4-positive regulatory T cells as described in Example 6-6.

Fig. 33. In vitro cytotoxic activity of anti-CTLA4 switching antibody SW1389ART5+ACT1 against CTLA4-positive regulatory T cells as described in Example 6-6.

Fig. 34. In vitro cytotoxic activity of anti-CTLA4 switching antibody SW1610 ART5+ACT1 against CTLA4-positive regulatory T cells as described in Example 6-6.

Fig. 35. In vitro cytotoxic activity of anti-CTLA4 switching antibody SW1610 ART5+ACT1 against CTLA4-positive regulatory T cells as described in Example 6-6.

Description of Embodiments of the Present Invention The methods and procedures described or referred to herein are generally understood and commonly used by those skilled in the art, for example, the commonly used methodologies described in the book:

Sambrook et al., “Molecular Cloning: A Laboratory Manual,” Ze ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; book: “Current Protocols in Molecular Biology”, ed. F.M. Ausubel et al., eds., 2003; series "Methods in Enzymology", published by Academic Press, Inc: PCR 2: A Practical Approach, ed. M.J. MacPherson, B.D. Hames, G.R.

I Taylor, 1995, ed. Harlow and Lane, 1988, Antibodies, A Laboratory Manual, and Animal Cell Culture, ed. R.I. Freshney, 1987; "Introduction to Cell and Tissue Culture", ed. J.P. Mather, P.E. Roberts, 1998, Plenum Press; "Cell and Tissue Culture: Laboratory Procedures", ed. A. Doyle, J.B. Griffiths, D. G. Newell, 1993-8, J. Wiley and Sons; "Handbook of Experimental Immunology", ed. D.M. Weir and C.C. Blackwell; "Gene Transfer Vectors for Mammalian Cells", ed. J.M. Miller and M.P. Calos, 1987; "PCR: The Polymerase Chain Reaction", ed. Mullis et al., 1994; "Current Protocols in Immunology", ed. J.E. Coligan et al., 1991; Short Protocols in Molecular Biology, Wiley and Sons, 1999; "Immunobiology", ed. S. A. Janeway and R. Travers, 1997; "Antibodies", ed. P. Finch, 1997; Antibodies: A Practical Approach, ed. D. Catty., IRL Press, 1988-1989; "Monoclonal Antibodies: A Practical Approach", ed. P. Shepherd and C. Dean, Oxford University Press, 2000; "Using Antibodies: A Laboratory Manual", ed. E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); "The Antibodies", ed. M. Zanetti and J. D. Capra, Harwood Academic Publishers, 1995; "Cancer: Principles and Practice of Oncology", ed. V.T. DeVita et al., J.B. Lippincott Company, 1993.

I. Definitions.

Unless otherwise specified, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention relates. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons, New York, 1994; March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th ed., John Wiley & Sons, New York, 1992), provide those skilled in the art with a general understanding of many of the terms used in the present invention. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

For purposes of interpretation of this specification, the following definitions apply and, where appropriate, terms used in the singular may also refer to the plural and vice versa. It should be noted that the terminology used in this description is intended

- 8 045931 is for the purpose of describing specific embodiments of the present invention only and is not intended to limit the present invention. If any definition set forth below is inconsistent with any document incorporated herein by reference, the definition set forth below shall control.

A human acceptor framework, for the purposes of this document, is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. A human acceptor framework derived from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence as it, or it may contain changes in the amino acid sequence. In some embodiments of the present invention, the number of amino acid substitutions 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 of the present invention, the human VL acceptor framework is identical in sequence to the human VL immunoglobulin framework sequence or the human consensus framework sequence.

Antibody-dependent cell-mediated cytotoxicity or ADCC - Antibody-dependent cell-mediated cytotoxicity refers to a form of cytotoxicity in which secreted immunoglobulin bound to Fc receptors (FcR) present on certain cytotoxic cells (eg, NK cells, neutrophils and macrophages) activates these cytotoxic effector cells specifically bind to the target cell bearing the antigen and subsequently kill the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, express only FcyRIII, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression on hematopoietic cells is presented in Table. 3 at page 464 in Ravetch and Kinet, Annu. Rev. Immunol 1991, 9:457-492. To evaluate the ADCC activity of a test molecule, an in vitro ADCC assay can be performed, for example, as described in US 5500362, US 5821337 or US 6737056 (Presta). Useful effector cells for such assays include PBMC and NK cells. Alternatively or additionally, the ADCC activity of the test molecule can be assessed in vivo, for example, in an animal model, such as the model described in Clynes et al., PNAS (USA), 1998, 95:652-656.

Cytotoxic activity includes, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) as defined above, complement-dependent cytotoxicity (CDC) as defined below, and T cell-mediated cytotoxic activity. CDC activity refers to the cytotoxic activity of the complement system. On the other hand, ADCC activity means an activity in which an antibody binds to an antigen present on the cell surface of a target cell, and the effector cell further binds to the antibody, and thereby the effector cell damages the target cell. Whether an antibody of interest has ADCC activity and whether an antibody of interest has CDC activity can be measured by known methods (see, for example, Current Protocols in Immunology, Chapter 7, Immunologic Studies in people, published by Coligan et al. 1993).

The term neutralizing activity refers to the activity of an antibody to inhibit some biological activity by binding to a molecule involved in the biological activity. In some embodiments of the present invention, the biological activity is provided by binding between the ligand and the receptor. In some embodiments of the present invention, the antibody inhibits binding between a ligand and a receptor by binding to the ligand or receptor. Antibodies that have this neutralizing activity are called neutralizing antibodies. The neutralizing activity of a particular test substance can be measured by comparing the biological activity in the presence of the ligand between conditions in the presence and absence of the test substance.

The term antibody-dependent cellular phagocytosis or ADCP refers to the process in which either the entire cell or a portion of it coated with an antibody is engulfed by phagocytic immune cells (for example, macrophages, neutrophils and dendritic cells) that bind to the Fc region of an immunoglobulin.

The term binding activity refers to the overall strength of all non-covalent interactions between one or more binding sites of a molecule (eg, antibody) and its binding partner (eg, antigen). In the present invention, binding activity is not strictly limited to a 1:1 interaction between members of a binding pair (eg, antibody and antigen). For example, when members of a binding pair reflect a 1:1 monovalent interaction, binding activity refers to the intrinsic binding affinity (affinity). When a member of a binding pair is capable of both monovalent and polyvalent binding, the binding activity is the sum of the strengths of each binding. The binding activity of molecule X to its partner Y can usually be represented by the dissociation constant (KD) or the amount of binding of the compound of interest per unit amount of ligand. Binding activity can be measured by conventional methods known in the art, including those described in the present invention. Specific examples to illustrate embodiments of active binding measurement

- 9 045931 details are described below.

An affinity matured antibody refers to an antibody with one or more changes in one or more hypervariable regions (HVR) compared to a parent antibody that does not have such changes, such changes resulting in an improvement in the affinity of the antibody for the antigen.

The terms anti-CTLA-4 antibody and antibody that binds CTLA-4 refer to an antibody that is capable of binding CTLA-4 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent for targeting CTLA-4. 4. In one embodiment of the present invention, the extent of binding of an anti-CTLA-4 antibody to an unrelated non-CTLA-4 protein is less than about 10% of the binding of the antibody to CTLA-4, as measured, for example, by a radioimmunoassay (RIA). . In some embodiments of the present invention, an antibody that binds to CTLA-4 has a dissociation constant (KD) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0. 01 nM or less, or 0.001 nM or less (for example, 10 ^-8 M or less, for example, from 10 ^-8 M to 10 ^-13 M, for example, from 10 ^-9 M to 10 ^-13 M). In some embodiments, the anti-CTLA-4 antibody binds to a CTLA-4 epitope that is conserved among CTLA-4 species.

The term antibody is used in the broadest sense in the present invention, and it covers various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) and antibody fragments, provided that they exhibit the desired antigen-binding activity .

The term antibody fragment refers to a molecule, other than the intact antibody, that contains a portion of the intact antibody that binds 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 (eg scFv); and multispecific antibodies formed from antibody fragments.

An antibody that binds to the same epitope as a control antibody refers to an antibody that blocks the control antibody from binding to its antigen in a competition assay, e.g., by 50% or more, and/or the control antibody blocks the antibody from binding to its antigen in competitive analysis, for example, by 50% or more. An example of a competitive analysis is presented in this document.

Autoimmune disease refers to a non-cancerous disease or disorder that arises from and is directed against a person's own tissues. Autoimmune diseases in the present invention specifically exclude malignant or cancerous diseases or conditions, especially 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 reactions such as inflammatory skin diseases, including psoriasis and dermatitis (eg, atopic dermatitis); systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS adult respiratory distress syndrome); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma, and other conditions involving T cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE - systemic lupus erythematosus) (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes mellitus (for example, type I diabetes mellitus or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, commonly found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; pernicious anemia (Addison's disease); diseases accompanied by leukocyte diapedesis; inflammatory disease of the central nervous system (CNS); multiple organ damage syndrome; hemolytic anemia (including, but not limited to, cryoglobulinemia or anemia with a positive Coombs test); myasthenia gravis; diseases mediated by the antigen-antibody complex; glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathies; Reiter's disease; muscle stiffness syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP - immune thrombocytopenic purpura) or autoimmune thrombocytopenia.

The terms cancer and malignant refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include breast cancer and liver cancer.

- 10 045931

The term complement-dependent cytotoxicity or CDC - complement-dependent cytotoxicity refers to a mechanism of cell death induction in which the effector Fc domain of an antibody bound to a target activates a series of enzymatic reactions leading to the formation of holes in the membrane of the target cell. Typically, the antigen-antibody complex formed on the target cell binds to and activates the complement component C1q, which in turn activates the complement cascade and leads to the death of the target cell. Moreover, complement activation can also lead to the deposition of complement components on the surface of the target cell, leading to binding to complement receptors (eg, CR3) on leukocytes and thereby promoting ADCC.

The term chemotherapy agent refers to a chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN (registered trademark)); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, meturedopa and uredopa; ethylene imines and methylol melamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethyl melamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL (registered trademark)); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including synthetic analogue of topotecan (HYCAMTIN (registered trademark)),

CPT-11 (irinotecan, CAMPTOSAR (registered trademark)), acetylcamptothecin, scopolectin and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (eg, calicheamicin, especially calicheamicin gammaP and calicheamicin omegaP (see, for example, Nicolaou et al., Angew. Chem Intl. Ed. Engl., 1994, 33: 183-186); CDP323, oral alpha-4 integrin inhibitor; dinemycin, including dinemycin A; esperamycin; and neocarzinostatin chromophore and related chromoprotein enediine antibiotic chromophores), aclacinomycins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carubicin, carminomycin, carcinophylline, chromomycins, dactinomycin , daunorubicin , detorubicin, 6-diazo-5-oxo-R-oxo-R. doxorubicin (including ADRIAMYCIN (registered trademark)), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal injection of doxorubicin HCl (DOXIL (registered trademark)), liposomal doxorubicin TLC D-99 (MYOCET (registered trademark) trademark)), pegylated liposomal doxorubicin (CAELYX (registered trademark) and deoxydoxorubicin), epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porphyromycin, puromycin, kelamicin, rhodorubi qing , streptorubicin, streptozorubicin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR (registered trademark)), tegafur (UFTORAL (registered trademark)), capecitabine (XELODA (registered trademark)), epothilone and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancytabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; adrenal medications such as aminoglutethimide, mitotane, trilostane; a folic acid replenisher such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defamine; demecolcine; diaziquon; elfornitine; elliptinium acetate; epothilone; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamole; nitraerin; pentostatin; phenomet; pirarubicin; Losoxantrone; 2ethylhydrazide; procarbazine; PSK Polysaccharide Complex (registered trademark) (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurine A, roridin A and anhydrine); urethane; vindesine (ELDISINE (registered trademark), FILDESIN (registered trademark)); dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (Ara-C); thiotepa; taxoids, for example, paclitaxel (TAXOL (registered trademark)), albumin-derived paclitaxel nanoparticle formulation (ABRAXANETM), and docetaxel (TAXOTERE (registered trademark)); chlorambucil; 6thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (eg, ELOXATIN (registered trademark)) and carboplatin; vinca alkaloids, which prevent

- 11 045931 polymerization of tubulin to form microtubules, including vinblastine (VELBAN (registered trademark)), vincristine (ONCOVIN (registered trademark)), vindesine (ELDISINE (registered trademark), FILDESIN (registered trademark)) and vinorelbine ( NAVELBINE (registered trademark)); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN (registered trademark)); bisphosphonates such as clodronate (eg, BONEFOS (registered trademark) or OSTAC (registered trademark)), etidronate (DIDROCAL (registered trademark)), NE-58095, zoledronic acid/zoledronate (ZOMETA (registered trademark)), alendronate (FOSAMAX (registered trademark)), pamidronate (AREDIA (registered trademark)), tiludronate (SKELID (registered trademark)) or risedronate (ACTONEL (registered trademark)); troxacitabine (analogue of cytosine 1,3-dioxolane nucleoside); antisense oligonucleotides, especially those that inhibit gene expression in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE vaccine (registered trademark) and gene therapy vaccines such as ALLOVECTIN vaccine (registered trademark), LEUVECTIN vaccine (registered trademark) and VAXID vaccine (registered trademark); topoisomerase 1 inhibitor (eg, LURTOTECAN (registered trademark)); rmRH (for example, ABARELIX (registered trademark)); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT (registered trademark), Pfizer); perifosine, COX-2 inhibitor (eg, celecoxib or etoricoxib), proteasome inhibitor (eg, PS341); bortezomib (VELCADE (registered trademark)); CCI-779; tipifarnib (R11577); sorafenib, ABT510; a Bcl-2 inhibitor such as oblimersen sodium (GENASENSE (registered trademark)); pixantron; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE (registered trademark)); farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing agents; as well as combinations of two or more of the above agents, such as CHOP, an acronym for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone; and FOLFOX, an acronym for the treatment regimen of oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin.

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 and the remainder of the heavy and/or light chain is derived from another source or species.

An antibody class refers to the type of constant domain or constant region that its heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.

In the present invention, the term cytotoxic agent refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (eg, ²¹¹ At, ¹³¹ 1, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb and Lu radioactive isotopes); chemotherapeutic agents or drugs (eg, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various chemotherapeutic agents described above.

Effector cells refer to leukocytes that express one or more FcRs and perform effector functions. In some embodiments of the present invention, the cells express at least FcyRIII and perform ADCC effector function(s). 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 a native source, such as blood. In some embodiments of the present invention, the effector cells may be human effector cells.

Effector functions refer to the biological activity that is attributed to the F region of an antibody and is dependent on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibodyosis

- 12 045931 dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cell-mediated phagocytosis (ADCP); down-regulation of cell surface receptors (eg, B-cell receptor); and B cell activation.

The term epitope includes any determinant capable of binding to an antibody. An epitope is the region of an antigen that is bound to the antibody targeting the antigen and includes specific amino acids that directly contact the antibody. Epitope determinants may include reactive surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and/or specific charge characteristics. In general, antibodies specific for a particular target antigen preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

The term Fc receptor or FcR refers to a receptor that binds to the Fc region of an antibody. In some embodiments of the present invention, the FcR is a naturally occurring human FcR. In some embodiments, the FcR binds an IgG antibody (receptor gamma), and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibitory receptor), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 1997, 15: 203-234). FcRs are discussed, for example, in the publications of Ravetch and Kinet, Annu. Rev. Immunol 1991, 9:457-492; Capel et al. Immunomethods 1994, 4: 25-34; de Haas et al., J. Lab. Clin. Med. 1995, 126: 330-341). Other FcRs, including those that will be identified in the future, fall within the term FcRs of the present invention.

The term Fc receptor or FcR also includes the neonatal FcRn receptor, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 1976, 117:587; Kim et al., J. Immunol. 1994, 24:249 ) and regulation of immunoglobulin homeostasis. Methods for measuring FcRn binding are known (see, for example, Ghetie and Ward, Immunol. Today 1997, 18(12): 592-598; Ghetie et al., Nature

Biotechnology, 1997, 15(7): 637-640; Hinton et al., J. Biol. Chem. 2004, 279(8):6213-6216; WO 2004/92219 (Hinton et al.)).

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

The term Fc region is used in the present invention to define the C-terminal region of an immunoglobulin heavy chain that contains at least part of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment of the present invention, the Fc region of the human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) Fc region may or may not be present. Unless otherwise noted herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also called 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.

The term Fc region-containing antibody refers to an antibody containing an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or C-terminal glycine-lysine (residues 446-447) Fc regions can be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Accordingly, a composition containing an antibody having the Fc regions of the present invention may contain an antibody with G446K447, with G446 and without K447, with all G446-K447 removed, or a mixture of the three types of antibodies described above.

The term variable region or variable domain refers to the heavy or light chain domain of an antibody that is involved in the binding of the antibody to an antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody typically have a similar structure, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al. , Kuby Immunology, 6th ed., 2007, WH Freeman and Co., p. 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a specific antigen may be

- 13 045931 are separated using the VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 1993, 150: 880-887; Clarkson et al., Nature 1991, 352: 624-628.

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

The terms full-length antibody, intact antibody, and full-length antibody are used interchangeably in the present invention to refer to an antibody having a structure substantially similar to that of a native antibody, or having heavy chains that contain an F region, as defined herein.

The functional Fc region has the effector function of the native sequence Fc region. Examples of effector functions include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor; BCR), etc. Such effector functions typically require association of an F region with a binding domain (eg, an antibody variable domain) and can be assessed using various assays, as described, for example, in the definitions provided herein.

A human antibody is an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or obtained from another source that uses human antibody repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes a humanized antibody containing non-human antigen-binding moieties.

The human consensus framework is a framework that represents the most frequently occurring amino acid residues in a number of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is made from a subset of variable domain sequences. Typically, the subset of sequences is such a subset as described in: Kabat et al., Sequences of Proteins of Immunological Interest, vol. 1-3, 5th ed., NIH Publication 91-3242, 1991, Bethesda, Maryland. In one embodiment of the present invention, the VL subgroup is the kI subgroup, as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III, as in Kabat et al., supra.

A humanized antibody refers to a chimeric antibody containing non-human HVR amino acid residues and human FR amino acid residues. In some embodiments of the present invention, a humanized antibody may contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of the non-human antibody, and all or virtually all FRs correspond to human antibody FRs. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. A humanized form of an antibody, for example, a non-human antibody, refers to an antibody that has been humanized.

The term hypervariable region or HVR, as used in the present invention, refers to each of the regions of an antibody variable domain that are hypervariable in sequence (complementarity determining regions or CDRs) and/or form structurally defined loops (hypervariable loops) and/or contain residues in contact with antigen (antigenic contacts). Typically, antibodies include six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Examples of HVR in the present invention include:

(a) hypervariable loops located on 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. 1987, 196:901-917);

(b) CDRs located 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, 1991, Bethesda, MD;

(c) antigen 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., 1996, 262: 732-745); and (d) combinations (a), (b) and/or (c), including amino acid residues HVR 46-56 (L2), 47-56 (L2), 4856 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

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

An immunoconjugate is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

An isolated antibody is an antibody that has been separated from its components

- 14 045931 of its natural environment. In some embodiments of the present invention, the antibody is purified to greater than 95% or 99% purity, as determined, for example, 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, for example, Flatman et al., J. Chromatogr. In, 2007, 848: 79-87.

Isolated nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain nucleic acid molecules, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location that differs from its natural chromosomal location.

An isolated antibody-encoding nucleic acid refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in the host cell.

The term vector as used in the present invention refers to a nucleic acid molecule capable of expanding copies of another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid structure, as well as a vector incorporated into the genome of the host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as expression vectors.

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, as well as the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny, regardless of the number of passages. The offspring may not be completely identical in nucleic acid content to the parent cell, and may contain mutations. Progeny mutants having the same function or biological activity as those screened or selected in the initially transformed cell are included herein.

The term monoclonal antibody as used in the present invention refers to an antibody obtained from a population of essentially homogeneous antibodies, i.e. The individual antibodies composing the population are identical and/or bind the same epitope, with the exception of possible variant antibodies, for example those containing natural mutations or those arising during preparation of the monoclonal antibody, such variants usually being present in minute quantities. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against one antigen determinant. Thus, the modifier monoclonal indicates that the antibody is derived from an essentially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by various methods, including, but not limited to, hybridoma method, recombinant DNA methods, phage display methods and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for producing monoclonal antibodies are described in the present invention.

The term naked antibody refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or radiolabel. The naked antibody may be present in the pharmaceutical composition.

The term native antibodies refers to naturally occurring immunoglobulin molecules with different structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains linked by a disulfide bond. From the N- to C-terminus, each heavy chain has a variable region (VH), also called a heavy chain variable domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Likewise, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). An antibody light chain can be classified into one of two types, called kappa (k) and lambda (λ), based on the amino acid sequence of its constant domain.

The term native Fc region sequence contains an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. The native sequence human Fc region includes the native sequence human IgG1 Fc region (non-A and A allotypes); Fc region of human IgG2 with native sequence; Human IgG3 F region with native sequence; and the Fc region of human IgG4 with the native sequence, as well as their natural variants.

- 15 045931

The Fc region variant contains an amino acid sequence that differs from the native sequence Fc region sequence due to at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution relative to the Fc region of the native sequence or the Fc region of the parent polypeptide, for example, from about one to about ten amino acid substitutions, preferably from about one to about five amino acid substitutions in the native Fc region sequence or in the Fc region of the parent polypeptide. The Fc region variant of the present invention preferably has at least about 80% homology to 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.

The concept of percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after sequence alignment and the introduction of spaces if necessary to achieve maximum percent identity sequence and without considering any conservative substitutions as part of sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

The ALIGN-2 sequence comparison computer program was originated by Genentech, Inc., and the source code is registered, along with user documentation, with the United States Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright No. TXU510087. The ALIGN-2 program is freely available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program must be compiled for use on the UNIX operating system, including the digital version of UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

In situations where ALIGN-2 is used to compare amino acid sequences, the percentage amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A that has or contains a certain % amino acid sequence identity with a given amino acid sequence B, or against it), is calculated as follows:

100-fold fraction X/Y, where X is the number of amino acid residues found identical by the ALIGN-2 sequence alignment program when aligning A and B by that program, and where Y is the total number of amino acid residues in B. It should be noted that if the length amino acid sequence A is not equal in length to amino acid sequence B, then the % identity of amino acid sequences A and B will not equal the % identity of amino acid sequences B and A. Unless specifically stated otherwise, all percent amino acid sequence identity values used in the present invention are obtained as described in the previous paragraph using the ALIGN-2 computer program.

The term pharmaceutical composition refers to a preparation that is in a form that provides effective biological activity of the active ingredient it contains, and that does not contain additional components that are unacceptably toxic to the subject for whom the drug may be prescribed.

The concept of individual or subject refers to mammals.

Mammals include, but are not limited to, domestic animals (for example, cows, sheep, cats, dogs and horses), primates (for example, humans and other primates such as monkeys), rabbits and rodents (for example, mice and rats). In some embodiments of the present invention, the individual or subject is a human.

The term pharmaceutically acceptable carrier refers to an ingredient of the pharmaceutical composition, other than the active ingredient, that is non-toxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative.

The term effective amount of an agent, such as a pharmaceutical composition, refers to an amount effective in doses and over periods of time necessary to achieve the same

- 16 045931 desired therapeutic or preventive result.

The term package insert is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information regarding the indications, use, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

The term CTLA-4 as used in the present invention refers to any native CTLA4 from any vertebrate animal, including mammals such as primates (eg, humans) and rodents (eg, mice and rats), unless otherwise noted. The term includes full-length, full-length CTLA-4, as well as any form of CTLA-4 resulting from cellular processing. The term also covers naturally occurring variants of CTLA-4, such as splice variants or allelic variants. The amino acid sequence of typical human CTLA-4 is shown in SEQ ID NO: 214, the amino acid sequence of mouse CTLA-4 is shown in SEQ ID NO: 247, the amino acid sequence of monkey CTLA-4 is shown in SEQ ID NO: 248, and the amino acid sequence of the extracellular domain of CTLA-4 4 human is shown in SEQ ID NO: 28. In the present invention, CTLA-4 may also be described as CTLA4.

The term regulatory T cells (Treg) refers to a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and suppress autoimmune diseases. These cells typically suppress the proliferation of effector T cells. The most studied Treg cells are those expressing CD4, CD25, and Foxp3 (CD4 ⁺ CD25 + Treg cells). These Treg cells are different from helper T cells. Several different methods are used to identify and monitor Treg cells. When measured by expression of CD4 and CD25 (CD4+ CD25+ cells), Treg cells constitute about 5 to about 10% of the mature CD4+ T cell subpopulation in mice and humans, while about 1 to about 2% Tregs can be measured in whole blood . Identification and monitoring can be accomplished by further measuring Foxp3 expression (CD4 ⁺ CD25 + Foxp3 ⁺ cells). In addition, the absence or low level of CD127 expression in combination with the presence of CD4 and CD25 can be used as another marker. Treg cells also express high levels of CTLA-4 and GITR. Tregs can also be identified using the methods described in the examples below.

As used herein, the term substantially similar, substantially equal, or substantially the same refers to a sufficiently high degree of similarity between two numerical values (e.g., one associated with an antibody of the present invention and the other associated with a reference/comparative antibody) such that one skilled in the art area can determine the difference between two values of negligible or no biological and/or statistical significance in the context of the biological characteristic measured by the specified values (eg, KD value).

As used herein, the term treatment (and its grammatical variation such as treat) refers to a clinical intervention in an attempt to alter the natural history of a disease in an individual undergoing treatment, and may be carried out either prophylactically or during the treatment of a clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating a disease state, remission, or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of a disease or to slow the progression of a disease.

The term tumor refers to all neoplastic cell growth and proliferation, malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms cancer, malignant, cell proliferative disorder, proliferative disorder and tumor are not mutually exclusive as used herein.

The term tumor tissue means tissue containing at least one tumor cell. Tumor tissue typically consists of a population of tumor cells that form the bulk of the tumor (parenchyma), and connective tissues and blood vessels that exist between these cells and support the tumor (stroma). The difference between them is obvious in some cases, but in others they are confused. Tumor tissues may be infiltrated by immune cells and the like. On the other hand, non-tumor tissue means tissue other than the tumor tissue(s) in a living body. Healthy/normal tissues that are not in a disease state are typical examples of non-tumor tissues.

II. Compositions and compositions.

One aspect of the present invention is based in part on anti-CTEA antibodies and their use. In some embodiments, the present invention provides antibodies that bind to CTLA-4. The antibodies of the present invention are useful, for example, in the diagnosis or treatment of cancer.

- 17 045931

A. Examples of aHTu-CTLA-4 antibodies.

In one aspect of the present invention, isolated antibodies are provided that bind to CTLA-4. In some embodiments, the anti-CTLA-4 antibody of the present invention has CTLA-4 binding activity depending on the concentration of the adenosine-containing compound. In some embodiments of the present invention, CTLA-4 binding activity is greater in the presence of an adenosine-containing compound compared to activity in the absence of an adenosine-containing compound. In another embodiment of the present invention, CTLA-4 binding activity is higher in the presence of a high concentration of an adenosine-containing compound compared to that in the presence of a low concentration of an adenosine-containing compound. In other embodiments of the present invention, the difference in binding activity to CTLA-4 is different, for example, two-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, 1 x 10 ³ times or more, 2 x 10 ³ times or more , 3x 10 ³ times or more, 5x 10 ³ times or more, 1x 10 ⁴ times or more, 2x 10 ⁴ times or more, 3x 10 ⁴ times or more, 5x 10 ⁴ times or more or 1x 10 ⁵ times or more.

In some embodiments of the present invention, the binding activity of an anti-CTLA-4 antibody can be expressed by a KD (dissociation constant) value. In another embodiment of the present invention, the KD value of the anti-CTLA-4 antibody in the presence of an adenosine-containing compound is less than in the absence of an adenosine-containing compound. In another embodiment of the present invention, the KD value of the anti-CTLA-4 antibody in the presence of a high concentration of an adenosine-containing compound is less than in the presence of a low concentration of an adenosine-containing compound. In another embodiment of the present invention, the difference in KD values of the anti-CTLA-4 antibody is different, for example, two-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, ^1x103 times or more, ^2x103 times or more, 3x10 ³ times or more, 5x10 ³ times or more, 1x 10 ⁴ times or more, 2x10 ⁴ times or more, 3x 10 4 times or more, 5x 10 ^{4 times} or more, or 1x 10 ⁵ times or more. The KD values of anti-CTLA-4 antibodies in the presence of an adenosine-containing compound or in the presence of a high concentration of an adenosine-containing compound may be, for example, 9x10 ^-7 M or less, 8x10 ^-7 M or less, 7x10 ^-7 M or less, 6x10 ^-7 M or less, 5x10 ^-7 M or less, 4x10 ^-7 M or less, 3x10 ^-7 M or less, 2x10 ^-7 M or less, 1 x 10 ^-7 M or less, 9x10 ^-8 M or less, 8x10 ^{- 8} M or less, 7x10 ^-8 M or less, 6x10 ^-8 M or less, 5x10 ^-8 M or less, 4x10 -8 M or less, 3x10 ^-8 M or less, ^{2x10 -8 M} or less, 1 x 10 ^-8 M or less, 9x10 ^-9 M or less, 8x10 ^-9 M or less, 7x10 ^-9 M or less, 6x10 ^-9 M or less, 5x10 ^-9 M or less, 4x10 ^-9 M or less, 3x10 ^{- 9} M or less, 2x10 ^-9 M or less, 1x 10 ^-9 M or less, 9x10 ^-10 M or less, 8x10 -10 M or less, 7x10 ^-10 M or less, ^{6x10 -10 M} or less, 5x10 ^{- 10} M or less, 4 x 10 ^-10 M or less, 3 x 10 ^-10 M or less, 2 x 10 ^-10 M or less, or 1 x 10 ^-10 M or less. The KD values of anti-CTLA4 antibodies in the absence of an adenosine-containing compound or in the presence of a low concentration of an adenosine-containing compound may be, for example, 1x10 ^-8 M or more, 2x10 ^-8 M or more, 3x10 ^-8 M or more, 4x10 ^-8 M or more, 5x 10 ^-8 M or more, 6x 10 ^-8 M or more, 7x 10 ^-8 M or more 8x 10 ^-8 M or more, 9x10 ^-8 M or more, 1 x 10 ^-7 M or more, 2x10 ^-7 M or more, 3x10 ^-7 M or more, 4x10 ^-7 M or more, 5x 10 ^-7 M or more, 6x 10 ^-7 M or more, 7x 10 ^-7 M or more, 8x 10 ^-7 M or more, 9x 10 ^-7 M or more, 1x 10 ^-6 M or more, 2x10 ^-6 M or more 3x10 -6 M or more, 4x10 ^-6 M or more, ^{5x10 -6 M} or more, 6x 10 ^-6 M or more, 7x 10 ^-6 M or more, 8x 10 ^-6 M or more, or 9x 10 ^-6 M or more.

In another embodiment of the present invention, the binding activity of an anti-CTLA-4 antibody may be represented by a kd (dissociation rate constant) value instead of a KD value.

In another embodiment of the present invention, the binding activity of an anti-CTLA-4 antibody can be represented by the amount of CTLA-4 binding per unit amount of antibody. For example, in surface plasmon resonance analysis, the amount of binding of an antibody immobilized on a sensor chip and the amount of binding of an antigen further bound thereto are measured as a resonance unit (RU). The value obtained by dividing the amount of antigen binding by the amount of antibody binding can be defined as the amount of antigen binding per unit amount of antibody. Specific methods for measuring and calculating such binding amounts are described in the examples below. In some embodiments of the present invention, the amount of CTLA-4 binding in the presence of an adenosine-containing compound is higher than in the absence of an adenosine-containing compound. Alternatively, in another embodiment of the present invention, the amount of CTLA-4 binding in the presence of a high concentration of adenosine-containing compound is greater than in the presence of a low concentration of adenosine-containing compound. In another embodiment of the present invention, the difference in CTLA-4 binding amount is different, for example, two-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold, or more, 50 times or more, 100 times or more, in

- 18 045931

200 times or more, 300 times or more, 500 times or more, 1x 10 ³ times or more, 2x 103 times or more, 3x 103 times or more, 5x 103 times or more, 1x 10 ⁴ times or more, 2x 10 ⁴ times or more, 3x 10 ⁴ times or more, 5x 10 ⁴ times or more, or 1x 10 ⁵ times or more. The amount of CTLA-4 bound in the presence of an adenosine-containing compound or in the presence of a high concentration of an adenosine-containing compound may 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 more than, 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 amount of CTLA-4 bound in the absence of an adenosine-containing compound or in the presence of a low concentration of an adenosine-containing compound may 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.

In some embodiments of the present invention, the KD values, kd values, binding amount values, and the like described in the present invention are measured or calculated by performing a surface plasmon resonance assay at 25°C or 37°C (see, for example, Example 3 in this specification).

Any concentration of adenosine-containing compound may be selected as long as a difference in the binding activity of the anti-CTLA-4 antibody is detected. In some embodiments of the present invention, a high concentration may include, for example, 1 nM or more than 1 nM, 3 nM or more than 3 nM, 10 nM or more than 10 nM, 30 nM or more than 30 nM, 100 nM or more than 100 nM. nM, 300 nM or more than 300 nM, 1 µM or more than 1 µM, 3 µM or more than 3 µM, 10 µM or more than 10 µM, 30 µM or more than 30 µM, 100 µM or more than 100 µM, 300 µM or more than 300 µM , 1 mM or more than 1 mM, 3 mM or more than 3 mM, 10 mM or more than 10 mM, 30 mM or more than 30 mM, 100 mM or more than 100 mM, 300 mM or more than 300 mM and 1 M or more than 1 M. In another embodiment, the high concentration in the present invention may be a sufficient amount at which the corresponding anti-CTLA4 antibody exhibits maximum binding activity. In one embodiment of the present invention, the high concentration may be 1 μM, 10 μM, 100 μM, 1 mM, or a sufficient amount at which the corresponding anti-CTLA-4 antibody exhibits maximum binding activity. In some embodiments of the present invention, the low concentration may include, for example, 1 mM or less than 1 mM, 300 μM or less than 300 μM, 100 μM or less than 100 μM, 30 μM or less than 30 μM, 10 μM or less than 10 μM. µM, 3 µM or less than 3 µM, 1 µM or less than 1 µM, 300 nM or less than 300 nM, 100 nM or less than 100 nM, 30 nM or less than 30 nM, 10 nM or less than 10 nM, 3 nM or less than 3 nM , 1 nM or below 1 nM, 300 pM or below 300 pM, 100 pM or below 100 pM, 30 pM or below 30 pM, 10 pM or below 10 pM, 3 pM or below 3 pM and 1 pM or below 1 pM. Alternatively, the low concentration may be the concentration at which the corresponding anti-CTLA-4 antibody exhibits minimal binding activity. The case where the significant concentration is zero (in the absence of an adenosine-containing compound) can also be selected as a low concentration embodiment. In one embodiment, the lower concentration here may be 1 mM, 100 μM, 10 μM, 1 μM at which the corresponding anti-CTLA-4 antibody exhibits minimal binding activity, or no adenosine compound. In another embodiment of the present invention, the ratio of high concentration to low concentration may be selected as follows: for example, 3 times or more, 10 times or more, 30 times or more, 100 times or more, 300 times, or more, 1x103 times or more, 3x103 times or more, 1x 10 ⁴ times or more, 3x10 ⁴ times or more, 1x 10 ⁵ times or more, 3x10 ⁵ times or more, 1x 10 ⁶ times or more, 3x10 ⁶ times or more , 1x 10 ⁷ times or more, 3x10 ⁷ times or more, 1 108 times or more, 3x10 ⁸ times or more, 1x 10 ⁹ times or more, 3x10 ⁹ times or more, 1x 10 ¹⁰ times or more, 3x10 ¹⁰ times or more than, 1x 10 ¹¹ times or more, 3x 10 ¹¹ times or more, or 1x 10 ¹² times or more.

In one embodiment of the present invention, the lower concentration may be 1 mM, 100 μM, 10 μM, 1 μM at which the corresponding anti-CTLA-4 antibody exhibits minimal binding activity, or in the absence of an adenosine compound. In another embodiment of the present invention, the ratio of high concentration to low concentration may be selected as follows: for example, 3 times or more, 10 times or more, 30 times or more, 100 times or more, 300 times, or more, 1x103 times or more, 3x103 times or more, 1x 10 ⁴ times or more, 3x10 ⁴ times or more, 1x 10 ⁵ times or more, 3x10 ⁵ times or more, 1x 10 ⁶ times or more, 3x 10 ⁶ times or more, 1x 107 times or more, 3x 107 times or more, 1 x 108 times or more, 3x10 ⁸ times or more, 1x 10 ⁹ times or more, 3x10 ⁹ times or more, 1x 10 ¹⁰ times or more, 3x 10 ¹⁰ times or more, 1x 10 ¹¹ times or more, 3x 10 ¹¹ times or more, or 1 x 10 ¹² times or more.

In another embodiment of the present invention, the anti-CTLA-4 antibodies of the present invention also have binding activity to adenosine-containing compounds. Quantity

- 19 045931 The binding activity of an adenosine-containing compound per unit amount of anti-CTA-4 antibody can be calculated using the above method and used as the binding activity of the antibody to the adenosine-containing compound. Specific methods for measuring and calculating such binding amounts are described in the examples below. The amount of binding amount of adenosine-containing compound per unit amount of anti-CTLA-4 antibody of the present invention may 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.

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

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

In another embodiment of the present invention, the anti-CTLA-4 antibodies of the present invention compete 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) by binding to CTLA- 4. In another embodiment of the present invention, the anti-CTLA-4 antibodies of the present invention bind to the same epitope as ABAM004. When there is an excess of anti-CTLA-4 antibodies, the binding of ABAM004 to CTLA-4 may be reduced, for example, by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more , 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more , 90% or more or 95% or more. This description provides examples of competition analyses.

In another embodiment of the present invention, the anti-CTLA-4 antibodies of the present invention exhibit cytotoxic activity against cells expressing CTLA-4. When CTLA-4 is expressed on the surface of a target cell and an anti-CTLA-4 antibody binds to it, the cell can be damaged. Cell damage may be caused by effector cells bound to the antibody, such as antibody-dependent cellular cytotoxicity (ADCC) activity and antibody-dependent cellular phagocytosis (ADCP) activity, or it may be caused by complements bound to the antibody, such as complement-dependent cytotoxicity (CDC). In another embodiment, the damage may be caused by a cytotoxic agent (eg, a radioisotope or chemotherapeutic agent) conjugated to an antibody, such as an immunoconjugate. Cytotoxicity in the present invention may include the effects of inducing cell death, suppressing cell proliferation and impairing cellular functions. When anti-CTLA-4 antibody is

- 20 045931 is present in sufficient quantity that it can cause damage, 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 cells expressing CTLA-4. Measurement of such cytotoxic activity can be performed by comparison with the measurement in the absence of antibody or in the presence of a negative control antibody. Examples of cytotoxicity assays are provided herein.

In another embodiment of the present invention, the anti-CTLA-4 antibodies of the present invention exhibit neutralizing activity against CTLA-4. CTLA-4 is known to function by interacting with its ligand, CD80 (B7-1) or CD86 (B7-2). In some embodiments, anti-CTLA-4 antibodies inhibit the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2). When the anti-CTLA-4 antibody is present in sufficient quantity, it can inhibit the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2), for example, by 10% or more, 15% or more, 20% or more 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 more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Measurement of such inhibitory activity can be performed by comparison with the measurement in the absence of antibody or in the presence of a negative control antibody. Specific methods for measuring neutralizing activity are provided herein.

In another embodiment of the present invention, the anti-CTLA-4 antibodies of the present invention bind to CTLA-4 derived from multiple animal species. Examples of animal species may include mammals, such as humans, monkeys, mice, rats, hamsters, guinea pigs, rabbits, pigs, cattle, goats, horses, sheep, camels, dogs and cats. In some embodiments of the present invention, anti-CTLA-4 antibodies bind to CTLA-4 obtained from humans and from other species (eg, monkeys, mice and rats). The amino acid sequence of human CTLA-4 is presented in SEQ ID NO: 214, the amino acid sequence of monkey CTLA-4 is presented in SEQ ID NO: 247, and the amino acid sequence of mouse CTLA-4 is presented in SEQ ID NO: 248. The amino acid sequences of CTLA-4 obtained from other animal species can also be suitably determined by methods known to those skilled in the art.

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

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

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

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

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 containing amino

- 21 045931 acid sequence SEQ ID NO: 224; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 225; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 226; (e) an HVR-L2 sequence containing the amino acid sequence of SEQ ID NO: 227; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 228.

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

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

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

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

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

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

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

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 containing the amino acid sequence SEQ ID NO: 104; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 116; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 115.

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

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

In another aspect, the present invention provides an antibody comprising (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (i) HVR-H1 comprising the amino acid sequence SEQ ID NO: 105 , (ii) HVRH2 containing the amino acid sequence of SEQ ID NO: 106, and (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two, or all three HVR VL sequences selected from (i) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133.

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

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

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

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

In another aspect, the present invention provides an antibody comprising the sequences of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2,

- 23 045931 containing the amino acid sequence SEQ ID NO: 108; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 121; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 153.

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

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

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

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

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

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

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

In another aspect, the present invention provides an antibody comprising the sequences: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 107; (b) HVR-H2,

- 24 045931 containing the amino acid sequence SEQ ID NO: 112; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133.

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

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

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

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

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

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

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

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 containing amino

- 25 045931 acid sequence SEQ ID NO: 112; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133.

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

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

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

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

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

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

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 containing the amino acid sequence of SEQ ID NO: 109; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133.

In some embodiments of the present invention, any one or more amino acids of the anti-CTLA-4 antibody as defined above are replaced at the following HVR positions:

- 26 045931 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.

In some embodiments of the present invention, the substitutions are conservative, as provided in the present invention. In some embodiments of the present invention, any one or more substitutions may be made as a 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; NUT; 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.

All possible combinations of the above substitutions are covered by the consensus sequences 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.

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

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

In another aspect, the present invention provides an anti-CTLA-4 antibody that contains a light chain variable domain (VL) whose sequence is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identical to the amino acid sequence of SEQ ID NO: 11. In certain embodiments of the present invention, a VL sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g. , conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-CTLA-4 antibody containing this sequence retains the ability to bind CTLA-4. In certain embodiments of the present invention, a total of 1 to 10 amino acids

- 27 045931 lot, up to 11, up to 12, up to 13, up to 14 or up to 15 amino acids are substituted, inserted and/or deleted in SEQ ID NO: 11. In certain embodiments of the present invention, the substitutions, insertions or deletions are outside the HVR (i.e. ie in FR). Optionally, the anti-CTLA-4 antibody contains the VL sequence of SEQ ID NO: 11, including post-translational modifications of this sequence. In one embodiment of the present invention, the VL contains one, two or three HVR regions selected from: (a) HVR-H1 containing the amino acid sequence SEQ ID NO: 100, (b) HVR-H2 containing the amino acid sequence SEQ ID NO: 101, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO:102. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

In another aspect, the present invention provides an anti-CTLA-4 antibody that contains a light chain variable domain (VL) whose sequence is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identical to the amino acid sequence of SEQ ID NO: 149. In some embodiments of the present invention, a VL that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-CTLA-4 antibody containing this sequence retains the ability to bind CTLA-4. In certain embodiments of the present invention, a total of 1 to 10 amino acids, up to 11, up to 12, up to 13, up to 14, or up to 15 amino acids are substituted, inserted, and/or deleted in SEQ ID NO: 149. In certain embodiments of the present invention, the substitutions , insertions or deletions are outside the HVR (i.e., in the FR). Optionally, the αanti-CTLA-4 antibody contains VL in SEQ ID NO: 149, including post-translational modifications of this sequence. In one embodiment of the present invention, the VL contains one, two or three HVR regions selected from: (a) HVR-H1 containing the amino acid sequence SEQ ID NO: 130, (b) HVR-H2 containing the amino acid sequence SEQ ID NO: 117, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO:133. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

In another aspect, the present invention provides an anti-CTLA-4 antibody that contains VH, as in any of the embodiments of the present invention presented above, and VL, as in any of the embodiments of the present invention presented above. In one embodiment, the antibody contains VH and VL in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody contains VH and VL in SEQ ID NO: 98 and SEQ ID NO: 99, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody contains VH and VL in SEQ ID NO: 83 and SEQ ID NO: 97, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody contains VH and VL in SEQ ID NO: 86 and SEQ ID NO: 134, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody comprises VH and VL in SEQ ID NO: 136 and SEQ ID NO: 95, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody comprises VH and VL in SEQ ID NO: 140 and SEQ ID NO: 146, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody comprises VH and VL in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody comprises VH and VL in SEQ ID NO: 140 and SEQ ID NO: 147, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody comprises VH and VL in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of these sequences. In one embodiment, the antibody contains VH and VL in SEQ ID NO: 136 and SEQ ID NO: 149, respectively, including post-translational modifications of these sequences. In another aspect, the present invention provides a heteromeric anti-CTLA-4 antibody, which antibody contains at least two different variable regions selected from the variable regions containing the VH and VL sequences presented above. In one embodiment of the present invention, the antibody comprises VH and VL in SEQ ID NO: 140 and SEQ ID NO: 146, respectively, and VH and VL in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of these sequences. In one embodiment of the present invention, the antibody comprises VH and VL in SEQ ID NO: 140 and SEQ ID NO: 147, respectively, and VH and VL in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of these sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy or light chain to pyroglutamic acid

- 28 045931 by pyroglutamylation.

When the amino acid at the N-terminus of the heavy chain or light chain of the anti-CTA-4 antibody provided in the present invention is glutamine, this amino acid can be replaced by glutamate. When the amino acid at the N-terminus of the heavy chain or light chain of the anti-CTLA-4 antibody provided in the present invention is glutamate, this amino acid can be replaced by glutamine.

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

In yet another aspect of the present invention, the anti-CTLA-4 antibody in accordance with any of the above embodiments of the present invention is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment of the present invention, the anti-CTLA-4 antibody is an antibody fragment, for example, an Fv fragment, Fab, Fab', scFv, diabody or F(ab')2 fragment. In another embodiment of the present invention, the antibody is a full length antibody, for example, an intact IgG1 antibody, an intact IgG4 antibody, or an antibody of another class or isotype of the present invention.

In another aspect of the present invention, the antibodies of the present invention contain constant regions. The constant regions may be heavy chain constant regions (including Fc regions), light chain constant regions, or both. In some embodiments of the present invention, the Fc region is native. Examples of heavy chain constant regions derived from native antibodies may include, for example, heavy chain constant regions such as human IgG1 (SEQ ID NO: 249), human IgG2 (SEQ ID NO: 250), human IgG3 (SEQ ID NO: 251) and human IgG4 (SEQ ID NO: 252). Additionally, in other examples, heavy chain constant regions may include heavy chain constant regions of SEQ ID NOs: 82 and 158. Examples of light chain constant regions derived from native antibodies may include, for example, light chain constant regions such as the κ chain human (SEQ ID NOs: 33, 63 and 159) and human λ chain (SEQ ID NOs: 53 and 87).

In another embodiment of the present invention, the variant Fc region is a variant Fc region obtained by adding amino acid changes to the Fc region of the native sequence. In some embodiments of the present invention, the variant Fc region has increased binding activity to at least one Fcy receptor selected from the group consisting of FcYRIa, FcYRIIa, FcYRIIb and FcYRIIIa, compared to the Fc region of the native sequence. In other embodiments of the present invention, the variant Fc region has increased binding activity to FcYRIIa and FcYRIIIa compared to the native sequence Fc region. Examples of heavy chain constant regions containing such a variant Fc region include, for example, the heavy chain constant regions listed in Table 1. 26-30, and heavy chain constant regions SEQ ID NOs: 31, 32, 41-46, 65, 66, 81, 207, 239, 253-271, 276, 277 and 278.

The native sequence Fc region typically consists of a homodimer consisting of two identical polypeptide chains. In some embodiments of the present invention, the Fc region variant may be a homodimer consisting of polypeptide chains with the same sequence, or a heterodimer consisting of polypeptide chains different from each other. Likewise, heavy chain constant regions containing the Fc region can be homodimeric, consisting of polypeptide chains with the same sequence, or heterodimeric, consisting of polypeptide chains with different sequences. Examples of heteromeric heavy chain constant regions include, for example, heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 31 and 32; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 43 and 44; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 45 and 46; heavy chain constant regions containing polypeptide

- 29 045931 chains SEQ ID NO: 254 and 256; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 257 and 258; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 259 and 260; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 261 and 263; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 262 and 264; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 265 and 267; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 266 and 268; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 269 and 270; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 271 and 81; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 65 and 66; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 239 and 207; heavy chain constant regions containing polypeptide chains of SEQ ID NOs: 259 and 276; and heavy chain constant regions containing the polypeptide chains of SEQ ID NOs: 65 and 278.

In another aspect, an anti-CTLA-4 antibody in accordance with any of the above embodiments of the present invention may include any of the features, alone or in combination, as described in sections 1-7 below.

1. Antibody binding activity.

In some embodiments of the present invention, c as provided in the present invention is represented by a dissociation constant (KD) of 10 μM or less, 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (eg, 10-8M or less, eg, 10-8M to ^10-13M , eg, ^10-9 to ^10-13M ).

In one embodiment of the present invention, binding activity is determined by a radiolabeled antigen binding assay (RIA). In yet another embodiment of the present invention, the RIA is performed with a Fab version of the test antibody and its antigen. For example, the binding affinity of a Fab in solution to an antigen is measured by equilibrating the Fab with a minimal concentration of ( ^|: 5[)-labeled antigen in the presence of a titration series of unlabeled antigen, and then capturing the bound antigen using a plate coated with anti-Fab antibody (see, for example, Chen et al., J Mol Biol 1999, 293: 865-881). To determine assay conditions, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) are coated overnight with 5 μg/ml anti-Fab capture antibody (Capel Labs) in 50 mM sodium carbonate (pH 9.6) and then block with 2% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (TBS) for two to five hours at room temperature (approximately 23°C). In a non-adsorption plate (Nunc, catalog no. 269620), 100 pM or 26 pM [IgC antigen is mixed with serial dilutions of the test Fab (for example, according to the evaluation of the anti-VEGF antibody, Fab-12, in the Presta publication with et al., Cancer Res. 1997, 57: 4593-4599). The Fab of interest is then incubated overnight; however, incubation may be continued for a longer period (eg, about 65 hours) to ensure equilibrium is achieved. The mixtures are then transferred to a capture plate for incubation at room temperature (eg, one hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in TBS. When the plates are dry, 150 μl/well of scintillator (MICROSCINT-20TM; Packard) is added and the plates are counted on a TOPCOUNTTM gamma counter (Packard) for ten minutes. Concentrations of each Fab producing less than or equal to 20% of maximum binding were selected for use in competitive binding assays.

In one embodiment of the present invention, the binding activity of an antibody is measured using a ligand capture assay using surface plasmon resonance as the measurement principle, for example, using BIACORE (registered trademark) T200 or BIACORE (registered trademark) 4000 (GE Healthcare , Uppsala, Sweden). BIACORE (registered trademark) Control Software is used to operate the device. In one embodiment of the present invention, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions, and a carboxymethyldextran-coated sensor chip (GE Healthcare, Uppsala, Sweden) is immobilized with the ligand capture molecule, for example, anti-tag antibody (anti-tag antibody, anti-IgG antibody and protein A). The ligand capture molecule is diluted with 10 mM sodium acetate solution at the appropriate pH and administered at the appropriate flow rate and injection time. The binding activity assay is carried out using a buffer containing 0.05% polysorbate 20 (also known as TWEEN (registered trademark)-20) as assay buffer, at a flow rate of 10 to 30 µl/min and at an assay temperature of preferably 25 °C or 37°C. When an assay is performed by allowing the ligand capture molecule to capture the antibody as a ligand, a serial dilution of the antigen or Fc receptor prepared with assay buffer (analyte) is administered after the target amount of antibody has been captured by antibody injection. When the assay is performed by allowing the ligand capture molecule to capture the antigen or Fc receptor as a

- 30 045931 ganda, a serial dilution of an antibody prepared with an assay buffer (analyte) is administered after capturing the target amount of antigen or Fc receptor by injection, antigen or Fc receptor.

In one embodiment of the present invention, the results are analyzed using evaluation software BIACORE (registered trademark). Kinetic parameters are calculated by simultaneously fitting binding and dissociation sensorgrams using a 1:1 binding model, allowing the calculation of the binding rate (kon or ka), dissociation rate (koff or kd) and the equilibrium dissociation constant (KD). When binding activity is weak, especially when dissociation is fast and kinetic parameters are difficult to calculate, the equilibrium dissociation constant (KD) can be calculated using a steady state model. As another parameter of binding activity, the amount of analyte binding per unit amount of ligand can be calculated by dividing the amount of analyte binding at a particular concentration (RU) by the amount of ligand captured (RU).

2. Antibody fragments.

In certain embodiments of the present invention, the provided antibody is an antibody fragment. 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. A review of certain passages is provided in Hudson et al., Nat. Med. 2003, 9:129-134. For a review of scFv fragments, see, for example, Pluckthiin in: The Pharmacology of Monoclonal Antibodies, ed. Rosenburg and Moore, (Springer-Verlag, New York), 1994, vol. 113:269-315; see also WO 93/16185 and US 5571894 and US 5587458. For a discussion of Fab and F(ab') ₂ fragments containing salvage receptor binding epitope residues and having an increased half-life in vivo, see US 5869046.

Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, for example, EP 404097; WO 1993/01161; Hudson et al., Nat. Med. 2003, 9: 129-134; Hollinger et al., Proc. Natl. Acad. Sci. USA 1993, 90: 6444-6448. Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. 2003, 9: 129-134.

Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments of the present invention, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, for example, US 6,248,516 B1).

Antibody fragments can be produced by a variety of methods, including, but not limited to, proteolytic cleavage of the intact antibody, as well as production by recombinant host cells (eg, E. coli or phage), as described herein.

3. Chimeric and humanized antibodies.

In some embodiments of the present invention, the antibody provided is a chimeric antibody. Certain chimeric antibodies are described, for example, in US 4816567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 1984, 81: 6851-6855. In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region derived from a mouse, rat, hamster, rabbit, or primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a class switched antibody in which the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

In some embodiments of the present invention, the 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. Typically, a humanized antibody contains one or more variable domains in which the HVRs, eg, CDRs (or portions thereof) are derived from a non-human antibody, and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody will also optionally contain at least a portion of the human constant region. In some embodiments of the present invention, certain FR residues in a humanized antibody are replaced with corresponding residues of a non-human antibody (eg, an antibody from which the HVR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

Humanized antibodies and methods for their production are discussed, for example, in the works of Almagro and Fransson, Front. Biosci. 2008, 13:1619-1633; Riechmann et al., Nature 1988, 332: 323-329; Queen et al., Proc. NatlAcad. Sci. USA 1989, 86:10029-10033; US 5821337, US 7527791, US 6982321 and US 7087409; Kashmiri et al., Methods 2005? 36:25-34 (describing the implementation of the specificity determining region (SDR)); Padlan, Mol. Immunol. 1991, 28:489-498 (update description); Dall'Acqua et al., Methods, 2005, 36:43-60 (describing FR shuffling); Osbourn et al., Methods, 2005, 36:61-68; Klimka et al., Br. J Cancer 2000, 83:252-260 (describing a directed selection approach for FR shuffling).

Human wireframes that can be used for humanization include, but are not limited to: wireframes selected using the best fit method

- 31 045931 (see, for example, Sims et al., J. Immunol. 1993, 151:2296); framework regions derived from a human antibody consensus sequence of a particular subset of light or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 1992, 89:4285; Presta et al., J Immunol, 151: 2623 (1993)); mature (somatically mutated) human or germline human frameworks (see, for example, Almagro and Fransson, Front. Biosci. 2008, 13:1619-1633); framework regions obtained from screening FR libraries (see, for example, Vasa et al., J. Biol. Chem. 1997, 272:10678-10684; Rosok et al., J. Biol. Chem. 1996, 271:22611 -22618).

4. Human antibodies.

In some embodiments of the present invention, the antibody provided is a human antibody. Human antibodies can be obtained using various methods known in the art. Human antibodies are generally described in the publications of van Dijk and van de Winkel, Curr. Opin. Pharmacol. 2001, 5: 368-374; and Lonberg, Curr. Opin. Immunol. 2008, 20:450-459.

Human antibodies can be produced by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact human variable region antibodies in response to antigenic stimulation. Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci, or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are usually inactivated. For a review of methods for producing human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 2005, 23: 1117-1125. See also, for example, US 6,075,181 and US 6,150,584, which describe XENOMOUSE™ technology; US 5,770,429 describing HuMab technology (registered trademark); US 7041870 describes K-M MOUSE technology (registered trademark) and US 2007/0061900 describes VelociMouse technology (registered trademark). Human variable regions from intact antibodies generated by such animals can be further modified, for example, by combining with another human constant region.

Human antibodies can also be produced by hybridoma-based methods. Human myeloma and murine-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies (see, for example, Kozbor J. Immunol, 1984, 133:3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, 1987, pp. 51- 63, Marcel Dekker, Inc., New York); Boerner et al., J. Immunol., 1991, 147: 86). Human antibodies produced using human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 2006, 103:35573562. Additional methods include those described, for example, in US 7189826 (production of human IgM monoclonal antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 2006, 26(4):265268 (description of hybridomas between different human cells). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 2005, 20(3):927-937; and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 2005, 27(3): 185-191.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from phage display libraries of human origin. Such variable domain sequences can then be combined with the desired human constant domain. Methods for selecting human antibodies from antibody libraries are described below.

5. Antibodies developed from libraries.

Antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for constructing phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are considered, for example, in the publications of Hoogenboom et al., in the book: Methods in Molecular Biology 2001, 178: 1-37, ed. O'Brien et al., Human Press, Totowa, NJ; as well as those described, for example, in the works of McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 1991, 352: 624-628; Marks et al., J. Mol. By1. 1992, 222: 581-597; Marks and Bradbury, in: Methods in Molecular Biology 2003, 248:161-175, ed. Lo, Human Press, Totowa, New Jersey; Sidhu et al., J. Mol. Biol. 2004, 338(2): 299-310; Lee et al., J. Mol. Biol. 2004, 340(5): 1073-1093; Fellowes, Proc. Natl. Acad. Sci. USA 2004, 101(34): 12467-12472; Lee et al., J. Immunol. Methods 2004, 284(1-2): 119-132.

In some phage display methods, the VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for the presence of antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol 1994, 12: 433-455. Phage typically displays antibody fragments as either single chain Fv fragments (scFv) or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to an immunogen without the need to construct hybridomas. Alternatively, the naive repertoire can be cloned (eg from humans) to provide a single source of antibodies to a wide range of foreign as well as self antigens without any immunization, as described by Griffiths et al., EMBO J, 1993, 12: 725

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734. Finally, naïve libraries can also be prepared synthetically by cloning unrearranged V gene segments from stem cells and using random sequence PNR primers to encode highly variable CDR3 regions and perform the rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol . Biol., 1992, 227: 381-388. Patent publications describing human antibody phage libraries include, for example: US 5750373, US 2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US 2007/0160598, US 2007/0237764, US 2007 /0292936 , US 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments in the present invention.

6. Multispecific antibodies.

In some embodiments of the present invention, the antibody provided in the present invention is a multispecific antibody, for example, a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different sites. In some embodiments of the present invention, one of the binding specificities is for CTLA-4 and the other is for any other antigen. In some embodiments of the present invention, bispecific antibodies can bind to two different epitopes of CTLA-4. Bispecific antibodies can also be used to localize cytotoxic agents to cells that express CTLA-4. Bispecific antibodies can be produced as full-length antibodies or antibody fragments.

Methods for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two pairs of immunoglobulin heavy and light chains having different specificities (see Milstein and Cuello, Nature 1983, 305: 537), WO 93/08829, Traunecker et al., EMBO J. 1991, 10: 3655), and projection-to-recess design (see, for example, US 5731168). Multispecific antibodies can also be produced by engineering electrostatic control effects to create Fc heterodimeric antibody molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments thereof (see, for example, US 4676980, and Brennan et al., Science, 1985, 229:81); the use of leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol, 1992, 148(5): 1547-1553); the use of diabody technology to produce bispecific antibody fragments (see, for example, Hollinger et al., Proc. Nail. Acad. Sci. USA, 1993, 90: 6444-6448); and using single chain Fv dimers (scFv) (see, for example, Gruber et al., J. Immunol, 1994, 152: 5368); and preparing trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 1991, 147:60.

Also included are engineered antibodies with three or more functional antigen binding sites, including octopus antibodies (see, for example, US 2006/0025576A1).

The antibody or fragment thereof of the present invention also includes a Dual Acting Fab (DAF) containing an antigen binding site that binds to CTLA-4 as well as another different antigen (see US 2008/0069820, for example).

7. Antibody variants.

In some embodiments of the present invention, variants of the amino acid sequence of the antibodies provided in the present invention are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Variants of the amino acid sequence of an antibody can be obtained by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletions, insertions and substitutions can be used to produce the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

A. Variants of substitutions, insertions and deletions.

In some embodiments, the present invention provides antibody variants having one or more amino acid substitutions. Sites of interest for replacement mutagenesis include HVR and FR. Conservative substitutions are shown in table. 1 under the heading Preferred Substitutions. More significant changes are presented in table. 1 under the heading Examples of Substitutions and as further described below with respect to classes of amino acid side chains. Amino acid substitutions can be introduced into the antibody of interest, and the products are screened for the desired activity, such as maintaining/improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

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Table 1

Initial balances Examples of substitutions Preferred Substitutions Ala (A) Val; Leu; lie Val Arg(R) Lys; Gin; Asn Lys Asn(N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His(H) Asn; Gin; Lys; Arg Arg He(I) Leu; Val; Met; Ala; Ph; Norleucine Leu Leu (L) Norleucine; lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met(M) Leu; Ph; lie Leu Phe (F) Trp; Leu; Val; lie; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Ph; Thr; Ser Phe Val(V) lie; Leu; Met; Ph; Ala; Norleucine Leu

Amino acids can be divided into groups according to the general properties of their side chains:

(1) hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu) and isoleucine (Ile);

(2) neutral and hydrophilic: cysteine (Cys), series (Ser), threonine (Thr), asparagine (Asn) and glutamine (Gln);

(3) acidic: aspartate (Asp) and glutamate (Glu);

(4) basic: histidine (His), lysine (Lys) and arginine (Arg);

(5) residues that affect chain orientation: glycine (Gly) and proline (Pro); and (6) aromatic: tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe). Non-conservative substitution refers to the replacement of a member of one of these classes by a member of another class.

One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Typically, the resulting variants selected for further study have modifications (eg, improvements) in certain biological properties (eg, increased affinity, reduced immunogenicity) compared to the parent antibody and/or substantially retain certain biological properties of the parent antibody. An exemplary replacement option is an affinity matured antibody that can be readily produced, for example, using phage display-based affinity maturation methods, such as those described in the present invention. Briefly, one or more HVR residues are mutated and the antibody variant is displayed on phage and screened for specific biological activity (eg, binding affinity).

Changes (eg, substitutions) can be made to the HVR, for example to improve antibody affinity. Such changes can be made at HVR hotspots, that is, at residues encoded by codons that undergo mutations at high rates during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 2008, 207: 179-196), and/or residues that contact the antigen, wherein the resulting VH or VL variant is tested for binding affinity. Affinity maturation by generating and iterating from secondary libraries has been described, as described, for example, by Hoogenboom et al., Methods in Molecular Biology, 2001, 178:1-37, ed. O'Brien et al., Human Press, Totova, New Jersey. In some affinity maturation embodiments of the present invention, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (eg, error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves HVR-targeting approaches in which multiple HVR residues (e.g., 4–6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, e.g. using alani

- 34 045931 new scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In some embodiments of the present invention, substitutions, insertions or deletions may occur in one or more HVRs as long as such changes do not significantly reduce the ability of the antibody to bind antigen. For example, conservative changes can be made to the HVR (eg, conservative substitutions as provided in the present invention) that do not significantly reduce binding affinity. Such changes may, for example, be beyond antigen-contacting residues in HVR regions. In some embodiments of the present invention, in the VH and VL variants provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

A useful method for identifying residues or regions of an antibody that may be targeted for mutagenesis is called alanine scanning mutagenesis, as described by Cunningham and Wells, Science, 1989, 244: 1081-1085. In this method, a residue or group of target residues (eg, charged residues such as arg, asp, his, Lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (eg, alanine or polyalanine) to determine whether the interaction of the antibody with antigen. Additional substitutions can be introduced at amino acid locations that exhibit functional sensitivity to the original substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be analyzed to identify points of contact between the antibody and the antigen. Such contact residues and adjacent residues can be selected or excluded as candidates for replacement. The options can be tested to determine whether they contain the desired properties.

Amino acid sequence insertions include fusions to the amino and/or carboxy terminus of sequences of varying lengths, from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of an antibody molecule include fusion of an enzyme (eg, the antibody targeting enzyme ADEPT) or polypeptide that increases the plasma half-life of the antibody to the N- or C-terminus of the antibody.

b. Glycosylation variants.

In some embodiments, the antibody of the present invention is modified to increase or decrease the degree of glycosylation of the antibody. The addition or removal of glycosylation sites in an antibody can be conveniently accomplished by changing the amino acid sequence such that one or more glycosylation sites are created or removed.

If an antibody contains an Fc region, the carbohydrate associated with it may be altered. Native antibodies produced by mammalian cells typically contain a branched, biantennary oligosaccharide that is typically N-linked to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 1997, 15: 26-32. The oligosaccharide may 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 of the present invention, modifications to the oligosaccharide in the antibody of the present invention can be made to create antibody variants with certain improved properties.

In one embodiment, the present invention provides antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody may be from 1 to 80%, from 1 to 65%, from 5 to 65%, or from 20 to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain of Asn297 relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures) as measured by MALDI-TOF mass spectrometry as described e.g. in WO 2008/077546. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located approximately +/- 3 amino acids upstream or downstream of position 297, that is, between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, patent publications US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to defucosylated or fucose-deficient antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki et al., J. Mol. Biol. 2004, 336: 1239-1249; Yamane-Ohnuki et al., Biotech. Bioeng. 2004, 87: 614. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 1986, 249: 533-545; US Pat Appl 2003/0157108 A1, Presta L.; WO 2004/056312 A1, Adams et al.,

- 35 045931 especially example 11), and cell lines with knockout, for example, the alpha-1,6-fucosyltransferase gene, FUT8, CHO cells with knockout (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 2004, 87: 614; Kanda Y. et al., Biotechnol. Bioeng., 2006, 94(4): 680-688; and WO 2003/085107).

In addition, antibody variants with bisected oligosaccharides are contemplated, for example, in which the biantennary oligosaccharide attached to the F region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); US 6602684 (Umana et al.); and US 2005/0123546 (Umana et al.). Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function; they are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju S); and WO 1999/22764 (Raju S.).

c. Variants of the Fc region.

In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody of the present invention, thereby producing a variant Fc region. The Fc region variant may comprise a human Fc region sequence (eg, human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (eg, substitution) at one or more amino acid positions.

In some embodiments, the present invention provides a variant antibody that possesses some, but not all, effector functions, making it a desirable candidate for applications in which the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are not needed. or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/exhaustion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody does not bind to FcyR (thus likely to have no ADCC activity) but retains the ability to bind FcRn. The primary cells for mediating ADCC, NK cells, express only FcyRIII, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression on hematopoietic cells is summarized in Table. 3 on page 464 in Ravetch and Kinet, Annu. Rev. Immunol. 1991, 9: 457-492. Non-limiting examples of in vitro assays for assessing the ADCC activity of a test molecule are described in US 5500362 (see, for example, Hellstrom I. et al., Proc. Nat'l Acad. Sci. USA 1986, 83:7059-7063) and Hellstrom I. et al., Proc. Nat'lAcad. Sci. USA 1985, 82:1499-1502; US 5821337 (see Bruggemann M et al., J. Exp. Med. 1987, 166: 1351-1361). Alternatively, non-radioactive assay methods may be used (see, for example, the ACT1™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.; and the CytoTox 96 (Registered Trademark) Non-Radioactive Cytotoxicity Assay (Promega, Madison) , Wisconsin. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the test molecule can be assessed in vivo, for example, in an animal model, e.g. described by Clynes et al., Proc. Nat'l Acad. Sci. USA 1998, 95:652-656. C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and therefore does not have CDC activity. ., for example, the binding ELISA C1q and C3c in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 1996, 202:163; Cragg M.S. et al., Blood2003, 101:1045-1052; Cragg M.S., Glennie M.J., Blood 2004, 103: 2738-2743). FcRn binding and determination of clearance/half-life in vivo can also be performed using methods known in the art (see, for example, Petkova S.B. et al., Int'l. Immunol. 2006, 18(12): 1759-1769) .

Antibodies with reduced effector function include antibodies with a substitution of one or more residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (US 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions from amino acid positions 265, 269, 270, 297 and 327, including the so-called Fc DANA mutant with residues 265 and 297 replaced by alanine (US Pat. No. 7,332,581).

Several antibody variants with increased or decreased FcR binding have been described (see, for example, US 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 2001, 9(2):6591-6604).

In some embodiments of the present invention, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333 and/or 334 of the Fc region (EU residue numbering).

In some embodiments of the present invention, changes occur in the F region, resulting in altered (i.e., increased or decreased) binding of C1q and/or K complement dependent cytotoxicity (CDC), for example, as described in US 6194551, WO 99/51642, Idusogie et al., J. Immunol. 2000, 164:4178-4184.

Antibodies with an extended half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 1976, 117:587; Kim et al., J. Immunol., 1994, 24:249), described in US 2005/0014934 A1 (Hinton et al.).

- 36 045931

These antibodies contain an Fc region with one or more substitutions that increase the binding of the Fc region to FcRn. Such Fc variants include variants with substitutions in one or more residues of the Fc region from among: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, replacing residue 434 of the Fc region (US 7371826).

See also Duncan and Winter, Nature 1988, 322:738-740; US 5648260; US 5624821; WO 94/29351, which describes other examples of Fc region variants.

d. Antibody variants processed with cysteine.

In some embodiments of the present invention, it may be advisable to process antibodies with cysteine, for example, thioMAB, in which one or more residues of the antibody are replaced by cysteine residues. In certain embodiments of the present invention, the substituted residues occur in accessible regions of the antibody. By replacing these residues with cysteine, the reactive thiol groups are thus located in accessible regions of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described later in the present invention . In some embodiments of the present invention, any one or more of the following residues may be replaced by cysteine: V205 (Kabat numbering) light chain; A118 (EU numbering) heavy chain; and S400 (EU numbering) heavy chain Fc region. Antibodies processed using cysteine can be prepared as described in EU 7521541.

d. Antibody derivatives.

In some embodiments, the antibody of the present invention may be further modified to contain additional non-protein moieties that are known in the art and readily available. Fragments suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Examples of such 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 copolymer /maleic anhydride, polyamino acids (either homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (for example, glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have an advantage in production due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can be varied, and if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymers used for derivatization can be determined based on the specific properties or function of the antibody that is to be improved, whether the antibody derivative will be used in therapy under certain conditions, etc.

In another embodiment, the present invention provides antibody-non-protein moiety conjugates that can be selectively heated by radiation. In one embodiment of the present invention, the non-protein portion is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 2005, 102: 11600-11605). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells, but which heat the non-protein portion to a temperature that kills cells proximal to the antibody-non-protein portion.

B. Recombinant methods and compositions.

Antibodies can be produced by recombination and using compositions such as those described in US Pat. No. 4,816,567. In one embodiment, the present invention provides an isolated nucleic acid encoding an anti-CTLA-4 antibody described in the present invention. Such a nucleic acid may encode an amino acid sequence comprising VL and/or an amino acid sequence comprising VH of an antibody (eg, antibody light and/or heavy chains). In another embodiment, the present invention provides one or more vectors (eg, expression vectors) containing such a nucleic acid. In another embodiment, the present invention provides a host cell containing said nucleic acid. In one such embodiment of the present invention, the host cell contains (eg, has been transformed): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL antibody and an amino acid sequence comprising a VH antibody, or (2) a first vector containing a nucleic acid encoding an amino acid sequence including a VL antibody, and a second vector containing a nucleic acid encoding an amino acid sequence including a VH antibody. In one embodiment of the present invention, the host cell is eukaryotic, for example, a Chinese hamster ovary (CHO) cell or a lymphoid cell

- 37 045931 (for example, Y0, NSO, Sp2/0 cells). In one embodiment, the present invention describes a method for producing an anti-CTLA-4 antibody, which includes culturing a host cell containing a nucleic acid encoding the antibody as described above under conditions suitable for expression of the anti-CTLA-4 antibody, and optionally isolating the antibody from the host cell (or host cell culture medium).

To recombinantly produce an anti-CTLA-4 antibody, the nucleic acid encoding the antibody, for example as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding antibody heavy and light chains).

Host cells suitable for cloning or expression of vectors encoding antibodies include prokaryotic or eukaryotic cells described in the present invention. For example, antibodies can be produced by bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5648237, US 5789199 and US 5840523 (see also Charlton, Methods in Molecular Biology, 2003, volume 248, pp. 245-254, edited by Lo V.K. .C, Humana Press, Totowa, NJ, which describes the expression of antibody fragments in E. coli). Once expressed, the antibody can be isolated from the bacterial cell biomass as a soluble fraction and can be further purified.

In addition to prokaryotes, suitable hosts for the cloning or expression of antibody-encoding vectors include eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been humanized, resulting in the production of an antibody with a glycosylation pattern that is partially or completely corresponds to that of humans. See Gerngross, Nat. Biotech. 2004, 22:1409-1414; Li et al., Nat. Biotech. 2006, 24:210-215.

Suitable host cells for expression of the glycosylated antibody are also obtained from multicellular organisms (invertebrates and vertebrates). Examples include invertebrate cells, including insects, and plant cells. Numerous strains of baculoviruses have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cells can be host cells. See, for example, US 5959177, US 6040498, US 6420548, US 7125978 and US 6417429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

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

Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (SC) or intraperitoneal (IP) injections of the appropriate antigen and adjuvant. May conjugate the appropriate antigen to a protein that is immunogenic in the immunized species, e.g., snail limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine residues), Nhydroxysuccinimide ( through lysine residues), glutaraldehyde, succinic anhydride, SOCl ₂ or R ¹ N=C=NR, where R and R ¹ are different alkyl groups.

Animals (usually non-human mammals) are immunized against the antigen, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant, and the solution is administered intradermally into several places. After a month, the animals are re-injected with 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection into the

- 38 045931 several places. After 7-14 days, blood is taken from the animals and the antibody titer in the serum is analyzed. Animals are given booster injections until the titer reaches a plateau. Preferably, the animal is revaccinated with a conjugate of the same antigen, but conjugated to a different protein and/or using a different cross-linking reagent. Conjugates can also be produced in recombinant cell culture as protein fusions. In addition, aggregating agents such as alum are useful to enhance the immune response.

Monoclonal antibodies are obtained from a population of essentially homogeneous antibodies, i.e., the individual antibodies making up the population are identical, except for possible natural mutations and/or post-translational modifications (eg, isomerization, amidation), which may be present in minor quantities. Thus, the modifier monoclonal indicates that the antibody is not a mixture of individual antibodies.

For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 1975, 256(5517):495-497. In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to identify 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.

The immunizing agent typically comprises an antigenic protein or a fusion thereof. Typically, either peripheral blood lymphocytes (PBL) are used if cells of human origin are preferred, or spleen cells or lymph node cells if other mammalian species are preferred. The lymphocytes are then fused with the immortalized cell line using a suitable coupling agent such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, 1986, pp. 59-103, Academic Press).

Immortalized cell lines are typically transformed mammalian cells, especially rodent, bovine and human myeloma cells. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma culture medium will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that prevent the growth of HGPRT-deficient cells.

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

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

Once hybridoma cells are identified that produce antibodies with the desired specificity, affinity and/or activity, clones can be subcloned using limiting dilution procedures and grown by standard methods (Goding publication, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 media. In addition, hybridoma cells can be grown in vivo as tumors in mammals.

Monoclonal antibodies secreted by the subclones are suitably isolated from culture medium, ascites fluid or serum using conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Antibodies can be obtained by immunizing an appropriate host animal against the antigen. In one embodiment of the present invention, the antigen is a polypeptide containing the full-length CTLA-4 antigen. In one embodiment of the present invention, the antigen is a polypeptide containing soluble CTLA-4. IN

- 39 045931 In one embodiment of the present invention, the antigen is a polypeptide containing a region corresponding to amino acids at position 97 (Glu) to position 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). In one embodiment of the present invention, the antigen is a polypeptide containing a region corresponding to amino acids at position 99 (Met) to position 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). Also included in the present invention are antibodies obtained by immunizing an animal against an antigen. The antibodies may include any of the features, alone or in combination, as described above in the Examples of Anti-CTLA-4 Antibodies section.

B. Research.

Anti-CTLA-4 antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activity using various assays known in the art.

1. Binding and other studies

In another aspect of the present invention, the antibody is tested for antigen binding activity, for example, using known methods such as ELISA, Western blotting, surface plasmon resonance, etc.

In another aspect of the present invention, competition assays can be used to identify an antibody that competes with the anti-CTLA-4 antibodies described in the present invention (for example, the anti-CTLA-4 antibodies described in Table 4, Table 9, Table 14 and Table 19) for binding to CTLA-4. In some embodiments of the present invention, if such a competing antibody is present in excess, binding of the reference antibody to CTLA-4 is prevented (e.g., reduced) by at least 10, 15, 20, 25, 30, 35, 40, 45, 50. 55, 60, 65, 70, 75 or higher. In some examples, binding is prevented by at least 80, 85, 90, 95% or greater. In some embodiments of the present invention, such a competitive antibody binds to the same epitope (for example, a linear or conformational epitope) that is bound by the anti-CTLA-4 antibodies described in the present invention (for example, the anti-CTLA-4 antibodies described in Table 4, table 9, table 14 and table 19). Detailed standard methods for mapping the epitope to which an antibody binds are presented in Morris Epitope Mapping Protocols in Methods in Molecular Biology vol. 66, ed. Humana Press, Totowa, NJ.

In an example of a competition assay, immobilized CTLA-4 is incubated in a solution containing a first labeled antibody that binds to CTLA-4 and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to CTLA-4. The second antibody may be present in the supernatant of the hybridoma. As a control, immobilized CTLA-4 was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions allowing binding of the first antibody to CTLA-4, excess unbound antibody is removed and the amount of label bound to the immobilized CTLA-4 is measured. If the amount of label bound to immobilized CTLA-4 is significantly reduced in the test sample compared to the control sample, this indicates that the second antibody is competing with the first antibody for binding to CTLA-4. See Antibodies: A Laboratory Manual, 1988, chapter 14, ed. E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

2. Activity research.

In one aspect of the present invention, assays are provided to identify anti-CTLA-4 antibodies having biological activity. Biological activities may include, for example, cell proliferation inhibitory activity, cytotoxic activity (eg, ADCC/CDC activity and ADCP activity), immunostimulatory activity, and CTLA-4 inhibitory activity. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In some embodiments, the antibody of the present invention is tested for such biological activity.

In some embodiments, an antibody of the present invention is tested for its ability to inhibit cell growth or proliferation in vitro. Cell growth or proliferation inhibition assays are well known in the art. Certain cell proliferation assays, such as the cell killing assays described herein, measure cell viability. One such assay is the CellTiter-GloTM Luminescent Cell Viability Assay, which is commercially available from Promega (Madison, WI). This assay determines the number of viable cells in a culture based on the quantification of ATP present, an indication of metabolically active cells. See Crouch et al. J. Immunol. Meth. 1993, 160:81-88, US 6602677. The assay can be performed in 96- or 384-well format, making it suitable for automated high-throughput screening. See Cree et al. AntiCancer Drugs 1995, 6:398-404. The assay procedure involves adding a single reagent (CellTiter-Glo Reagent (registered trademark)) directly to the cultured cells. This leads to cell lysis and generation of luminescent

- 40 045931 signal caused by 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 using a luminometer or a CCD camera imaging device. The luminescence output is expressed in relative light units (RLU - relative light units).

Another test for cell proliferation 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. See, for example, Mosmann, J. Immunol. Meth., 1983, 65:55-63; Zhang et al., Cancer Res. 2005, 65:3877-3882.

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 are transfected with a nucleic acid encoding CTLA-4.

In one aspect of the present invention, the described anti-CTLA-4 antibody is tested for its ability to inhibit cell growth or proliferation in vivo. In some embodiments, the anti-CTLA-4 antibody is tested for its ability to inhibit tumor growth in vivo. In vivo model systems, such as xenograft models, can be used in such testing. In an exemplary xenograft system, human tumor cells are administered to a suitable immunocompromised non-human animal, such as an athymic nude mouse. The antibody of the present invention is administered to an animal. The ability of an antibody to inhibit or reduce tumor growth is measured. In some embodiments of the present invention relating to the above xenograft system, the human tumor cells are tumor cells from a human patient. Such xenograft models are commercially available from Oncotest GmbH (Friberg, Germany). In some embodiments of the present invention, human tumor cells are administered to a suitably immunocompromised animal, other than a human, by subcutaneous injection or by transplantation into a suitable site, such as mammary adipose tissue.

It will be appreciated that any of the above studies can be performed using the immunoconjugate of the present invention in place of or in addition to the anti-CTLA-4 antibody.

A typical assay for measuring the ADCC activity of a therapeutic antibody is based on a ⁵¹ Cr release assay and includes the following steps: labeling target cells with [ ⁵¹ Cr]Na ₂ CrO ₄ ; opsonization of target cells expressing antigen on the cell surface with an antibody; combining opsonized radiolabeled target cells with effector cells in a suitable ratio in a microtiter plate in the presence or absence of a test antibody; incubating the cell mixture preferably for 16 to 18 hours, preferably at 37°C collecting the supernatant; and analysis of radioactivity in the supernatant sample. The cytotoxicity of the test antibody is then determined, for example, using the following equation: degree of specific cytotoxicity (%) = (radioactivity in the presence of antibody - radioactivity in the absence of antibody) / (maximum radioactivity - radioactivity in the absence of antibody) x 100. The graph can be constructed by changing the ratio of cells -targets and effector cells or antibody concentration.

To assess complement activation, a complement-dependent cytotoxicity (CDC) assay can be performed, as described, for example, in Gazzano-Santoro et al., J. Immunol. Methods, 1996, 202:163. Briefly, various concentrations of the polypeptide variant and human complement are diluted in buffer. Cells expressing the antigen to which the variant polypeptide binds are diluted to a density of approximately 1 x 106 cells/ml. A mixture of variant polypeptide, dilute human complement, and antigen-expressing cells is added to a flat-bottomed 96-well tissue culture plate and incubated at 37° C. and 5% CO ₂ for 2 hours to stimulate complement-mediated cell lysis. 50 μl of Alamar Blue dye (Accumed International) was then added to each well and incubated at 37°C overnight. Absorbance is measured using a 96-well fluorimeter with excitation at 530 nm and emission at 590 nm. The results are presented in relative fluorescence units (RFU). Sample concentrations can be calculated from a standard curve, and the percentage of activity relative to the non-variant polypeptide is reported for the variant polypeptide of interest.

One method for assaying ADCP activity may include: coating bioparticles such as FITC-labeled E. coli (Molecular Probes) or Staphylococcus aureus-FITC with a test antibody; formation of opsonized particles; adding the above opsonized particles to THP-1 effector cells (a monocytic cell line available from ATCC) at a ratio of 1:1, 10:1, 30:1, 60:1, 75:1 or 100:1 to induce FcYR-mediated phagocytosis ; it is preferable to incubate cells and E. coli-FITC/antibody at 37°C for 1.5 hours; after incubation, adding trypan blue dye to the cells (preferably at room temperature for

- 41 045931 two to three minutes) to quench the fluorescence of bacteria that have not penetrated the cells and are attached to the outer surface of the cells; transfer cells into FACS buffer (eg, 0.1% BSA and 0.1% sodium azide in PBS) for fluorescence analysis of THP-1 cells using FACS (eg, BD FACS Calibur). To assess the extent of ADCP, a gate is preferably installed on THP-1 cells and the average fluorescence intensity is measured. In the most preferred embodiments of the present invention, the ADCP assay is performed using E. coli-FITC in media (control); E. coliFITC and THP-1 cells (used as FcYR-independent ADCP activity); and E. coli-FITC, THP-1 cells and test antibody (used as FcYR-dependent ADCP activity).

The cytotoxic activity of an antibody typically involves binding of the antibody to the cell surface. Expression of the antigen on the surface of the target cell can be suitably confirmed by methods known to those skilled in the art, such as FACS.

Immune activation can be detected using cellular or humoral immune responses as indicators. Immune activation includes, but is not limited to, increasing the expression of cytokines (e.g., IL-6, G-CSF, IL-12, TNFa and ZFNy) or their receptors, stimulating the proliferation of immune cells (e.g., B cells, T cells, NK, macrophages and monocytes), increased activation state, increased functions and increased cytotoxic activity. In particular, T cell activation can be detected by measuring increased expression of activation markers such as CD25, CD69 and ICOS. For example, patients receiving the anti-CTLA4 antibody, ipilimumab, are known to experience an increase in ICOS+ CD4+ T cells in the peripheral blood after administration, and this is considered to be an effect of activation of the systemic immune status by the administration of the anti-CTLA-4 antibody (Cancer Immunol Res., 2013, 1(4): 229-234).

Activation of T cells requires not only stimulation through the antigen receptor (TCR), but also ancillary stimulation through CD28. When CD28 on the surface of a T cell binds to B7-1 (CD80) or B7-2 (CD86) on the surface of an antigen-presenting cell, an auxiliary signal is transmitted to the T cell, and the T cell is then activated. On the other hand, CTLA-4 is expressed on the surface of activated T cells. Because CTLA-4 binds to CD80 and CD86 with greater affinity than CD28, it interacts with CD80 and CD86 more than CD28, resulting in suppression of T cell activation.

Based on this mechanism of action, CTLA-4 inhibitory activity can be measured as the activity of inhibiting the binding of CTLA-4 to CD80 or CD86. In one embodiment of the present invention, the assay for measuring CTLA-4 inhibitory activity includes the following steps: allowing the purified CTLA-4 protein to bind to a support, such as a microtiter plate or magnetic beads; adding test antibody and labeled soluble CD80 or CD86; washing out unbound components; and quantification of bound labeled CD80 or CD86. Whether the test antibody cross-reacts with CD28 or not can be confirmed by performing a similar assay in which CD28 is used instead of CTLA-4. Moreover, in another embodiment of the present invention, a functional assay that detects T cell activation as described above can also be used to measure CTLA-4 inhibitory activity. For example, when a test antibody having CTLA-4 inhibitory activity is added to a system in which T cell activation is measured by stimulating a population of T cells with cells expressing CD80 or CD86, T cell activation is further enhanced.

D. Immunoconjugates.

The present invention also provides immunoconjugates comprising an anti-CTLA-4 antibody conjugated herein to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active bacterial toxins, fungi, plants, or animals, or their fragments) or radioactive isotopes.

In one embodiment of the present invention, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including, but not limited to, a maytansinoid (see US Pat. Nos. 5,208,020, US 5,416,064, and European patent EP 0425235 B1); auristatin, such as monomethyl auristatin molecules DE and DF (MMAE and MMAF) (see US 5635483, US 5780588 and US 7498298); dolastatin; calicheamicin or a derivative thereof (see US 5712374, US 5714586, US 5739116, US 5767285, US 5770701, US 5770710, US 5773001 and US 5877296; Hinman et al., Cancer Res. 1993, 53: 3336-33 42; Lode s et al., Cancer Res. 1998, 58: 2925-2928); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 2006, 13: 477-523; Jeffrey et al., Bioorganic & Med. Chem. Letters 2006, 16: 358-362; Torgov et al. et al., Bioconj. Chem. 2005, 16: 717-721; Nagy et al., Proc. Natl. Acad. Sci. USA 2000, 97: 829-834; Dubowchik et al., Bioorg. & Med. Chem. Letters 2002 , 12: 1529-1532; King et al., J Med Chem 2002, 45:4336-4343; US 6630579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel; trichothecene; and CC1065.

- 42 045931

In another embodiment of the present invention, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria toxin A chain, non-binding active diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modezzan A chain, alpha-sarcin, Aleuritesfordii proteins, diantine proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, chicken, carotene, Saponaria officinalis inhibitor, G alanine, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes.

In another embodiment of the present invention, the immunoconjugate contains an antibody described in the present invention, conjugated to a radioactive atom to form a radioconjugate. Various radioactive isotopes 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 Pb and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, such as Tc-99sh or ¹²³ I, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Antibody-cytotoxic agent conjugates can be prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane ( IT), bifunctional imidoester derivatives (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6diisocyanate) and biactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 1987, 238:1098. Carbon-141-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for radionuclide-antibody conjugation. See WO 94/11026. The linker may be a cleavable linker that facilitates release of the cytotoxic drug into the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 1992, 52: 127-131; US 5,208,020).

Immunoconjugates or ADCs in the present invention include, but are not limited to, those made 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 sulfoSMPB, as well as SVSB (succinimidyl (4-vinyl sulfone) benzoate), which are commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL, USA).

D. Methods and compositions for diagnostics and research.

In certain embodiments of the present invention, 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 prospecting includes quantitative or qualitative discovery. In certain embodiments of the present invention, the biological sample includes a cell or tissue, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, brain fluid, amniotic fluid, ascitic fluid, milk, colostrum , breast secretion, lymph fluid, urine, sweat, tear fluid, gastric juice, synovial fluid, ascitic fluid, ocular fluid and mucus.

In one embodiment, the present invention provides an anti-CTLA-4 antibody for use in a diagnostic or research method. In yet another aspect, the present invention provides a method for detecting the presence of CTLA-4 in a biological sample. In certain embodiments of the present invention, the method includes contacting a biological sample with an anti-CTLA-4 antibody as described herein, under conditions allowing the anti-CTLA-4 antibody to bind to CTLA-4, and determining whether a complex is formed between anti-CTLA-4 antibody and CTLA-4. This method can be performed in vitro or in vivo. In one embodiment of the present invention, an anti-CTLA-4 antibody is used to select subjects suitable for anti-CTLA-4 antibody therapy, for example, where CTLA-4 is a biomarker for patient selection.

The antibody of the present invention can be used, for example, to test the state of the immune response and diagnose immune system dysfunction.

In certain embodiments, the present invention provides anti-CTLA-4 labeled antibodies. Labels include, but are not limited to, tags or moieties that are directly detectable (e.g., fluorescent, chromophore, electron dense, hemil

- 43 045931 minescent and radioactive tags), as well as fragments such as enzymes or ligands that are detected indirectly, for example through an enzymatic reaction or molecular interaction. Examples of labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, for example , firefly luciferase and bacterial luciferase (US 4737456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g. glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogen aza , heterocyclic oxidases such as uricase and xanthine oxidase coupled to an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin tags, bacteriophage tags, stable free radicals and others.

E. Pharmaceutical compounds.

Pharmaceutical compositions of anti-CTLA-4 antibodies, as described in the present invention, are prepared by mixing such antibody, having the desired degree of purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 1980, 16th edition, edited by Osol A.) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (for example, Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Examples of pharmaceutically acceptable carriers in the present invention further include interstitial drug dispersing agents such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), for example, soluble human hyaluronidase glycoprotein PH-20 such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Some representative sHASEGPs and methods of use, including rHuPH20, are described in patent publications US 2005/0260186 and US 2006/0104968. In one aspect of the present invention, sHASEGP is combined with one or more glycosaminoglycanases, such as chondroitinases.

Examples of lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006/044908, the latter formulations including histidine acetate buffer.

The composition of the present invention may also contain more than one active ingredient if necessary for the particular indication being treated, preferably with additional activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose.

The active ingredients can be encapsulated in microcapsules prepared, for example, by coacervation or interfacial polymerization methods, e.g. microcapsules of hydroxymethylcellulose, gelatin or poly(methyl methacrylate), respectively, for colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such methods are described in the book: Remington's Pharmaceutical Sciences, 1980, 16th ed., edited by Osol A.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of molded articles such as films or microcapsules.

Formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filter membranes.

G. Therapeutic methods and compositions.

Any of the anti-CTLA-4 antibodies provided in the present invention can be used in therapeutic methods.

In one aspect of the present invention, anti-CTLA-4 antibodies are provided for use as a medicine. In other aspects, an anti-CTLA-4 antibody is provided for use in the treatment of tumors. In certain embodiments of the present invention

- 44 045931 provide an anti-STA-4 antibody for use in a method of treating tumors. In certain embodiments, the present invention provides an anti-CTLA-4 antibody for use in a method of treating an individual with a tumor, comprising administering to the individual an effective amount of a ηη™-Ο^Α-4 antibody. In one such embodiment of the present invention, the method further includes administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described above. In other embodiments, the present invention relates to a nnti-STEAM antibody used to damage cells. In certain embodiments, the present invention provides an anti-CTLA-4 antibody for use in a method of damaging cells in an individual, comprising administering to the individual an effective amount of an anti-CTLA-4 antibody to damage cells. In other embodiments, an anti-CTLA-4 antibody is provided for immune activation. In certain embodiments, the present invention provides an anti-CTLA-4 antibody used in a method of activating the immune system in an individual, comprising administering to the individual an effective amount of an anti-CTLA-4 antibody to activate the immune system. The individual in any of the above embodiments of the present invention is preferably a human.

In some embodiments of the present invention, the tumor is a solid tumor. In a solid tumor, tumor cells typically proliferate to form a population, and the tumor tissue is composed primarily of these cells. In addition, tumor tissues of living organisms are often infiltrated by immune cells, such as lymphocytes, which also form part of the tumor tissues. In some embodiments of the present invention, tumor tissues are infiltrated by immune cells, especially regulatory T cells (Treg). In one embodiment of the present invention, cell damage is caused by ADCC activity, CDC activity, or ADCP activity. In one embodiment of the present invention, cells expressing CTLA-4 on their cell surface are damaged. In another embodiment of the present invention, the cells to be damaged are Treg cells. In certain embodiments of the present invention, Treg cells that have infiltrated tumor tissue are damaged. In one embodiment of the present invention, immunity is activated by damage to Treg cells (immunosuppression by Treg cells is reversed). In other embodiments, the present invention activates immunity (in particular, antitumor immunity) in tumor tissues. In some embodiments of the present invention, the immune activation is T cell activation.

In other aspects of the present invention, the degree of drug exposure produced by the anti-CTLA-4 antibody of the present invention varies depending on the human tissue. In some embodiments of the present invention, the degree of drug effect varies depending on the concentration of the adenosine-containing compound in the tissue. In other embodiments of the present invention, the effect is enhanced in tissue with a high concentration of the adenosine-containing compound compared to tissue with a low concentration of the adenosine-containing compound. Tissues with a high concentration of adenosine-containing compounds include, for example, tumor tissues. Tissues with low concentrations of adenosine-containing compounds include, for example, non-tumor tissues such as healthy tissues. In some embodiments of the present invention, immunity is more strongly activated in tumor tissue than in non-tumor tissue. Such differences in response need not be observed for all doses of anti-CTLA-4 antibodies, but should only be observed for a certain range of doses. In another embodiment of the present invention, immunity is activated at a lower dose in tumor tissue than in non-tumor tissue. Moreover, in another embodiment of the present invention, the therapeutic effect is observed at a lower dose than that at which the side effect is observed. In certain embodiments of the present invention, the therapeutic effect is to produce an antitumor effect (eg, tumor regression and induction of cell death or inhibition of tumor cell growth) and the side effect is the development of an autoimmune disease (including damage to healthy tissue due to excessive immune responses).

In another aspect of the present invention, the degree of drug effect produced by the anti-CTLA-4 antibody of the present invention varies depending on whether it has CTLA-4 binding activity that is dependent on an adenosine-containing compound (i.e. changes according to the concentration of the adenosine-containing compound). 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 of the present invention, a control anti-CTLA-4 antibody is an antibody that does not have concentration-dependent CTLA-4 binding activity of the adenosine-containing compound. In some embodiments of the present invention, an antibody that does not have CTLA-4 binding activity dependent on the concentration of an adenosine-containing compound is an antibody in which the difference in CTLA-4 binding activity in the presence and absence of

- 45 045931 compound differs, for example, by less than two times, less than 1.8 times, less than 1.5 times, less than 1.3 times, less than 1.2 times or less than 1 ,1 time. It is desirable that the anti-CTCL-4 antibody of the present invention and the control anti-CTCL-4 antibody have substantially equal CTLA-4 binding activity in the presence of a sufficient amount of adenosine-containing compound.

In some aspects of the present invention, the anti-STCL-4 antibody of the present invention and the control anti-CTCL-4 antibody as drugs differ in their effects. In certain embodiments of the present invention, they differ in their action as drugs in tissue with a low concentration of the adenosine-containing compound. Tissues with low concentrations of adenosine-containing compounds include, for example, non-tumor tissues, ie normal tissues. Anti-STCL-4 antibodies may also be provided in the form of a pharmaceutical composition containing the antibody. In some embodiments of the present invention, in tissue with a low concentration of an adenosine-containing compound, the anti-CTLA-4 antibody of the present invention exhibits a low level of immune activation compared to a control anti-CTLA-4 antibody. In some embodiments of the present invention, in tissue with a low concentration of adenosine-containing compound, the doses of the anti-STCL-4 antibody of the present invention required to activate the immune system are high compared to the doses of the control anti-CTCL-4 antibody. In some embodiments of the present invention, in tissue with a low concentration of an adenosine-containing compound, the anti-CTCL-4 antibody of the present invention exhibits low levels of side effects compared to a control antibody. In some embodiments of the present invention, in tissue with a low concentration of an adenosine-containing compound, the doses of the anti-STCL-4 antibody of the present invention at which the side effect is observed are high compared to the doses of the control anti-CTCL-4 antibody. Such differences in response need not be observed in all tissues (eg, in all tissues with low concentrations of the adenosine-containing compound), but should be observed only in some tissues. In some embodiments of the present invention, the side effect is an autoimmune disease (including damage to normal tissues due to an excessive immune response).

In some aspects of the present invention, the anti-STCL-4 antibody of the present invention and the control anti-CTCL-4 antibody as drugs have substantially equal effects. In certain embodiments of the present invention, they exhibit substantially equal activity as drugs in tissue with a high concentration of the adenosine-containing compound. Tissues with a high concentration of adenosine-containing compounds include, for example, tumor tissues. Anti-CTCL-4 antibodies may also be provided in the form of pharmaceutical compositions containing the antibody. In some embodiments of the present invention, in tissue with a high concentration of an adenosine-containing compound, the anti-STCL-4 antibody of the present invention and the control anti-CTCL-4 antibody exhibit substantially equal levels of immune activation. In some embodiments of the present invention, in tissue with a high concentration of an adenosine-containing compound, the anti-STCL-4 antibody of the present invention and the control anti-CTCL-4 antibody exhibit equal levels of immune activation at substantially the same dose. In some embodiments, in tissue with a high concentration of an adenosine-containing compound, the anti-STCL-4 antibody of the present invention and the control anti-CTCL-4 antibody exhibit substantially the same level of therapeutic effect. In some embodiments, in tissue with a high concentration of an adenosine-containing compound, the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody exhibit the same level of therapeutic effect at substantially the same dose. In certain embodiments of the present invention, the therapeutic effect is the manifestation of an antitumor effect (eg, tumor regression and induction of cell death or inhibition of tumor cell growth).

In certain embodiments of the present invention, the tumor is selected from the group consisting of breast cancer and liver cancer.

In yet another aspect, the present invention provides for the use of an anti-CTCL-4 antibody in the manufacture or preparation of a medicinal product. In one embodiment of the present invention, the medicament is for the treatment of tumors. In another embodiment of the present invention, the drug is for use in a method of treating a tumor, comprising administering to an individual having a tumor an effective amount of the drug. In one such embodiment of the present invention, the method further includes administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below. In another embodiment of the present invention, the drug is intended to destroy cells. In another embodiment of the present invention, the drug is for use in a method of destroying cells in an individual, comprising administering to the individual an effective amount of the drug to destroy cells. In another embodiment, on

- 46 045931 of the invention, the drug is intended to activate the immune system. In another embodiment of the present invention, the drug is for use in a method of activating the immune system in a subject, comprising administering to the subject an effective amount of the drug to activate the immune system. The individual in any of the above embodiments of the present invention may be a human.

In yet another aspect, the present invention provides a method for treating cancer. In one embodiment of the present invention, the method includes administering to an individual with such a tumor an effective amount of a ηη™^ΤΕΑ-4 antibody. In one such embodiment of the present invention, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. The individual in accordance with any of the above embodiments may be a human.

In yet another aspect, the present invention provides a method for damaging cells in an individual. In one embodiment of the present invention, the method includes administering to an individual an effective amount of an anti-CTLA-4 antibody to damage cells. In another aspect, the present invention provides a method for activating the immune system in an individual. In one embodiment of the present invention, the method includes administering to a subject an effective amount of an anti-CTLA-4 antibody to activate the immune system. In one embodiment of the present invention, the term individual means a person.

In yet another aspect, the present invention provides pharmaceutical compositions comprising any of the anti-CTLA-4 antibodies provided herein, for example, for use in any of the above therapeutic methods. In one embodiment of the present invention, the pharmaceutical composition includes any of the anti-CTLA-4 antibodies provided herein and a pharmaceutically acceptable carrier. In one embodiment, the present invention provides a pharmaceutical composition for use in the treatment of tumors. In one embodiment, the present invention provides a pharmaceutical composition for use in cell destruction. In one embodiment, the present invention provides a pharmaceutical composition for use in stimulating the immune system. In another embodiment of the present invention, the pharmaceutical composition contains any of the anti-CTLA-4 antibodies provided in the present invention and at least one additional therapeutic agent, for example, as described below.

In another aspect, the present invention provides methods for preparing a drug or pharmaceutical composition (eg, for use in any of the above therapeutic methods) comprising admixing any of the anti-CTLA-4 antibodies provided herein with a pharmaceutically acceptable carrier. In one embodiment of the present invention, the method of producing a drug or pharmaceutical composition further includes adding at least one additional therapeutic agent to the drug or pharmaceutical composition.

The antibodies of the present invention can be used in therapy either alone or in combination with other agents. For example, an antibody of the present invention can be co-administered with at least one additional therapeutic agent. In certain embodiments of the present invention, the additional therapeutic agent is an immune checkpoint inhibitor, an EGFR inhibitor, a HER2 inhibitor, or a chemotherapeutic agent. The immune checkpoint inhibitor may be, for example, an anti-CTLA-4 inhibitor, an anti-PD-i inhibitor, an anti-PD-L! inhibitor, an anti-PD-L2 inhibitor, an anti-TGM-3 inhibitor, an anti-LAG- 3, anti-TIGIT inhibitor, anti-BTLA inhibitor and anti-VISTA inhibitor. The anti-CTLA-4 inhibitor may include, for example, ipilimumab and tremelimumab. Anti-PD-1 inhibitors include, for example, nivolumab and pembrolizumab. Anti-PD-L inhibitor! may be, for example, atezolizumab, durvalumab and avelumab. The anti-PD-12 inhibitor may be, for example, an anti-PD-L2 inhibitory antibody. The anti-TGM-3 inhibitor may be, for example, an anti-TGM-3 inhibitory antibody. The anti-LAG-3 inhibitor may be, for example, an anti-LAG-3 inhibitory antibody. The anti-TIGIT inhibitor may be, for example, an anti-TIGIT inhibitory antibody. The anti-BTLA inhibitor may be, for example, an anti-BTLA inhibitory antibody. The anti-VISTA inhibitor may be, for example, an anti-VISTA inhibitory antibody. EGFR inhibitors may include, for example, cetuximab, panitumumab, nimotuzumab, necitumumab and zalutumumab. HER2 inhibitors may include, for example, trastuzumab, trastuzumab emtansine and pertuzumab.

Such combination therapies mentioned above include combination administrations (where two or more therapeutic agents are included in the same or separate formulations) and separate administrations, wherein the antibodies of the present invention may be administered before, simultaneously and/or after administration additional therapeutic agent or agents. In one embodiment of the present invention, administration of the anti-CTLA-4 antibody and administration of the additional therapeutic agent occur over a period of about one month, or for about one, two, or three weeks, or for every about one, two, three, four, five or six

- 47 045931 days. The antibodies of the present invention can also be used in combination with radiation therapy.

The antibody of the present invention (and any additional therapeutic agent) can be administered by any suitable route, including parenteral, intrapulmonary and intranasal administration, and, if necessary for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be accomplished by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. The present invention contemplates various dosing regimens, including, but not limited to, single or multiple administrations at different times, bolus administration and pulse infusions.

The antibodies of the present invention must be formulated, dosed and administered in accordance with good medical practice. Factors to be considered in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the particular patient, the cause of the disorder, the site of delivery of the agent, the route of administration, the schedule of administration and other factors known to the practitioner. The antibody need not be included in one or more agents currently used for the prevention or treatment of the disorder in question. The effective amount of such other agents depends on the amount of antibodies present in the formulation, the type of disorder or treatment, and other factors discussed above. They are typically used at the same dosages and routes of administration as described herein, or from about 1 to 99% of the dosages described herein, or at any dosage and route of administration that are empirically/clinically determined to be appropriate.

For the prevention or treatment of a disease, the appropriate dosage of an antibody of the present invention (when used alone or in combination with one or more or other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether antibody for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, as well as the opinion and decision 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, approximately 1 mcg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg) of antibody may be the initial recommended dose to administer to the patient, whether e.g. or more separate administrations or continuous infusion. One typical daily dose may vary from 1 mcg/kg to 100 mg/kg or more, depending on the factors mentioned above. With repeated administrations over several days or longer, depending on the condition, treatment is usually continued until the desired suppression of disease symptoms occurs. One typical dose of antibody may range from about 0.05 to about 10 mg/kg. Thus, the patient can be administered one or more doses of about 0.5, 2.0, 4.0 or 10 mg/kg (or any combination thereof). Such doses may be administered intermittently, for example, every week or every three weeks (for example, such that the patient receives from about two to about twenty or, for example, about six doses of the antibody). An initial higher loading dose may be administered followed by one or more lower doses. The progress of this therapy can be easily monitored using conventional methods and tests.

It will be appreciated that any of the above formulations or therapeutic methods can be carried out using the immunoconjugate of the present invention instead of or in addition to the anti-CTLA-4 antibody.

3. Products.

In another aspect, the present invention provides an article containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above. The manufactured article includes a container and a label or package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV bags, etc. Containers can be made from various materials such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective for the treatment, prevention and/or diagnosis of a condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a pierceable stopper hypodermic needle). At least one active ingredient in the composition is an antibody of the present invention. The label or package insert states that the composition is used to treat a specific condition. In addition, the article of manufacture may include (a) a first container containing a composition that contains an antibody of the present invention; and (b) a second container containing a composition containing an additional cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the present invention may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively or additionally, the article may further comprise a second (or third) cone

- 48 045931 container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. In addition, it may include other materials desirable from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.

It is understood that any of the above industrial products may include an immunoconjugate of the present invention instead of or in addition to an anti-CTLA-4 antibody.

Polypeptides containing a variant Fc region.

In one aspect of the present invention, isolated polypeptides containing a variant Fc region are described. In some entities, the polypeptides are antibodies. In some cases, the polypeptides are Fc fusion proteins. In certain embodiments of the present invention, the Fc region variants contain at least one amino acid residue change (e.g., substitution) compared to the corresponding sequence in the Fc region of the native sequence or a comparable sequence variant (all of which may be referred to as the original Fc region in the present invention ). The native sequence Fc region typically consists of a homodimer consisting of two identical polypeptide chains. The amino acid changes in the Fc region variants of the present invention can be introduced into either one of the two polypeptide chains of the original Fc region or into both polypeptide chains.

In some aspects of the present invention, Fc region variants are provided whose function is altered from that of the original Fc region. In some aspects, the Fc region variants of the present invention have increased Fcy receptor binding activity compared to the parent Fc region. In certain embodiments of the present invention, variants of the Fc regions of the present invention have increased binding activity to at least one Fcy receptor selected from the group consisting of FcYRIa, FcYRIIa, FcYRIIb and FcYRIIIa, compared to the parent Fc region. In some embodiments of the present invention, the Fc region variants of the present invention have increased FcYRIIa binding activity. In some embodiments of the present invention, the Fc region variants of the present invention have increased FcYRIIIa binding activity. In other embodiments of the present invention, the Fc region variants of the present invention have increased binding activity to FcYRIIa and FcYRIIIa.

In some embodiments of the present invention, the Fc region variants of the present invention comprise at least one amino acid change at at least one position selected from the group consisting of EU numbering positions 234, 235, 236, 298, 330, 332 and 334 . Alternatively, amino acid changes such as those described in WO 2013/002362 and WO 2014/104165 can be similarly used in the present invention.

In certain embodiments of the present invention, the binding activity of the original Fc region and variant Fc regions can be represented by a KD (dissociation constant) value. In one embodiment of the present invention, the ratio [KD value of the original Fc region for FcγRIIa]/[KD value of the variant Fc region for FcYRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more than, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more . In other embodiments of the present invention, FcYRIIa may be FcYRIIa R or FcYRIIa H, or both receptors. In one embodiment of the present invention, the ratio [binding activity of the original Fc region to FcγRIIIa]/[binding activity of the variant Fc region to FcYRIIIa] is equal to, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1 x 10 ³ or more, 2 x 10 ³ or more, 3 x 10 ³ or more, or 5 x 10 ³ or more . In other embodiments of the present invention, FcYRIIIa may be FcYRIIIa F or FcYRIIIa V, or both.

In one embodiment of the present invention, the KD values of Fc region variants for FcYRIIa are, for example, 1.0 x 10 ^-6 M or less, 5.0 x ^{10 -7} M or less, 3.0 x ^{10 -7} M or less, 2.0 x 10 ^{- 7} M or less, 1.0x10 ^-7 M or less, 5.0x10 ^-8 M or less, 3.0x10 ^-8 M or less, 2.0x10 ^-8 M or less, 1.0x10 ^-8 M or less, 5.0x10 ^-9 M or less, 3.0x10 ^-9 M or less, 2.0x10 ^-9 M or less, or 1.0x10 ^-9 M or less. In other embodiments of the present invention, FcYRIIa may be FcYRIIa R or FcYRIIa H, or both. In one embodiment of the present invention, the KD values of Fc region variants for FcYRIIa are, for example, 1.0 x 10 ^-6 M or less, 5.0 x ^{10 -7} M or less, 3.0 x ^{10 -7} M or less, 2.0 x 10 ^{- 7} M or less, 1.0x10 ^-7 M or less, 5.0x10 ^-8 M or less, 3.0x10 ^-8 M or less, 2.0x10 ^-8 M or less, 1.0x10 ^-8 M or less, 5.0x10 ^-9 M or less, 3.0x10 ^-9 M or less, 2.0x10 ^-9 M or less, 1.0x10 ^-9 M or less, 5.0x10 ^-10 M or less, 3.0x10 ^-10 M or less, 2.0x10 ^-10 M or less, or 1.0x10 ^-10 M or less. In other embodiments of the present invention, FcYRIIIa may be FcYRIIIa F or FcYRIIIa V, or both.

- 49 045931

In another embodiment of the present invention, the binding activity of the original and variant Fc regions can be represented by a kd (dissociation rate constant) value instead of a KD value.

In another embodiment of the present invention, the binding activity of the original and variant Fc regions can be represented by the amount of Fcy receptor binding per unit amount of Fc region. For example, in surface plasmon resonance analysis, the bound amount of Fc region immobilized on a sensor chip and the bound amount of Fcy receptor further bound thereto are measured as a resonance unit (RU). The value obtained by dividing the amount of Fcy receptor binding by the amount of Fc region binding can be determined as the amount of Fcy receptor binding per unit amount of Fc region. Specific methods for measuring and calculating such binding amounts are described in the examples below. In some embodiments of the present invention, the ratio [amount of variant Fc region bound to FcγRIIa]/[amount of parent Fc region bound to FcYRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 40 or more, or 50 or more. In some embodiments of the present invention, the ratio [amount of variant Fc region bound to FcγRIIIa]/[amount of parent Fc region bound to FcYRIIIa] 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, 1x 10 ³ or more, 2x 10 ³ or more, 3x 10 ³ or more, 5x 10 ³ or more.

In some embodiments of the present invention, KD values, kd values, binding amount values, and others described in the present invention are measured or calculated by performing surface plasmon resonance analysis at 25° C. or 37° C. (see, for example, Example 6 in this description).

In some aspects of the present invention, the variant Fc region of the present invention has improved selectivity for activating and inhibitory Fcy receptors compared to the parent Fc region. In other words, the binding activity of the Fc region variant of the present invention to the activating Fcy receptor is significantly higher than the binding activity to the inhibitory Fcy receptor compared to the parent Fc region. In certain embodiments of the present invention, the activating Fcy receptor is at least one Fcy receptor selected from the group consisting of FcYRIa, FcYRIIa R, FcYRIIa H, FcYRIIIa F, and FcYRIIIa V, and the inhibitory Fcy receptor is FcYRIIb. In some embodiments of the present invention, the Fc region variants of the present invention have improved selectivity among FcYRIIa and FcYRIIb. In some embodiments of the present invention, the Fc region variant of the present invention has improved selectivity among FcYRIIIa and FcYRIIb. In other embodiments of the present invention, the variant Fc region of the present invention has improved selectivity among FcYRIIa and FcYRIIb, as well as among FcYRIIIa and FcYRIIb.

In some embodiments of the present invention, the variants of the Fc regions of the present invention contain at least one amino acid change at at least one position selected from the group consisting of positions 236, 239, 268, 270 and 326 according to EU numbering. Alternatively, the amino acid changes described in WO 2013/002362 and WO 2014/104165 can be similarly used in the present invention.

In certain embodiments of the present invention, the binding activity of the parent Fc region and variants thereof can be represented by a KD (dissociation constant) value. Embodiments of the present invention regarding binding activity to FcYRIIa and FcYRIIIa are described above. In one embodiment of the present invention, the ratio [KD value of the original Fc region for FcγRIIb]/[KD value of the variant Fc region for FcYRIIb] 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 of the present invention, the binding activity of the original and variant Fc regions can be represented by a kd (dissociation rate constant) value instead of a KD value.

In another embodiment of the present invention, the binding activity of the parent Fc region and its variants may be represented by the above amount of Fcy receptor binding per unit amount of Fc region. In some embodiments of the present invention, the ratio [amount of variant Fc region bound to FcγRIIb]/[amount of parent Fc region bound to FcYRIIb] 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 of the present invention, the amount of Fc region variant binding to FcYRIIb 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

- 50 045931 less.

In some aspects of the present invention, the variant Fc region of the present invention has improved stability compared to the original Fc region. In certain embodiments of the present invention, the stability is thermodynamic stability. The thermodynamic stability of a polypeptide can be judged, for example, by using the Tm value as an indicator. Tm values can be measured using methods known to those skilled in the art, such as circular dichroism (CD), differential scanning calorimeter (DSC), and differential scanning fluorimetry (DSF). In one embodiment of the present invention, in an embodiment of the Fc region of the present invention, the Tm value of the CH2 region is increased by 0.1 degrees or more, 0.2 degrees or more, 0.3 degrees or more, 0.4 degrees or more, 0 .5 degrees or more, 1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees or more, or 10 degrees or more compared to the original Fc region.

In some embodiments of the present invention, the Fc region variants of the present invention contain at least one amino acid change at at least one position selected from the group consisting of positions 250 and 307 according to EU numbering. Alternatively, the amino acid changes described in WO 2013/118858 can be similarly used in the present invention.

In some aspects of the present invention, the variant Fc region of the present invention consists of two polypeptide chains with different sequences from each other. In a further aspect of the present invention, a variant of the Fc region of the present invention promotes heterodimerization between the first polypeptide and the second polypeptide. When a heterodimeric protein is produced by recombination, it is preferred that peptide chains that are different from each other are preferably associated to form a heterodimer, and non-identical polypeptide chains are preferentially associated to form a homodimer. Whether heterodimerization in an Fc region variant is beneficial or not can be judged, for example, by separating homodimers and heterodimers from the resulting Fc region variants using a method such as chromatography and by determining the proportion of each component.

In some embodiments of the present invention, the variants of the Fc regions of the present invention contain at least one amino acid change at at least one position selected from the group consisting of positions 349, 356, 366, 368, 407 and 439 in accordance with EU numbering . Alternatively, the amino acid changes described in WO 2006/106905 and WO 1996/027011 can be similarly used in the present invention.

In some aspects of the present invention, the Fc region variant of the present invention has increased FcRn binding activity at acidic pH. In some embodiments of the present invention, an acidic pH means a pH value between 4.0 and 6.5. In other embodiments of the present invention, the acidic pH is at least one value 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 embodiments of the present invention, the acidic pH is pH 5.8.

In some embodiments of the present invention, the variants of the Fc regions of the present invention contain at least one amino acid change at at least one position selected from the group consisting of positions 428, 434, 436, 438 and 440 according to EU numbering. Alternatively, the amino acid changes described in WO 2016/125495 can be similarly used in the present invention.

In one aspect of the present invention, a variant Fc region of the present invention contains at least one amino acid change at at least one position selected from the group consisting of positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, 407, 428, 434, 436, 438, 439 and 440 according to EU numbering.

In some aspects of the present invention, the variant Fc region of the present invention contains amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326 and 334 according to EU numbering. In yet another aspect, the variant Fc region comprises amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, consistent with EU numbering in the first polypeptide of the original Fc region, and (ii) positions 236, 250, 270, 298, 307, 326 and 334 according to EU numbering in the second polypeptide of the original Fc region.

In another aspect of the present invention, the Fc region variant of the present invention further comprises an amino acid change at position 332 in accordance with the EU numbering. In yet another aspect of the present invention, the variant Fc region comprises an amino acid change at position 332 consistent with EU numbering in the first polypeptide of the original Fc region.

In another aspect of the present invention, the Fc region variant of the present invention further comprises an amino acid change at position 356 in accordance with the EU numbering. In yet another aspect of the present invention, the Fc region variant comprises an amino acid change in polo

- 51 045931 gene 356 in accordance with the EU numbering in the first polypeptide of the original Fc region.

In another aspect of the present invention, the Fc region variant of the present invention further comprises an amino acid change at position 366 in accordance with the EU numbering. In yet another aspect of the present invention, the variant Fc region comprises an amino acid change at position 366 corresponding to EU numbering in the first polypeptide of the original Fc region.

In another aspect of the present invention, the Fc region variant of the present invention further comprises an amino acid change at position 349 in accordance with the EU numbering. In yet another aspect of the present invention, the variant Fc region comprises an amino acid change at position 349 consistent with EU numbering in the first polypeptide of the original Fc region.

In another aspect of the present invention, the Fc region variant of the present invention further comprises an amino acid change at position 332 in accordance with the EU numbering. In yet another aspect of the present invention, the variant Fc region comprises an amino acid change at position 332 consistent with EU numbering in the second polypeptide of the original Fc region.

In another aspect of the present invention, the Fc region variant of the present invention further comprises an amino acid change at position 330 in accordance with the EU numbering. In yet another aspect of the present invention, the variant Fc region comprises an amino acid change at position 330 consistent with EU numbering in the second polypeptide of the original Fc region.

In another aspect of the present invention, the Fc region variant of the present invention further comprises an amino acid change at position 439 in accordance with the EU numbering. In yet another aspect of the present invention, the variant Fc region comprises an amino acid change at position 439 consistent with EU numbering in the second polypeptide of the original Fc region.

In another aspect of the present invention, the variant Fc region of the present invention further comprises amino acid changes at positions 366, 368 and 407 in accordance with EU numbering. In yet another aspect of the present invention, the variant Fc region comprises amino acid changes at positions 366, 368 and 407 in accordance with the EU numbering in the second polypeptide of the original Fc region.

In another aspect of the present invention, the Fc region variant of the present invention further comprises an amino acid change at position 356 in accordance with the EU numbering. In yet another aspect of the present invention, the variant Fc region comprises an amino acid change at position 356 consistent with EU numbering in the second polypeptide of the original Fc region.

In some aspects of the present invention, the Fc region variants of the present invention contain amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 334, 349, 356, 366, 368, and 407 by numbering EU. In yet another aspect of the present invention, the variant Fc region comprises amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349 and 366 in accordance with the EU numbering in the first polypeptide of the original Fc region and at (ii) positions 236, 250, 270, 298, 307, 326, 334, 356, 366, 368 and 407 according to EU numbering in the second polypeptide of the original Fc region.

In some aspects of the present invention, the Fc region variants of the present invention comprise amino acid changes at EU numbering positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 334, 349, 356, and 439. In other aspects of the present invention, the variant Fc region comprises amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326 and 356, consistent with EU numbering in the first polypeptide of the original Fc region and at (ii) positions 236, 250, 270, 298, 307, 326, 334 and 439 according to EU numbering in the second polypeptide of the original Fc region.

In some aspects of the present invention, the Fc region variants of the present invention contain amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368 and 407 according to EU numbering. In other aspects of the present invention, the variant Fc region comprises amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349 and 366 in accordance with the EU numbering in the first polypeptide of the original Fc -region and at (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 356, 366, 368 and 407 according to EU numbering in the second polypeptide of the original Fc region.

In some aspects of the present invention, the Fc region variants of the present invention contain amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 356 and 439 according to EU numbering. In other aspects of the present invention, the variant Fc region comprises amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326 and 356, consistent with EU numbering in the first polypeptide of the original Fc region and at (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, 334 and 439 according to EU numbering in the second polypeptide of the original F region.

In some aspects of the present invention, the Fc region variants of the present invention contain amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 349, 356, 366, 368, and 407 according to EU numbering. In other aspects of the present invention, the variant Fc region comprises amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 349 and 366, consistent with EU numbering in the first polypeptide of the original Fc -areas and in (ii)

- 52 045931 positions 236, 250, 270, 298, 307, 326, 332, 334, 356, 366, 368 and 407 according to EU numbering in the second polypeptide of the original Fc region.

In some aspects of the present invention, the Fc region variants of the present invention contain amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 356 and 439 according to EU numbering. In other aspects of the present invention, the variant Fc region contains amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332 and 356 in accordance with the EU numbering in the first polypeptide of the original Fc -region and at (ii) positions 236, 250, 270, 298, 307, 326, 332, 334 and 439 according to EU numbering in the second polypeptide of the original F region.

In some aspects of the present invention, the Fc region variants of the present invention contain amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 366, 368 and 407 according to numbering EU. In other aspects of the present invention, the Fc region variant comprises amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 366 in accordance with the EU numbering in the first polypeptide of the original Fc region and in (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 366, 368 and 407 according to EU numbering in the second polypeptide of the parent Fc region.

In some aspects of the present invention, the Fc region variants of the present invention contain amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 366, 368 and 407 according to EU numbering. In other aspects of the present invention, the variant Fc region comprises amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332 and 366, consistent with EU numbering in the first polypeptide of the original Fc -region and at (ii) positions 236, 250, 270, 298, 307, 326, 332, 334, 366, 368 and 407 according to EU numbering in the second polypeptide of the original Fc region.

In some aspects of the present invention, the Fc region variants of the present invention contain amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368 and 407 according to EU numbering. In other aspects of the present invention, the variant Fc region comprises amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349 and 366 in accordance with the EU numbering in the first polypeptide of the original Fc -region and at (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 356, 366, 368 and 407 according to EU numbering in the second polypeptide of the original Fc region.

In some aspects of the present invention, the Fc region variants of the present invention contain amino acid changes at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 349, 356, 366, 368, and 407 according to EU numbering. In other aspects of the present invention, the variant Fc region comprises amino acid changes at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 349 and 366, consistent with EU numbering in the first polypeptide of the original Fc -region and at (ii) positions 236, 250, 270, 298, 307, 326, 332, 334, 356, 366, 368 and 407 according to EU numbering in the second polypeptide of the original Fc region.

In other embodiments of the present invention, the Fc region variants of the present invention contain at least one amino acid change selected from the group consisting of: (i) Tyr or Phe at position 234, Gln at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Glu at position 332, Cys at position 349, Lys at position 356 and Trp at position 366, according to the EU numbering in the first polypeptide of the original Fc region; and (ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met or Lys at position 330, Asp or Glu at position 332, Glu at position position 334, Cys at position 356, Ser at position 366, Ala at position 368, Val at position 407 and Glu at position 439, according to EU numbering, in the second polypeptide of the original Fc region.

In other aspects, variants of the Fc regions of the present invention further comprise any of the amino acid changes (a)-(d) below:

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

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

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

In other embodiments, the present invention provides polypeptides comprising the amino acid sequence of any of SEQ ID NOs: 43-46, 65, 66, 81, 207, 239, 253-271, 276, 277, and 278.

The designation Fcy receptors (herein referred to as Fcy, FcyR, or FcgR receptors) refers to receptors that can bind to the Fc region of monoclonal antibodies IgG1, IgG2, IgG3, and IgG4, and virtually any member of the family of proteins encoded by the genes

- 53 045931 Fcy receptor. In humans, this family includes FcyRI (CD64), including the FcYRIa, FcYRIb, and FcyRIc isoforms; FcyRII (CD32), including isoforms FcYRIIa (including allotypes H131 (type H) and R131 (type R)), FcYRIIb (including FcYRIIb-1 and FcYRIIb-2) and FcyRIIc; and FcyRIII (CD16), including but not limited to the FcYRIIIa (including allotypes V158 and F158) and FcYRIIIb isoforms (including the FcYRIIIb-NA1 and FcYRIIIb-NA2 allotypes), and any human FcyR receptors, FcyR isoforms or allotypes yet to be discovered . FcYRIIb1 and FcYRIIb2 have been reported to be splice variants of human FcYRIIb. In addition, a splice variant called FcYRIIb3 has been reported (J Exp Med 1989, 170:1369-1385). In addition to these splice variants, human FcYRIIb includes all splice variants reported in NCBI, namely NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1 and NP_003992.3. In addition, human FcYRIIb includes all previously described genetic polymorphisms, as well as FcYRIIb (Arthritis Rheum. 2003, 48:3242-3252); Kono et al., Hum. Mol. Genet. 2005, 14:2881-2892; and Kyogoju et al., Arthritis Rheum. 2002, 46:1242-1254), and every genetic polymorphism that will be known in the future.

There are two allotypes of FcYRIIa: one in which the amino acid at position 131 of FcYRIIa is a histidine (type H), and the other in which the amino acid at position 131 is replaced by arginine (type R) (Warrmerdam, J. Exp. Med. 1990, 172: 19-25 ).

FcyR includes, but is not limited to, human, mouse, rat, rabbit and monkey-derived FcyR receptors and can be derived from any organism. Mouse FcyRs include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FcyRIII-2 (CD16-2) and any mouse FcyRs or FcyR isoforms.

The amino acid sequence of human FcyRI is shown in SEQ ID NO: 131 (NP_000557.1); the amino acid sequence of human FcYRIIa is provided 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 FcYRIIb is provided in SEQ ID NO: 151 (AAI46679.1), SEQ ID NO: 169 or SEQ ID NO: 172; the amino acid sequence of human FcYRIIIa is provided 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 FcYRIIIb is shown in SEQ ID NO: 178 (AAI28563.1).

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

The designation "original Fc region" in the present invention refers to the Fc region prior to the introduction of the amino acid change(s) described in the present invention. In some embodiments of the present invention, the original Fc region means the Fc region of the native sequence (or the Fc region of the native antibody). The antibody includes, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4) and IgM. The antibody may be obtained from a human or a monkey (eg, cynomolgus, rhesus, monkey, chimpanzee, or baboon). The native antibody may include naturally occurring mutations. A variety of allotypic IgG sequences due to genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH publication No. 91-3242, and any of them can be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356 to 358 (EU numbering) can be either DEL or EEM. In certain embodiments of the present invention, 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 of the present invention, the parent Fc region is an Fc region derived from the heavy chain constant region of SEQ ID NO: 82 or SEQ ID NO: 158. In other embodiments of the present invention, the parent Fc region may be an Fc region , obtained by adding amino acid change(s) other than the amino acid change(s) described in the present invention to the Fc region of the native sequence (Fc region of the reference sequence variant). The Fc region of the native sequence is usually a homodimer consisting of two identical polypeptide chains.

Additionally, amino acid changes made for other purposes can be combined with

- 54 045931 a variant of the Fc region described in the present invention. For example, substitutions may be added that improve FcRn binding activity (Hinton et al., J. Immunol. 2006, 176(1):346356; Dall'Acqua et al., J. Biol. Chem. 2006, 281(33) ): 23514-23524; Petkova et al., Intl. Immunol. 2006, 18(12): 1759-1769; Zalevsky et al., Nat. Biotechnol. 2010, 28(2): 157-159; WO 2006/019447 ; WO 2006/053301; WO 2009/086320), and amino acid substitutions to improve heterogeneity or stability of antibodies (WO 2009/041613). In another embodiment, polypeptides capable of promoting antigen clearance, which are described in WO 2011/122011, WO 2012/132067, WO 2013/046704 or WO 2013/180201, polypeptides with the property of specific binding to a target tissue, which are described in WO 2013/180200 , polypeptides with the property of multiple binding to multiple antigen molecules, which are described in WO 2009/125825, WO 2012/073992 or WO 2013/047752, can be combined with the Fc region variant described in the present invention. In another embodiment, in order to impart binding ability to other antigens, the amino acid changes described in EP 1752471 and EP 1772465 can be combined into the CH3 variant Fc region described in the present invention. In another embodiment, in order to increase plasma retention, amino acid changes that reduce the pI of the constant region (WO 2012/016227) can be combined with the Fc region variant described in the present invention. Alternatively, amino acid changes that increase the pI constant region (WO 2014/145159) can be combined with the Fc region variant described herein to promote cellular uptake. Alternatively, in order to promote elimination of a target molecule from plasma, amino acid changes that increase the pI of the constant region (WO 2016/125495) can be combined into a variant Fc region described in the present invention. In one embodiment of the present invention, such a change may include, for example, replacing at least one provision selected from the group consisting of provisions 311, 343, 384, 399, 400 and 413 according to the EU numbering. In another embodiment of the present invention, such a substitution may be an amino acid substitution of Lys or Arg at each position.

In addition, methods for producing heterodimerized antibodies that use the association of CH1 and CL antibodies and the association of VH and VL, which are described in WO 2011/028952, can also be used.

As with the method described in WO 2008/119353 and WO 2011/131746, it is also possible to use a method for producing heterodimerized antibodies by first preparing two types of homodimerized antibodies, incubating the antibodies under reducing conditions to dissociate them, and then re-associating them.

In addition, as with the method described in WO 2012/058768, a method for producing heterodimerized antibodies by adding changes to the CH2 and CH3 domains can also be used.

When two Fc region variant-containing polypeptides that have different amino acid sequences are co-expressed to produce polypeptides containing heterologous Fc region variants, polypeptides containing homologous Fc region variants are also typically produced as impurities. In such cases, polypeptides containing heterologous Fc region variants can be efficiently obtained by separating and purifying them from polypeptides containing homologous Fc region variants using known techniques. A method for efficiently separating and purifying heterodimerized antibodies from homodimerized antibodies using ion exchange chromatography by introducing amino acid changes into the variable regions of two types of antibody heavy chains to create differences in isoelectric points between homodimerized antibodies and heterodimerized antibodies has been reported (WO 2007/114325). Another method for purifying heterodimerized antibodies using Protein A chromatography has been reported by constructing a heterodimerized antibody containing two types of heavy chains derived from mouse IgG2a, which binds to Protein A, and rat IgG2b, which does not bind to Protein A.

Another method for purifying heterodimerized antibodies using Protein A chromatography has been reported by constructing a heterodimerized antibody containing two types of heavy chains derived from mouse IgG2a, which binds to Protein A, and rat IgG2b, which does not bind to Protein A (WO 1998/050431 and WO 1995/033844).

In addition, a heterodimerized antibody can be efficiently purified by Protein A chromatography by replacing amino acid residues at positions 435 and 436 (EU numbering), which are located in the Protein A binding site of the antibody heavy chain, with amino acids such as Tyr or His to produce a different binding affinity for protein A.

In the present invention, an amino acid change means any substitution, deletion, addition, insertion and modification, or a combination thereof. In the present invention, an amino acid change can be rephrased as an amino acid mutation.

The number of amino acid changes introduced into the Fc region is not limited. In certain embodiments of the present invention, there 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 more

- 55 045931 her, 18 or less or 20 or less.

In one aspect, the present invention relates to methods for producing a polypeptide containing a variant Fc region. In further aspects, the present invention provides methods for producing a polypeptide containing a variant Fc region whose function has been altered. In some entities, the polypeptides are antibodies. In some cases, the polypeptides are Fc fusion proteins. In certain embodiments of the present invention, these methods include introducing at least one amino acid substitution into the original Fc region. In certain embodiments of the present invention, these methods include: (i) producing polypeptide(s) comprising the original Fc region; and (ii) introducing at least one amino acid change in the original Fc region. In certain embodiments of the present invention, these methods may further include (iii) measuring the function of the polypeptide(s) containing the Fc region variant. The native Fc region usually consists of two identical polypeptide chains. Amino acid changes in the original Fc region can be introduced into either one of the two polypeptide chains of the original Fc region, or into both of the two polypeptide chains.

In another embodiment of the present invention, a method for producing polypeptide(s) containing a variant Fc region comprises: (i) producing one or more nucleic acids encoding polypeptides containing the original Fc region; (ii) introducing at least one mutation into the region(s) encoding the original Fc region in the nucleic acids; (iii) introducing the nucleic acids obtained in (ii) into a host cell; and (iv) culturing the cell described in (iii) such that the polypeptide(s) containing the Fc region variant are expressed. In certain embodiments of the present invention, the above methods may further comprise (v) isolating the polypeptide(s) containing the Fc region variant from the host cell culture described in (iv).

In certain embodiments of the present invention, the nucleic acids obtained in (ii) may be included in one or more vectors (eg, expression vectors).

In some embodiments of the present invention, the amino acid changes used in the production methods of the present invention are selected from any of the single amino acid changes that may be contained in the above-mentioned Fc region variants, combinations of single changes, or combined changes listed in Table. 26-30.

The Fc region can be obtained by re-eluting the fraction adsorbed on the Protein A column after partial digestion of monoclonal antibodies IgG1, IgG2, IgG3, IgG4 or the like using a protease such as pepsin. A protease is not particularly limited if it can cleave a full-length antibody such that Fab and F(ab')2 are produced in a limited manner by appropriately setting enzymatic reaction conditions such as pH, examples being pepsin and papain.

Polypeptides containing the Fc region variant of the present invention can be produced by other methods known in the art, in addition to the above production methods. Polypeptides comprising a variant Fc region produced by the production methods described herein are also included in the present invention.

The assay methods described in the present invention, or various measurement methods known in the art, can be used to identify or screen F region variants provided in the present invention, or to elucidate their physical or chemical properties or biological activity.

Assays for determining the binding activity of a polypeptide containing an Fc region variant to one or more FcR family receptors are described in the present invention or otherwise known in the art. Such binding assays include, but are not limited to, surface plasmon resonance analysis, Amplified Luminescent Proximity Homogeneous Assay (ALPHA) screening, ELISA, and fluorescence activated cell sorting (FACS) (Lazar et al., Proc. Natl. Acad. Sci. USA, 2006, 103(11): 4005-4010).

In one embodiment of the present invention, the binding activity of a polypeptide containing an Fc region variant to members of the FcR family can be measured using a surface plasmon resonance assay. For example, various FcRs as test compounds are reacted with a polypeptide containing a variant Fc region immobilized or captured on a sensor chip using known methods and reagents (eg, protein A, protein L, protein A/G, protein G, antibody with anti-A chain, antibodies with anti-chain, antigenic peptides and antigenic proteins). In another embodiment, FcRs can be immobilized or captured on a sensor chip, and a polypeptide containing a variant Fc region can be used as an analyte. As a result of such interactions, binding sensorgrams are obtained and, by analyzing them, the values of the dissociation constants (KD) of such bindings can be calculated. Moreover, the difference in the resonance unit (RU) value in the sensorgrams before and after interaction with FcR (i.e., the amount of FcR binding) can be used as an indicator of the binding activity of the polypeptide,

- 56 045931 including a variant of the Fc region to FcR. In addition, the adjusted value obtained by dividing the above amount of FcR binding by the difference in RU values in the sensorgrams before and after immobilization or capture of the Fc region variant-containing polypeptide on the sensor chip (i.e., the amount of Fc region variant-containing polypeptide ) (that is, the correction value is the amount of FcR binding per unit amount of polypeptide containing the Fc region variant) can be used as an indicator of binding activity.

In another aspect, the present invention provides pharmaceutical compositions containing a polypeptide comprising a variant Fc region as described herein. In one embodiment of the present invention, the above pharmaceutical compositions further contain pharmaceutically acceptable carriers.

Examples.

Shown below are examples of methods and compositions of the present invention. It should be appreciated that various other embodiments may be implemented in light of the general description mentioned above.

Example 0: A switch antibody concept that exhibits 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, antibody-dependent cytotoxic activity (ADCC activity) on CTLA4-expressing T cells has recently been reported to be important, and clearance of regulatory T cells in tumors and ADCC activity have been found to be important mechanisms of action for the antitumor effect of anti-CTLA4 antibodies.

In addition, it is known that the ADCC activity of IgG1 antibodies results from the induction of cytotoxic activity due to the binding of the antibody constant region to the FcyR of NK cells and macrophages, and antibodies with a constant region modified to enhance such binding induce a stronger cytotoxic effect, manifested as an antitumor effect .

On the other hand, the removal of regulatory T cells from the entire body has been reported to induce a systemic response similar to an autoimmune disease, and regulating the balance between cytotoxic activity to produce an antitumor effect and the systemic response is considered to be a very important task.

More specifically, the antibody is expected to exhibit more potent cytotoxic activity and be able to suppress systemic responses, provided that the antibody can tightly bind to regulatory T cells or exhausted T cells in the cancer microenvironment, exert a potent antitumor effect by removing regulatory T cells. cells or exhausted T cells for cytotoxic activity, and limit the response only to the cancer microenvironment. Antibodies with this mechanism of action have not yet been reported. Therefore, herein, we actually created and tested antibodies (CTLA4 switch antibodies) that act against CTLA4 only locally in the tumor and contain a constant region with enhanced binding to FcyRs expressed on NK cells and macrophages.

Example 1: Generation of antibodies that bind to antigens in the presence of ATP or its metabolite from a naive library and a rationally designed antibody library using phage display techniques.

Example 1-1. Preparing antigens to produce antibodies that bind to the antigen in the presence of a small molecule.

The biotinylated extracellular region of mouse CTLA4 (mCTLA4), the extracellular region of human CTLA4 (hCTLA4) and abatacept protein are obtained as antigens. Specifically, regarding the extracellular region of hCTLA4, the hCTLA4-His-BAP gene (SEQ ID NO: 1) was synthesized in which a His tag and a BAP tag are fused to the C-terminus of the extracellular region of hCTLA4, which are inserted into an animal expression plasmid. The antigen protein is expressed and purified using the following method. The prepared plasmid is introduced by lipofection into the FreeStyle 293-F cell line obtained from human embryonic kidney cells (Invitrogen), and the cells are seeded in a flask after suspension in FreeStyle 293 expression medium (Invitrogen) at a cell density of 1.33/106 cells/ml. Three hours after the introduction of the plasmid, biotin is added to a final concentration of 100 μM, cultivated in an incubator in a CO ₂ atmosphere (37 ° C, 8% CO ₂ , 125 rpm) for 4 days, and the antigen is purified from the culture supernatant according to a well-known method specialists in this field. The absorbance of the purified antigen solution at 280 nm is measured using a spectrophotometer. The concentration of the purified antigen is calculated from the obtained value using the extinction coefficient calculated by the PACE method (Protein Science, 1995, 4: 2411-2423). On the other hand, mCTLA4-His (Sino Biologies Inc. 50503-M08H, catalog number NP_033973.2), in which the His tag is fused to the extracellular region of mCTLA4, and abatacept (Alfresa Corporation), in which

- 57 045931 human IgG1 stant region fused to hCTLA4, biotinylated by amine coupling (PIERCE, catalog number 21329).

Example 1-2. Generation of antibodies that bind to mouse CTLA4 in the presence of small molecules from a naïve human antibody library by bead panning.

A human antibody phage display library consisting of a plurality of phages that represent different Fab domains of human antibody sequences is constructed in accordance with a method known to those skilled in the art using poly-A RNAs derived from human PBMCs as templates. , commercially available human poly-A RNA, etc.

From a constructed naïve human antibody phage display library, antibodies were selected whose binding activity to the extracellular region of mouse CTLA4 (mCTLA4) was altered in the presence and absence of the small molecule. More specifically, phages are collected that present antibodies that exhibit mCTLA4 binding activity and are captured on beads in the presence of a small molecule. Phages were isolated from phage eluate eluted from beads in the absence of the small molecule. In this production method, biotin-tagged mCTLA4 (mCTLA4-HisBiotin) was used as the antigen.

Phages obtained from E. coli cells carrying an engineered phagemid for phage display are purified using a general procedure. A phage library solution dialyzed against TBS is then obtained. Panning is carried out using antigen immobilized on magnetic beads. The magnetic beads used are NeutrAvidin-coated beads (Sera-Mag SpeedBeads, NeutrAvidin-coated beads) or streptavidin-coated beads (Dynabeads M-280 Streptavidin).

To efficiently produce a small molecule-dependent small molecule switch antibody that can act as a switch in cancerous tissues, panning was performed by referring to the method described in prior patent literature WO 2013/180200. This panning method enriches antibodies that bind to antigen in the presence of adenosine 5'-triphosphate (ATP) and ATP metabolites, but do not bind to antigen in the absence of ATP.

Example 1-3. Assessment of binding activity in the presence and absence of a small molecule using phage ELISA.

From single colonies of E. coli obtained in example 1-2, a culture supernatant containing phage is obtained in accordance with the generally accepted method (Methods Mol. Biol. 2002, 178:133-145). Culture supernatants obtained using NucleoFast 96 (MACHERY-NAGEL) are subjected to ultrafiltration. 100 μl of the supernatant of each culture is added to each well of NucleoFast 96 and centrifuged at 4500 g for 45 min to remove the duct. Add 100 µl of H ₂ O and wash again by centrifugation at 4500 g for 30 minutes. Then 100 μl of TBS is added and the mixture is left at room temperature for 5 min, after which the phage solutions contained in the supernatants are removed.

Purified phages to which TBS was added were analyzed by ELISA using the following procedure. A StreptaWell 96 microtiter plate (Roche) is coated with 100 μl of TBS containing mCTLA4-His-biotin overnight. After washing each well with TBST to remove mCTLA4-His-biotin not bound to the plate, the wells are blocked with 250 μl of 2% skim milk-TBS for 1 h or longer. After removing the 2% skim milk-TBS, prepared purified phages are added to each well and the plate is allowed to stand at room temperature for 1 hour, allowing the antibody-presenting phages to bind to the mCTLA4-His-biotin present in each well with or without 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 is added and the plate is incubated for 1 hour. After washing with TBST or ATP/TBST, color development reaction occurs. solution in each well to which one TMB solution (ZyMED) was added is stopped by adding sulfuric acid, and then the color change is measured by absorbance at 450 nm. The results confirmed multiple antibodies that bound mCTLA4 only in the presence of ATP. The phage ELISA results are shown in Table. 2. In this case, clones having an absorption coefficient higher than 0.2 in the presence of ATP are defined as positive, and clones having an absorption coefficient higher than 2 in the presence/absence of ATP are defined as positive clones possessing ATP-dependent antigen-binding ability (switching clones ). In this example, SM can be used as an acronym for small molecule such as ATP.

table 2

Total Number of ELISA-treated clones 192 Number of positive clones (absorbance >0.2) 103 Number of switching clones (SM± ratio>2) 28

Example 1-4. Producing antibodies that bind to an antigen in the presence of a small molecule

- 58 045931 from the library of rational design using ATP or its metabolite.

From a phage display library with rational antibody design generated in prior practice (WO 2015/083764), antibodies exhibiting antigen binding activity in the presence of ATP or an ATP metabolite (e.g. ADP, AMP, adenosine (ADO), etc.) were received. To obtain antibodies, phages presenting antibodies that demonstrate the ability to bind to antigen captured on beads in the presence of ATP or an ATP metabolite are collected, and the phages are then isolated from the bead elute in the absence of ATP or an ATP metabolite.

Phages are produced in the usual manner from E. coli carrying an engineered phagemid for phage display. A phage library solution is prepared by diluting the phage population with TBS, precipitated by adding 2.5 M NaCl/10% PEG to the E. coli culture solution in which the phages were obtained. BSA is then added to the phage library solution to a final concentration of 4%. Panning is carried out using antigen immobilized on magnetic beads. The magnetic beads used are NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated beads) or streptavidin-coated beads (Dynabeads M-280 Streptavidin). Biotinylated abatacept (Abatacept-Biotin) is used as an antigen.

To efficiently produce a small molecule-dependent small molecule antibody switch that can act as a switch in cancer tissue, panning to enrich antibodies that bind antigen in the presence of adenosine 5'-triphosphate (ATP) or an ATP metabolite, and do not bind antigen in the absence ATP or ATP metabolite is carried out with reference to the method shown in previous patent literature (WO 2015/083674).

Example 1-5. Evaluation of binding activity in the presence and absence of ATP or its metabolite using phage ELISA.

From single colonies of E. coli obtained as described above, a culture supernatant containing phage is obtained in accordance with the conventional method (Methods Mol. Biol. 2002, 178:133-145). The recovered culture supernatant was ultrafiltrated using NucleoFast 96 (MACHEREY-NAGEL). The duct is removed by centrifugation (4500 g, 45 minutes) NucleoFast 96 with the addition of 100 μl of culture supernatant to each well. NucleoFast 96 with the addition of 100 μl of H ₂ O to each well is washed again by centrifugation (4500 g, 30 min). Finally, 100 μl of TBS is added to obtain a phage solution contained in the supernatant of each NucleoFast 96 well, which is left at room temperature for 5 minutes.

TBS or TBS containing ATP or its metabolite (SM/TBS) is added to the purified phages, and the phages are subjected to ELISA analysis using the following procedure. A StreptaWell 96 microtiter plate (Roche) is coated overnight with 100 μl of TBS containing the biotin-labeled antigen (abatacept-biotin) prepared in Example 1-1. After free abatacept-biotin is removed by washing each well of the plate with TBST, the wells are blocked with 250 μl of 2% skim milk-TBS for 1 hour or longer. After removing the 2% skim milk-TBS, prepared purified phages are added to each well and the plate is left at 37°C for 1 hour, allowing the antibody-presenting phages to bind to the abatacept-biotin present in each well in the absence or presence of ATP or its metabolite. After each well of the plate is washed with TBST or TBST containing ATP or its metabolite (SM/TBST), HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS or SM/TBS is added and the plate is incubated. 1 hour

After washing with TBST or ATP/TBST, the color development reaction of the solution in each well to which one TMB solution (ZyMED) was added was stopped by adding sulfuric acid, and then the color change was measured by absorbance at 450 nm. The results confirmed multiple antibodies that bound mCTLA4 only in the presence of ATP. The results of the phage ELISA are shown in Table. 3. In this case, clones having an absorption coefficient above 2 in the presence of ATP are defined as positive, and clones having an absorption coefficient above 2 in the presence/absence of ATP or its metabolite are defined as clones having antigen-binding ability, which depends on ATP or its metabolite (clone switching).

Table 3

4th test 5th test Total Number of ELISA-treated clones 288 288 576 Number of positive clones (S/N >2) 157 159 316 4th test 5th test Total Number of switching clones (SM± ratio>2) 6 16 22

- 59 045931

Example 1-6. Analysis of the sequence of switching antibodies, the antigen-binding activity of which changes depending on the presence or absence of ATP and its metabolites.

Nucleotide sequences of genes amplified using specific primers lacPF (SEQ ID NO: 2) and GlseqR (SEQ ID NO: 3) from clones with antigen binding activity, subject to the presence of ATP and its metabolites as a result of phage ELISA analysis. As a result of the analysis, clones ABADh11-4_020, ABADh11-4086, ABADh124_014, ABADh12-5_001, ABADh12-5_046 and ABADh5-5_041 were obtained, which were found to have binding activity with biotin-labeled abatacept in the presence of ATP and its metabolites. The clone names were renamed and became ABAM001, ABAM002, ABAM003, ABAM004, ABAM005 and ABAM006, respectively (Table 4).

Table 4

Clones Antibodies VH SEQ ID NO VL SEQ ID NO ABADhll-4 020 ABAM001 4 5 ABADhll-4 086 ABAM002 6 7 ABADhl2-4 014 ABAM003 8 9 ABADhl2-5 001 ABAM004 10 eleven ABADhl2-5 046 ABAM005 12 13 ABADh5-5 041 ABAM006 14 15

Example 1-7. Expression and purification of switch antibodies whose antigen-binding activity varies depending on the presence and absence of ATP and its metabolites.

The genes encoding the variable regions ABAM001, ABAM002, ABAM003, ABAM004, ABAM005 and ABAM006, obtained from a rationally designed human phage library, are inserted into a human IgG1/Lambda animal expression plasmid. Antibodies are expressed using the following method. The resulting plasmid is introduced by lipofection into the FreeStyle 293-F cell line, obtained from human embryonic kidney cells (Invitrogen), which was suspended in the FreeStyle 293 expression medium (Invitrogen) at a cell density of 1.33 x 106 cells/ml, and seeded 3 ml/well in each well of 6-well plates. Antibodies are purified from culture supernatant cultured in a CO ₂ incubator (37°C, 8% CO ₂ , 90 rpm) for 4 days using rProtein A Sepharose™ Fast Flow medium (Amersham Biosciences) by a method known in the art. in this area. The absorbance of purified antibody solutions at 280 nm is measured using a spectrophotometer. From the obtained measured values, the concentration of purified antibodies is calculated using the extinction coefficient calculated by the PACE method (Protein Science, 1995, 4: 2411-2423).

Example 1-8. Evaluation of the binding activity of the obtained antibodies against hCTLA4 in the presence and absence of AMPs using IgG ELISA.

The resulting six antibodies ABAM001, ABAM002, ABAM003, ABAM004, ABAM005 and ABAM006 were analyzed by IgG ELISA. The buffers shown in table. 5, Prepare properly. Biotin-labeled human CTLA4 (hCTLA4-His-Biotin) is used as the antigen.

Table 5

Buffer Compound Wash buffer TSB, 0.1% Tween20 Blocking buffer TSB, 2% BSA Sample Buffer TSB, 1 mM AMP

First, a StreptaWell 96 microtiter plate (Roche) is coated with 100 μl of TBS containing hCTLA4-His-biotin at room temperature for 1 hour or longer. After each well of the plate is washed with wash buffer to remove hCTLA4-His-biotin not bound to the plate, the wells are blocked with 250 μl of blocking buffer for 1 hour or longer. To each well from which blocking buffer has been removed, add 100 μl of each purified IgG at a final concentration of 2.5 μg/ml in sample buffer containing AMP at a final concentration of 1 mM, and the plate is left at room temperature for 1 h. , 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, the plate containing HRP-conjugated anti-human IgG antibody (BIOSOURCE) diluted in sample buffer was added to each well for 1 hour. After washing with wash buffer, containing a small molecule, the color change reaction of the solution in each well to which the TMB solution (ZyMED) was added was stopped by adding sulfuric acid, and then the color development was measured by absorbance at 450 nm. Buffers containing the compositions shown in table were used as buffers. 5.

The measurement results are presented in table. 6. Crowded holes are given a score of 5.00. The results show that for all clones ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, the uptake in the absence of AMP was significantly lower than in the presence of AMP. This re

- 60 045931 The result confirms that all clones ABAM001, ABAM002, ABAM003, ABAM004, ABAM005 and ABAM006 have the property of altering antigen binding depending on the presence or absence of small molecules.

Table 6

Antibodies Absorbance at 450 nm Presence of a small molecule Lack of small molecule S/N ABAM001 5,000 3.455 1.45 ABAM002 5,000 0.139 35.97 ABAM003 5,000 3,180 1.57 ABAM004 5,000 0.643 7.78 ABAM005 0.303 0.069 4.46 ABAM006 2,995 0.776 3.86

Will accept 1-9. Evaluation of the effect of ATP and its metabolites on binding to human CTLA4 by surface plasmon resonance.

ABAM004 is further evaluated as a CTLA4 switch antibody.

Antigen-antibody interaction between ABAM004 and hCTLA4-His-BAP was analyzed using Biacore T200 (GE Healthcare). After ABAM004 is captured by the CM5 sensor chip (GE Healthcare), on which protein A/G (Pierce) is immobilized in an appropriate amount by amine coupling, it is allowed to interact with the hCTLA4-His-BAP antigen obtained in Example 1-1. TBS is used as the running buffer, and 10 mM glycine-HCl (pH 1.5) is used as the regeneration solution.

After capturing 1 µg/ml ABAM004 suspended in TBS, a solution containing 500 nM hCTLA4-His-BAP and 10 concentrations of ATP, ADP or AMP diluted to a concentration of 4 of 4000 µM, and 2 mM MgCl ₂ injected into each flow cell at a flow rate of 10 µl/min for 3 min. This 3 min is used as the hCTLA4-His-BAP binding phase. After completion of the binding phase, the working buffer is injected for 2 min, which is used as the dissociation phase of hCTLA4-His-BAP. After completion of the dissociation phase, the regenerating solution is introduced at a flow rate of 30 μl/min for 30 s. The above is defined as the ABAM004 binding activity measurement cycle. The amount of hCTLA-4-His-BAP binding that interacts with ABAM004 in the binding phase is adjusted for the amount of antibody captured. To analyze and plot the results obtained, Biacore T200 Evaluation Software Version: 2.0 and Microsoft Excel 2013 (Microsoft) are used.

Fig. 1 represents the amount of binding of ABAM004 and hCTLA4-His-BAP in the presence of ATP and its metabolites, obtained using the described measurement.

In fig. 1 confirms that ABAM004 has the property of binding to hCTLA4, using not only ATP, but also ATP metabolites as a switch. In addition, this antibody has been shown to have the strongest binding activity, especially in the presence of AMP.

Example 1-10. Assessment of antibody binding to human CTLA4-expressing cells.

A flow cytometer is used to evaluate changes in antigen-antibody interaction between ABAM004 and human CTLA4 in the presence and absence of AMP. CHO cells that stably express human CTLA4 (hCTLA4-CHO cells) are prepared at the appropriate concentration. At the same time, PBS containing 0.1% BSA (FACS buffer) is used for suspension. Antibody is added to 100 μl of cell solution to a final concentration of 10 mg/ml, then AMP is added to final concentrations of 0, 0.4, 4, 40, 200 and 1000 μM and left at 4°C for 30 min. The cell line is then washed with a wash buffer, which is a FACS buffer containing AMP at final concentrations of 0, 0.4, 4, 40, 200 and 1000 µM, followed by a FITC-labeled secondary antibody ((Goat F(ab'2) Anti-Human IgG Mouse ads-FITC, Beckman, catalog number 732598), and again left at 4°C for 30 minutes in the dark. After repeated washing, measurements were made and analyzed using a flow cytometer (FACS CyAnTM ADP). Results are shown. in Fig. 2.

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

Example 1-11. ADCC activity of a test antibody using human peripheral blood mononuclear cells (PBMCs) as effector cells.

The ADCC activity of the test antibody, which binds to antigen in an ATP-dependent manner, is measured according to the following method. The ADCC activity of the test antibody is simultaneously measured as follows using human peripheral blood mononuclear cells (hereinafter referred to as human PBMCs) as effector cells.

First, a human PBMC solution is prepared. Healthy volunteers (adult men) are taken

- 61 045931 50 ml of peripheral blood using a syringe already containing 200 μl of heparin solution 1000 IU/ml (Novo-heparin for injection 5000 IU, Novo Nordisk). Peripheral blood, diluted 2-fold with PBS(-), is divided into 4 aliquots and added to Leucosep lymphocyte separation tubes (Greiner Bio-One), which are centrifuged after a preliminary injection of 15 ml Ficoll-Paque PLUS. Separation tubes into which aliquots of peripheral blood were added are centrifuged at 2150 rpm for 10 minutes at room temperature, and then mononuclear cell fractions are separated. After washing the cells contained in the fractions once with RPMI-1640 (Nacalai Tesque) containing 10% FBS (hereinafter referred to as 10% FBS/RPMI), the cells are suspended in 10% FBS/RPMI to a cell density of 1x10 ⁷ cells/ml. The cell suspension is used as a human PBMC solution for subsequent experiments.

Then, as target cells, hCTLA4-CHO cells, obtained by forced expression of the extracellular region of human CTLA4 in CHO cells, were suspended and prepared in 10% FBS/RPMI to obtain 2x10 ⁵ cells/ml. In addition, AMP (Sigma) diluted to 4 mM with RPMI was used as the AMP solution for subsequent assays.

ADCC activity is assessed by the release of LDH (lactate dehydrogenase). First, 50 μl of an antibody solution prepared at each concentration (0, 0.04, 0.4, 4 and 40 μg/ml) was added to each well of a 96-well plate with a U-shaped bottom, and 50 μl were seeded into the wells. µl of target cells (1 x 10 ⁴ cells/well). In addition, 50 μl of AMP solution was added to each well and the mixture was left at room temperature for 15 min. Add 50 μl (5x10 cells/well) of human PBMC solution to each well, centrifuge the plate and then leave for 4 hours at 37°C in an incubator with 5% carbon dioxide. After completion of the reaction, 100 μl of the culture supernatant is collected and transferred into a 96-well plate for measurement, and then the catalyst (C) and dye solution (D) attached to the LDH detection kit (TaKaRa) are mixed 1:45, and 100 μl is added this mixture. After reacting at room temperature for 15 min, add 50 μl of 1H HCl to stop the reaction. Absorbance at 492 nm is measured and ADCC activity is assessed by LDH release. ADCC activity is determined based on the following formula:

ADCC activity (%) = {(A-D) - (C-D)} x 100/{(B-D)-(C-D)}

In the above formula, A denotes the average LDH activity (OD at 492 nm) in the wells to which each test antibody is added. B represents the average value of LDH activity (OD at 492 nm) in the wells to which 10 μl of 20% aqueous TritonX solution was added after the reaction. C means the average value of LDH activity (OD at 492 nm) 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 the target cells. D is the average LDH activity (OD at 492 nm) in wells containing only 10% FBS/RPMI. The test is carried out in duplicate and the average ADCC activity value (%) in the test is calculated, reflecting the ADCC activity of the test antibody. The results are presented in Fig. 3.

From the above results, it follows that the ABAM004 antibody has antigen-binding activity in the presence of AMP and has the ability to kill target cells by exhibiting ADCC activity.

Example 2 Analysis of the crystal structure of an anti-CTLA4 antibody having ATP-dependent binding properties

Example 2-1. X-ray crystallographic analysis of the anti-CTLA4-binding antibody ABAM004, which uses AMP as a switch.

For the hCTLA4-binding antibody ABAM004, which uses AMP as a switch and is derived from the library in Example 1, the crystal structures of the ABAM004 Fab fragment, the complex of the ABAM004 Fab fragment and AMP, and the complex of the ABAM004 Fab fragment, AMP, and the hCTLA4 extracellular domain were analyzed separately.

Example 2-2. Preparation of full length ABAM004 antibody for crystallization.

The preparation and purification of full length ABAM004 antibody for crystallization is accomplished by a method known to those skilled in the art.

Example 2-3. Preparation of Fab fragment for crystal structure analysis of ABAM004 Fab fragment.

The Fab fragment of the ABAM004 antibody is obtained by the usual method of restriction digestion with rLys-C protease (Promega, catalog number V1671) followed by loading into a protein A column (MabSelect SuRe, GE Healthcare), cation exchange column (HiTrap SP HP, GE Healthcare) and a gel filtration column (Superdex200 16/60, GE Healthcare) to remove Fc fragments. Fractions containing Fab fragments are pooled and stored at -80°C.

Example 2-4. Preparation of ABAM004 Fab fragment crystals.

The ABAM004 antibody Fab fragment for crystallization, purified by the method of Example 2-3, is concentrated to approximately 13 mg/ml and crystallized at 20°C by sessile drop vapor diffusion. The reservoir solution consists of 0.1 M MES, pH 6.5, 25% w/v. polyethylene glycol 4000. The resulting crystals are immersed in a solution of 0.08 M MES, pH 6.5, 20% w/v. polyethylene glycol 4000 and 20% eti

- 62 045931 lenglycol.

Example 2-5. Collection of X-ray diffraction analysis data on crystals of the Fab ABAM004 fragment and determination of the structure.

X-ray diffraction data is measured using the BL-17A radiation facility of the Photon Factory of the High Energy Accelerator Research Organization. During the measurement, the crystals were kept frozen continuously in a nitrogen stream at -178°C, a total of 360 x-ray diffraction images were obtained using a Quantum 270 CCD detector (ADSC) connected to the beam channel, rotating the crystals at a time by 0.5°. Determination of cell parameters, indexing of diffraction spots and processing of diffraction data from diffraction images is carried out using the Xia2 program (J. Appl. Cryst. 2010, 43:186-190), the XDS package (Acta. Cryst. 2010, D66:125-132. Scala (Acta. Cryst. 2006, D62:72-82) and results in diffraction intensity data with a resolution of up to 1.70 A. Crystallographic data statistics are presented in Table 7.

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

Table 7

Summary of X-ray diffraction data and statistical refinements

Data collected ABAM004fab ABAM004fab -AMP ABAM004fab -AMF-STKA4 Space group R42L Ρ4^2 Dimensions of one cell a, b, c (A) 64.27, 70.86, 79.59 135.75, 135.75, 66.04 72.29, 72.29,309.31 α,β,γ (°) 90.00, 90.00, 90.00, 90.00, 90.00, 90.00, 90.00, 90.00, 90.00, Resolution (A) 52.92-1.70 47.33-2.89 52.81-3.09 Number of total reflections 296585 181799 157732 Number of unique reflections 40500 14366 16037 Completeness (outer shell) 99.6 (98.8) 100.0 (100.0) 99.9 (99.9) YategeeCh extreme shell)(%) 5.8 (67.6) 17.3 (88.7) 14.1 (63.2) Treatment Resolution (A) 52.92-1.70 47.33-2.89 52.81-3.09 Number of reflections 38523 13658 15145 Ifactors ⁰ (Rfree ^B ) (%) 16.92 (21.22) 19.97 (25.62) 23.49 (63.2) rms deviation from ideal value bond length (A) 0.0128 0.0037 0.0038 bond angles (°) 1.7102 0.9275 0.8442

Example 2-6. Preparation of the ABAM004 Fab fragment from the full-length antibody to analyze the crystal structure of the complex of the ABAM004 Fab fragment with AMP, and the complex of the ABAM004 Fab fragment, AMP and hCTLA4.

The Fab ABAM004 fragment was prepared by conventional papain restriction digestion (Roche Diagnostics, catalog no. 1047825) followed by loading onto a protein A column (MabSelect SuRe, GE Healthcare), a cation exchange column (HiTrap SP HP, GE Healthcare) and a gel filtration (Superdex200 16/60, GE Healthcare) to remove Fc fragments. Fractions containing Fab fragments are pooled and stored at -80°C.

Example 2-7. Preparation of crystals of the complex of ABAM004 Fab fragment and AMP.

The ABAM004 Fab fragment for crystallization, purified by the method of Example 2-6, was concentrated to approximately 13 mg/ml, to which AMP was added to give a final concentration of 2 mM, and crystallization was carried out at 20° C. using the sessile drop vapor diffusion method. The reservoir solution consists of 0.1 M Morpheus 2 buffer, pH 7.5, 37.5% w/v. MPD_P1K_P3350, 10% Morpheus carboxylic acids (Morpheus, Molecular Dimensions).

Example 2-8. Collection of X-ray diffraction data on crystals of the complex of the ABAM004 Fab fragment with AMP and determination of the structure.

X-ray diffraction data were measured using the BL-1A radiation facility of the Photon Factory at the High Energy Accelerator Re

- 63 045931 search Organization). During the measurement, the crystals were kept frozen continuously in a nitrogen stream at -178°C, and 720 x-ray diffraction images were obtained using a Pilatus 2M detector (DECTRIS) connected to the beam, rotating the crystals 0.25° at a time . Determination of cell parameters, indexing of diffraction spots and processing of data from diffraction images is carried out using the Xia2 program (J. Appl. Cryst. 2010, 43:186-190), the XDS package (Acta. Cryst. 2010, D66:125-132) and Scala (Acta. Cryst. 2006, D62:72-82), and as a result diffraction intensity data were obtained up to a resolution of 1.70 A. The statistics of the crystallographic data are presented in Table. 7.

The structure is determined by molecular replacement using the Phaser program (J. Appl. Cryst. 2007, 40, 658-674). The Fab fragment search model is derived from the published crystal structure of Fab (PDB code: 4NKI). The model was built using the Coot program (Acta Cryst. 2010, D66: 486-501) and refined using the Refmac5 program (Acta Cryst. 2011, D67: 355-367). The crystallographic confidence factor (R) of diffraction intensity data from 47.33-2.89 A is 19.97%, the free R value is 25.62%. Structural refinement statistics are presented in Table. 7.

Example 2-9. Preparation of the extracellular domain of hCTLA4.

The extracellular domain of hCTLA4 is obtained by the usual method of restriction digestion of abatacept with endoproteinase Lys-C (Roche, catalog number 11047825001) followed by loading on a protein A column (MabSelect SuRe, GE Healthcare) and a gel filtration column (Superdex200 10/300, GE Healthcare) to remove Fc fragments. Fractions containing the extracellular domain of hCTLA4 were pooled and stored at -80°C.

Example 2-10. Preparation of a complex of ABAM004 Fab fragment, AMP and the extracellular domain of hCTLA4.

The extracellular domain of hCTLA4, purified by the method of Example 2-9, is mixed with the Fab fragment ABAM004, purified by the method of Example 2-6, in a molar ratio of 1.5:1, and AMP is added to it to obtain a final concentration of 2 mM. The complex is purified by gel filtration chromatography (Superdex200 10/300, GE Healthcare) using a column equilibrated with 25 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM AMP.

Example 2-11. Preparation of crystals of the complex of the ABAM004 Fab fragment, AMP and the extracellular domain of hCTLA4.

The purified complex is concentrated to approximately 8 mg/ml and crystallized at 20°C using the sessile drop vapor diffusion method combined with the seeding method. The reservoir solution consisted of 0.1 M Morpheus 1 buffer, pH 6.5, 37.5% w/v. M1K3350, 10% halogen (Morpheus, Molecular Dimensions).

Example 2-12. Collection of X-ray diffraction data on crystals of the complex of ABAM004 Fab fragment, AMP and hCTLA4 and determination of the structure.

X-ray diffraction data is measured using a BL32XU SPring-8. During the measurement, the crystals were kept frozen continuously in a nitrogen stream at -178°C, and a total of 180 x-ray diffraction images were obtained using a CCD MX-225HS detector (RAYONIX) connected to the beam, turning the crystals by 1 ,0° at a time. Determination of cell parameters, indexing of diffraction spots and processing of data from diffraction images is carried out using the Xia2 program (J. Appl. Cryst. 2010, 43:186190), the XDS package (Acta. Cryst. 2010, D66:125-132) and Scala ( Acta. Cryst. 2006, D62:72-82), and as a result, diffraction intensity data were obtained up to a resolution of 1.70 A. The statistics of crystallographic data are presented in table. 7.

The structure is determined by molecular replacement using the Phaser program (J. Appl. Cryst. 2007, 40, 658-674). The search model for Fab fragments is derived from the published crystal structure of Fab (PDB code: 4NKI), and the search model for the extracellular domain of hCTLA4 is derived from the published crystal structure of human CTLA4 (PDB code: 3OSK, J. Biol. Chem. 2011, 286: 6685-6696) . The model was built using the Coot program (Acta Cryst. 2010, D66: 486-501) and refined using the Refmac5 program (Acta Cryst. 2011, D67: 355-367). The crystallographic confidence factor (R) of diffraction intensity data from 52.81-3.09 A was 23.49%, and the free R value was 31.02%. Structural refinement statistics are presented in Table. 7.

Example 2-13. Determination of the site of interaction between ABAM004 and AMP.

The crystal structure showed that AMP is mainly recognized by the antibody heavy chain.

The adenine ring portion of AMP is recognized by heavy chain CDR1 and CDR3, and the ribose portion and phosphate group portion are recognized by CDR1 and CDR2.

In particular, as shown in FIG. 4, the adenine ring fragment of AMP is recognized by the side chains T33 belonging to CDR1 of the heavy chain and Y95, L100B and W100C belonging to CDR3, and the main chains G96 and M100A of the antibody. In particular, it was found that carbonyl oxygen atoms in the main chains of G96 and M100A form hydrogen bonds with N in position 6 of AMP, NH group

- 64 045931 the main chain amide W100C forms a hydrogen bond with the N at position 1, and the side chains Y95, L100B and W100C interact using the pi electrons of the adenine ring moiety, so that the antibody strictly recognizes the adenine ring moiety. The ribose moiety is recognized by each of the side chains of T33, belonging to heavy chain CDR1, and Y56 and Y58, belonging to CDR2, through van der Waals forces and through Y56 pi-electron interactions. In addition, the phosphate group moiety is recognized by each of the side chains T33 belonging to the heavy chain of CDR1, and S52, S52A and R53 belonging to CDR2, and the main chain of S52A. In particular, hydrogen bonds formed by the side chain of T33 and the NH amide group of the main chain of S52A with the phosphate group moiety, and van der Waals interactions of S52 and R53 are thought to play an important role in recognizing the phosphate group moiety. Fab amino acid residue numbering is based on the Kabat numbering scheme.

Example 2-14. Identification of ABAM004 epitopes.

In fig. 5 and 6 epitopes of the ABAM004 Fab contact region are mapped to the hCTLA4 crystal structure and amino acid sequence, respectively. The epitopes contain hCTLA4 amino acid residues containing one or more non-hydrogen atoms located within 4.2 A of any portion of the ABAM004 Fab or AMP in the crystal structure.

From the crystal structure it is clear that at least M3, E33, R35, T53, E97, M99, Y100, P101, P102, P103, Y104, Y105 and L106 of the antigen are recognized by CDR2 and CDR3 of the heavy chain, CDR1 and CDR3 of the light chains of the antibody and AMP. In particular, the loop consisting of the M99-Y104 fragment of the antigen is clearly recognized by the antibody because it is buried in the CDR loop of the antibody, and this loop is believed to play a major role in the recognition of the antigen by the antibody.

Example 2-15. AMP-dependent mechanism of antigen binding.

The antibody variable region is extracted from the crystal structure of ABAM004 Fab alone, the crystal structure of the complex of ABAM004 Fab and AMP, and the crystal structure of the ternary complex consisting of ABAM004 Fab, AMP and CTLA4; fig. 7 is an overlay of highlighted variable regions centered around the heavy chain. For AMP-dependent antigen binding, not only the direct interaction between AMP and CTLA4, shown in example 2-14, but also the structural change in the antibody associated with AMP binding is considered important.

As shown in FIG. 7, by comparing the crystal structure of one antibody with the crystal structure of the antibody to which AMP binds, it was found that the loop structures of the heavy chain CDR3 and the light chain CDR3, as well as the torsion angle between the heavy chain and the light chain in the antibody variable region, also changed. In addition, by comparing the crystal structure of the antibody to which AMP binds and the crystal structure of the ternary complex consisting of antibody, AMP and CTLA4, it is also recognized that the structures of the heavy chain CDR3 and light chain CDR1 are further modified by antigen-binding, antigen-dependent structural changes. On the other hand, since the structure of the light chain CDR3 loop and the twist angle between the heavy chain and the light chain have not changed, it is believed that AMP binding may change the structure of the antibody to a state similar to the structure at the time of antigen binding. Thus, it is believed that the structural changes associated with AMP binding are necessary for the formation of a suitable structure for antigen binding and play an important role in AMP-dependent antigen binding.

Example 3. Preparation of CTLA4-modified antibodies and assessment of their activity.

Example 3-1. Preparation of CTLA4-binding variants of the ABAM004 antibody with increased activity.

The amino acid sequence ABAM004 (VH SEQ ID NO: 10, VL SEQ ID NO: 11), derived from the human rational design phage library described in Example 1, is modified to reduce the CTLA4 binding activity of the sequence in the absence of ATP analogues and to increase binding activity Human CTLA4 in the presence of ATP analogues and to enhance binding to ATP and ATP analogues. To achieve this, variants were created with point mutations at residues expected to be involved in binding, based on the co-crystal structure of ABAM004 and AMP and the co-crystal structure of ABAM004, AMP and human CTLA4 obtained by the method described in Example 2 In addition, variants have also been created in which each amino acid contained in the CDR is replaced by Ala or Pro. Point mutation variants are measured using Biacore T200 or Biacore 4000 (GE Healthcare) for human CTLA4 binding activity (abatacept and hCTLA4-His-BAP) in the absence of ATP and in the presence of ATP, ADP or AMP to screen for mutations that enhance binding activity. Variants were generated by combining mutations that enhance binding activity, and the KD value was calculated using Biacore software. As a result, it was found that the introduction of substitutions H32A and S52aT into the heavy chain of ABAM004 and T24D, T26P and E50F into the light chain (according to Kabat numbering) enhances the binding ability of ABAM004. The variant is designated 04H0150/04L0072 (VH SEQ ID NO: 47, VL SEQ ID NO: 48).

Example 3-2. Measurement by surface plasmon resonance of the effect of ATP and its metabolites on the binding activity of human CTLA4 with ABAM004 and 04H0150/04L0072.

- 65 045931

First, the Biacore T200 measurement chip was created. The Biacore T200 temperature is set to 25°C and the flow rate is set to 10 μL/min. HBS-EP+ is used as a running buffer. A mixture of equal volumes of NHS (N-hydroxysuccinimide) and EDC (N-ethyl-K'(dimethylaminopropyl)carbodiimide) was added to the CM5 sensor chip (GE Healthcare) at a flow rate of 10 μl/min for 10 minutes to activate the flow cells. 25 μg/ml protein A/G (Pierce) suspended in 10 mM sodium acetate, pH 4.0 was then added and allowed to bind at 10 μl/min for 30 minutes. Excess active groups on the flow cells are then blocked by adding 1 M ethanolamine-HCl at 10 μL/min for 10 min.

The effect of ATP and its metabolites on the binding of the target antibody to human CTLA4 is then measured. The set temperature is 25°C and TBS is used as the running buffer. 10 mM glycine-HCl (pH 1.5) is used as the regeneration solution. Once the antibody suspended in TBS can be captured, each flow cell is injected with a TBS solution containing 500 nM hCTLA4-His-BAP, 10 concentrations of ATP, ADP, or AMP diluted in the usual ratio of 4 to 4000 μM and 2 mM MgCl ₂ at a flow rate of 10 µl/min for 3 min. This 3 min is used as the hCTLA4-His-BAP binding phase. After completion of the binding phase, the injection is switched to the running buffer for 2 minutes, which is used as the dissociation phase. After completion of the dissociation phase, the regenerating solution is introduced at a flow rate of 30 μl/min for 30 s; the above is considered to be a binding activity measurement cycle. The amount of hCTLA4-His-BAP binding that interacted with ABAM004 or 04H0150/04L0072 in the binding phase was adjusted to the amount of antibody captured. The results are shown in Fig. 8. In addition, when measuring the binding of ABAM004 and 04H0150/04L0072, the concentration of small molecules in the binding phase is maintained at 62.5 μM or 1 mM, and 8 concentrations of hCTLA4-His-BAP are diluted at the usual ratio of 2 of 2000 nM and used in the phase binding. The KD values obtained from hCTLA4-His-BAP binding amount analysis are shown in Table. 8. To analyze and plot data, use Biacore T200 software version: 2.0 and Microsoft Excel 2013 (Microsoft). A robust affinity model is used to calculate the KD value.

Table 8

Ligand Sample KD (M) SM (62.5 µM)* SM(ImM) 04H0150/04L0072 hCTLA4 AMP 6.9E-07 2,ZE-07 hCTLA4 ADP 4.2E-07 3.2E-07 hCTLA4 ATP 8DE-07 3.2E-07 ABAM004 hCTLA4 AMP 4,ZE-06 1.4E-06 hCTLA4 ADP 2.5E-06 1.5E-06 hCTLA4 ATP 5.5E-06 2,ZE-06

*SM refers to small molecules (ATP, ADP or AMP) used in this assay.

Example 3-3. Increasing binding capacity by making various changes

To create improved anti-CTLA4 antibodies, amino acid changes were specifically introduced into 04H0150/04L0072, which are the variable regions of the anti-human CTLA4 antibody produced in Example 3-1. Variants in which amino acid changes were made to each of the 18 amino acids except cysteine in all CDRs 04H0150 and 04L0072 were generated by methods known to those skilled in the art, such as PCR. Measurements of approximately 1200 variants designed to bind to human CTLA4 were performed using Biacore 4000 software. The A/G protein (Thermo Fisher Scientific) is immobilized on a Series S CM5 sensor chip (GE Healthcare) and antibodies are captured by the chip by interaction with a culture supernatant containing the antibody variant. A solution of human CTLA4 to which a small molecule (ATP, ADP, or AMP) has been added, or a solution of human CTLA4 to which no small molecule has been added, is then reacted with the antibody to evaluate the binding ability of the antibody to human CTLA4 in the presence or absence of the small molecule . The assay is performed at 25°C using Tris-buffered saline, 0.02% PS20 as running buffer.

Changes that increase binding to human CTLA4 in the presence of the small molecule and changes that decrease binding to human CTLA4 in the absence of the small molecule were identified using the method described above and combined to obtain anti-human CTLA4 antibodies showing better profiles. To the heavy chain gene, antibodies 04H0150-G1m (SEQ ID NO: 209), which has 04H0150 as the heavy chain variable region and Glm (SEQ ID NO: 82), in which the C-terminal Gly and Lys of human IgG1 are removed as the constant region heavy chain, changes detected by introducing comprehensive changes and changes in the framework are combined to obtain antibody heavy chain genes. For the antibody light chain 04L0072-lam1 (SEQ ID NO: 208) having 04L0072 as the light chain variable region and lam1 λ chain (SEQ ID NO: 87) as the human light chain constant region, the detected changes are combined to obtain the light chain genes antibody chains. In addition, a variant is also created in which the framework of the variable region and the constant region

- 66 045931 light chain regions are replaced by human κ chain sequence. For comparison, the MDX10D1H-G1m antibody heavy chain gene (SEQ ID NO: 210) containing the MDX10D1H heavy chain variable region (SEQ ID NO: 154) and the MDX10D1L-k0MT antibody light chain gene (SEQ ID NO: 211) having light chain variable region MDX10D1L (SEQ ID NO: 155) of the existing anti-human CTLA4 antibody described in WO 0114424. The antibodies are expressed and purified in a manner known to those skilled in the art by combining these genes to produce the anti-CTLA4 antibodies of interest. In table 9 lists the SEQ ID NOs of the heavy chain variable regions, light chain variable regions, heavy chain constant regions, light chain constant regions and hypervariable regions of the resulting antibodies.

In the present specification, antibodies are named according to the following rule: (heavy chain variable region) - (heavy chain constant region)/(light chain variable region) - (light chain constant region). For example, this means that if the name of an antibody is 04H0150G1m/04L0072-lam1, the heavy chain variable region of that antibody is 04H0150, the heavy chain constant region is G1m, the light chain variable region is 04L0072, and the light chain constant region is lam1.

Table 9 Amino acid sequences of heavy chains, light chains ____and their hypervariable regions (designated as SEQ ID NO_)_____

Antibody Variable region Constant region Hypervariable region (HVR) Heavy chain Light chain Heavy chain Light chain H1 H2 NZ L1 L2 L3 04Н0150-G1 m/04L0072-lsm1 98 99 82 87 103 104 102 116 117 115 04H1077-G1m/04L104Ham1 83 88 82 87 105 106 102 118 117 115 04H1077-G1 m/04L1063~lam1 83 89 82 87 105 106 102 116 117 133 04H1077-G1m/04L1027-lain1 83 90 82 87 105 106 102 119 117 115 04H1077-G1m/04L1034-lam1 83 91 82 87 105 106 102 120 117 115 04H1077-G1 m/04L1066-laml 83 92 82 87 105 106 102 121 117 115 04H1077-G1 m/04L1067-laml 83 93 82 87 105 106 102 122 117 115 Q4H1077-G1 m/04L1068-laml 83 94 82 87 105 106 102 118 117 133 04H1077-G1 m/04L1086-laml 83 97 82 87 105 106 102 122 117 133 04H107 7-G1 m/04L1305-kOMT 83 95 82 96 105 106 102 122 117 133 04H1206-G1 m/04L1086-lam1 84 97 82 87 105 106 102 122 117 133 04H1207-G1 m/04L1086-laml 85 97 82 87 107 106 102 122 117 133 04H1208-GW04L1086-lam! 86 97 82 87 107 108 102 122 117 133 04H1208-G1 m/04L1407-kOMT 86 134 82 96 107 108 102 121 123 153

Biacore T200 is used to measure the binding of the resulting antibodies to human CTLA4. The working buffer is 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl ₂ , 0.05% Tween 20 with the addition of ATP to the desired concentration, the analysis is carried out at 37°C. First, protein G (CALBIOCHEM) is immobilized on a CM3 S series sensor chip (GE Healthcare) and antibodies are captured by contacting the chip with an antibody solution prepared in an ATP-free running buffer. Next, by reacting with a solution of human CTLA4 prepared in a running buffer containing ATP to the desired concentration, or with a solution of human CTLA4 prepared in a running buffer not containing ATP, the binding ability of the anti-human CTLA4 antibody in the presence or absence of ATP is assessed. The chips were regenerated with 25 mM NaOH and 10 mM glycine-HCl (pH 1.5) and assayed by antibody recapture. The dissociation constant of each antibody relative to CTLA4 was calculated using Biacore T200 Evaluation Software 2.0. Specifically, the binding rate constant ka (L/mol/s) and the dissociation rate constant kd (1/s) are calculated by globally fitting the measured sensorograms using a 1:1 Langmuir binding model. From these values the dissociation constant KD (mol/l) is calculated. Alternatively, the dissociation constant KD (mol/L) is calculated from the steady state model. In addition, the amount of CTLA4 binding per unit amount of antibody is calculated by adjusting the amount of CTLA4 bound calculated from the sensogram obtained from the assay with the amount of antibody captured on the chip surface. In table Figure 10 shows the results of these analyses.

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Table 10

Analysis of altered antibody binding to human CTLA4.

Antibody Binding to human CTLA4 Kd for human CTLA4 (M) Without ATP ATP 1 µM ATP 1G)mk\-1 ATP 100 µM Without ATP ATP 10 µM ATP 100 µM MDX1001HG1 m/MDX10D1 LkOMT 0.189 0.189 0.181 0.170 4. ZE-08 4.9E-08 4.5E-08 04Н0150-G1 m/04L0072-lam1 0.001 0.001 0.010 0.056 N.A. N.A. 1.9E-06 04H1077-G1 m/04L1041 -Iam1 0.006 0.023 0.094 0.172 N.A. 7.3E-O7 1.4E-07 04H1077-G1 m/04L1063-lam1 0.001 0.003 0.029 0.105 N.A. N.A. 5.8E-07 04H1077-G1 m/04L1027-lam1 0.000 0.001 0.024 0.099 N.A. N.A. 6.7E-07 04H1077-G1 m/04L1034-lam1 0.004 0.007 0.041 0.114 N.A. N.A. 3.2E-07 04H1077-G1 m/04L1066-lam1 0.020 0.057 0.142 0.185 *6.9E-O6 2.8E-07 4.8E-08 04H1077-G1 m/04L1067-lam1 0.012 0.041 0.131 0.192 N.A. 4.0E-07 7.6E-08 04H1077 G1 m/04L1068lam1 0.005 0.018 0.080 0.165 N.A. 8.8E-07 1.5E-07 04H1077-G1 m/04LI 086-lam 1 0.0ΙΟ 0.031 0.106 0.181 N.A. 6.9E-07 I.IE-07 04H 1077-G1 m/04L1305-kCMT 0.018 0.046 0.128 0.185 ⁺ 6.5E-06 3.6E-07 5.6E-08 04H1206-G1 m/04L1086-lam1 0.010 0.029 0.103 0.179 N.A. 6.8E-07 1.2E-07 04H1207-G1 m/04L1086-lam1 0.013 0.047 0.137 0.196 N.A. Z.ZE-07 5.9E-08 04H1208-G1 m/04L1086-lam1 0.005 0.030 0.108 0.170 N.A. 4.5E-07 7.4E-08 04H1208-G1 m/04L1407-kOMT 0.138 0.175 0.200 0.214 *4.3E-07 2.3E-08 6.2E-09

N.A. The results are extremely low and do not allow us to determine the KD value.

* Dissociation constant KD (mol/l) is calculated using the steady state model.

The human CTLA4 binding value in the table indicates the amount of human CTLA4 antigen bound per unit amount of antibody when 1000 nM human CTLA4 is used at each of the listed ATP concentrations, and the KD for human CTLA4 (M) means the dissociation constant for human CTLA4 at each ATP concentration. The KD values noted in the table. 12 *, calculated using a stationary model. All variants obtained using 04H0150-G1m/04L0072-lam1 as the starting antibody were found to have increased binding in the presence of ATP compared to 04H0150-G1m/04L0072-lam1. In addition, both 04H0150-G1m/04L0072-lam1 and these variants exhibit increased binding at 10 μM ATP concentration compared to 1 μM concentration; the amount bound at 100 μM is even higher, thus demonstrating the dependence of binding to human CTLA4 on ATP concentration. On the other hand, comparison with MDX10D1H-G1m/MDX10D1L-k0MT did not show such ATP concentration-dependent binding to human CTLA4. Although 04H1077-G1m/04L1305-k0MT, in which the 04H1077-G1m/04L1086-lam1 light chain backbone and constant region are replaced by the human κ chain, has increased binding to human CTLA4 in the absence of ATP, in contrast to 04H1077-G1m/04L1086-lam1, ATP concentration-dependent binding was also enhanced. These results showed that the property of binding to human CTLA4 in an ATP-dependent manner is retained even when the sequence is replaced by a human κ chain sequence. Among the antibodies developed in the present invention, 04Η1077-01μΌ^1066-^1, 04H1077-G1m/04L1305-k0MT and 04H1207-G1m/04L1086-lam1 show almost the same binding activity as the existing anti-CTLA4 human antibody MDX10D1H-G1m/MDX10D1L -k0MT under conditions where ATP is present at 100 μM, and 04H1208-G1m/04L1407-k0MT exhibits stronger binding activity than MDX10D1HG1m/MDX10D1L-k0MT under conditions where ATP is present at 10 μM or more.

Then, among the antibodies presented in table. 10, assess the binding of 04H1077-G1m/04L1086lam1 and 04H1208-G1m/04L1407-k0MT to mouse CTLA4. For comparison, the antibody heavy chain gene hUH02-G1d (SEQ ID NO: 212), containing the heavy chain variable region hUH02 (SEQ ID NO: 156) of the mouse anti-CTLA4 antibody, and the antibody light chain gene hUL01-k0 (SEQ ID NO: 213) were obtained ) containing the hUL0l light chain variable region (SEQ ID NO: 157), the antibody is expressed, purified and used. The assay was performed using a Biacore T200 under the same conditions as the human CTLA4 binding measurement, except using mouse CTLA4 as a sample (Table 11). Mouse CTLA4 was prepared as follows.

The mouse CTLA4 His-tagged extracellular region gene (mCTLA4-His) (SEQ ID NO: 49) was synthesized and inserted into an animal expression plasmid. The prepared plasmid is introduced by lipofection into the FreeStyle 293-F line obtained from human embryonic kidney cells (Invitrogen), which is seeded into a flask after suspension in the FreeStyle 293 expression medium (Invitrogen) at a cell density of 1.33x106 cells/ml. The absorbance of the purified antigen solution at 280 nm is measured using a spectrophotometer. From the obtained measurement values, the concentration of the purified antigen is calculated using the extinction coefficient calculated by the PACE method (Protein Science, 1995,4,2411-2423).

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Table 11

Analysis of the binding of altered antibodies to mouse CTLA4

Antibody Binding to mouse CTLA4 K _D for CTLA4 mouse Without ATP ATP 1 µM ATP 10 µM ATP 100 µM ATP 10 µM ATP 100 µM hUH02-Gld/hUL01-k0 0.155 0.160 0.159 0.157 1.1E-07 Ι,ΙΕ-07 04H1077-Glm/04L1086- 1am 1 N.D. 0.001 0.024 0.093 4DE-06 5.9E-07 04H1208-Glm/04L1407kOMT 0.023 0.099 0.153 0.175 8.4E-08 1.6E-08

N.D. - indicators are too weak to determine binding.

The mouse CTLA4 binding value in the table means the amount of mouse CTLA4 binding per unit amount of antibody when mouse CTLA4 reacts at 1000 nM with each of the ATP concentrations, and the mouse CTLA4 KD (M) means the dissociation constant for mouse CTLA4 at each ATP concentration. It was found that hUH02-Gld/hUL01-k0 binds to mouse CTLA4 equally regardless of ATP concentration, while 04H1077-G1m-04L1086-lam1 and 04H1208G1m/04L1407-k0MT bind to mouse CTLA4 depending on ATP concentration. Compared to binding to human CTLA4 shown in Table. 10, the binding ability of 4H1077-G1m-04L1086lam1 to mouse CTLA4 is approximately 5 times weaker compared to the ability to bind to human CTLA4, and the ability of 04H1208-G1m/ 04L1407-k0MT to bind to mouse CTLA4 is approximately two times weaker compared to the ability to bind to human CTLA4. Human CTLA4 in the presence of 100 μM ATP.

Example 3-4. Generation of control anti-mCTLA4 antibody and switching anti-mCTLA4 antibody.

Create a control anti-mCTLA4 antibody (hUH02-mFa55/hUL01-mk1, abbreviation: mNSmFa55) and anti-CTLA4 switches (04H1077-mFa55/04L1086-m10r, abbreviation: SW1077-mFa55; and 04H1208-mFa55/04L 1407s-mk1, abbreviation: SW1208-mFa55). The mNS-mFa55 antibody uses the hUH02 heavy chain variable region (SEQ ID NO: 16) and the hUL01 light chain variable region (SEQ ID NO: 17), and the mFa55 mouse heavy chain constant region (SEQ ID NO: 18) is used as the constant regions ) and wild-type mouse light chain mk1 constant region (SEQ ID NO: 19). A mouse heavy chain constant region is also used, to which modifications have been added to enhance binding to the Fcy receptor. The antibody is expressed and purified in a manner known to those skilled in the art.

The SW1077-mFa55 antibody uses the heavy chain variable region 04H1077 (SEQ ID NO: 20) and the light chain variable region 04L1086 (SEQ ID NO: 21), and the mouse heavy chain constant region mFa55 (SEQ ID NO: 18) is used as the constant regions ) and the wild-type mouse light chain constant region m10r (SEQ ID NO: 22). A mouse heavy chain constant region is also used, to which modifications have been added to enhance binding to the Fcy receptor. The antibody is expressed and purified in a manner known to those skilled in the art.

The SW1208-mFa55 antibody uses the heavy chain variable region 04H1208 (SEQ ID NO: 23) and the light chain variable region 04L1407s (SEQ ID NO: 21), and the mouse heavy chain constant region mFa55 (SEQ ID NO: 18) is used as the constant regions ) and wild-type mouse light chain constant region mk1 (SEQ ID NO: 19). A mouse heavy chain constant region is also used, to which modifications have been added to enhance binding to the Fcy receptor. The antibody is expressed and purified in a manner known to those skilled in the art.

Example 3-6. Evaluation of neutralizing activity of CTLA4 switch antibody.

The neutralizing activity of the anti-CTLA4 switching antibody (SW1077-mFa55) obtained in Example 3-4 was assessed by a competitive enzyme-linked immunosorbent assay (ELISA). mCTLA4-Fc (SEQ ID NO: 25), in which the human constant region is linked to mCTLA4, is diluted to a concentration of 5 μg/ml (55 nM) with 0.1 M NaHCO ₃ , 0.05% NaN ₃ to prepare a solution of mCTLA4- Fc. 100 μl of each prepared mCTLA4-Fc solution was added to a 96-well plate, and the plate was left at 4°C overnight to immobilize mCTLA4-Fc on the surface of the plate. After washing 3 times with TBS, 0.1% Tween 20, 250 μl of BSA solution diluted to 2% TBS was added to each well to block the plate surface. The plate is then washed 3 times. mCD86-Fc-His (Sino Biologies Inc. 50068-M03H, accession number NP_062261.3), in which the human constant region and His tag are fused to mouse CD86, diluted with TBS to a final concentration of 55 nM, SW1077-mFa55 antibody solution, diluted to final concentrations of 6.25, 1.56, 0.390, 0.0977, 0.0061 and 0 µg/ml, and ATP solution diluted to final concentrations of 0, 1, 10 and 100 µM, each mixed so as to obtain a total of 100 µl, the mixture is added to each well and the plate is left at 37°C for 1 hour. Each well is then washed 3 times with TBS, 0.1% Tween 20, prepared

- 69 045931 adjusted so that it contains the same concentration of ATP as the solution added to each well. Then, 100 μl of anti-His-tag mAb-HRP-Direct (MBL Life Sciences) diluted 10,000 times with blocking buffer is added to each well so that the ATP concentration is the same as the solution added to each well. and the plate is left at 37°C for 1 hour. Each well is then washed 3 times with TBS, 0.1% Tween 20, prepared to contain the same concentration of ATP as the solution added to each well. Add 100 µl of TMB solution to each well and leave the plate at 37°C for 1 hour. Add 50 µl of 1 M H2SO to each well.4. to stop the reaction, and determine the absorbance at 450 nm using a microplate reader (Wako Sunrise).

The absorbance value in wells without antibodies at the same ATP concentration is taken as the degree of mCTLA4-mCD86 binding equal to 100%, and the extent to which the binding rate decreases with the addition of antibodies is assessed. The results are presented in Fig. 9.

The results indicate that the neutralizing activity of the SW1077 antibody against the mCTLA4-mCD86 interaction becomes stronger as the ATP concentration in the assay increases. These results confirmed that antibody SW1077 has ATP-dependent neutralizing activity.

Example 3-7. Efficacy of an anti-STEA4 antibody switch in a murine model of syngeneic tumor cell transplantation, increase/decrease of regulatory T cells (Treg) in the tumor, and alteration of a systemic response marker in the spleen.

Example 3-7-1. Generation of cell line and mouse model with transplanted syngeneic tumor line.

The cells used are mouse mammary cancer cell line FM3A, purchased from RIKEN. FM3A cells are maintained and passaged in RPMI 1640 medium (Sigma) containing 10% bovine serum (Thermo Fisher Scientific). SZN/HeN mice (7 weeks old, females) purchased from Charles River Laboratories, Japan, were used. FM3A cells are transplanted into the abdomen of mice under the skin, and the model is confirmed to be established when the volume of the transplanted tumor reaches a size of about 150 to about 300 mm ³ .

The volume of the transplanted tumor is calculated using the following formula:

Tumor volume=larger diameterxsmaller diameterxsmaller diameter/2.

Example 3-7-2. Obtaining medications for administration.

The drugs administered to the FM3A cell transplant models are the anti-mouse CTLA4 control antibody (mNS-mFa55) and the anti-C^A4 switch antibody (SW1208-mFa55) prepared in Example 3-4. Using His buffer (20 mM His-HCl, 150 mM NaCl, pH 6.0), solutions of mNS-mFa55 are prepared at concentrations of 0.0005 mg/ml, 0.005 mg/ml, 0.0125 mg/ml, 0.05 mg/ml, 0.5 mg/ml, 1.5 mg/ml and 5 mg/ml, and SW1208-mFa55 solution is prepared in concentrations of 0.005 mg/ml, 0.05 mg/ml, 0.5 mg/ml, 5 mg/ml and 25 mg/ml, respectively.

Example 3-7-3. Administration of drugs to measure antitumor effect.

On the 9th day after transplantation, mice were administered mNS-mFa55 at doses of 0.01 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 1 mg/kg, 10 mg/kg, 30 mg/kg and 100 mg/kg, and SW1208-mFa55 is administered at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg and 500 mg/kg, respectively. Prepared solutions for administration are administered at a dose of 20 ml/kg through the tail vein.

In table 12 details the drug treatment when measuring the antitumor effect.

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Table 12

Measuring the antitumor effect in the FM3A cell transplant model

Group Animals Medicine Dose Introduction Date of introduction 1 4 His buffer - To the tail vein 9th day after transplantation 2 4 mNS-mFa55 0.01 mg/kg To the tail vein 9th day after transplantation 3 4 mNS-mFa55 OD mg/kg To the tail vein 9th day after transplantation 4 4 mNS-mFa55 0.25 mg/kg To the tail vein 9th day after transplantation 5 4 mNS-mFa55 1 mg/kg To the tail vein 9th day after transplantation 6 4 mNS-mFa55 10 mg/kg To the tail vein 9th day after transplantation 7 4 mNS-mFa55 30 mg/kg To the tail vein 9th day after transplantation 8 4 mNS-mFa55 100 mg/kg To the tail vein 9th day after transplantation 9 4 SW1208mFa55 0.1 mg/kg To the tail vein 9th day after transplantation 10 4 SW1208mFa55 1 mg/kg To the tail vein 9th day after transplantation AND 4 SW1208mFa55 10 mg/kg To the tail vein 9th day after transplantation 12 4 SW1208mFa55 100 mg/kg To the tail vein 9th day after transplantation 13 4 SW1208mFa55 500 mg/kg To the tail vein 9th day after transplantation

Example 3-7-4. Evaluation of antitumor effect.

The antitumor effect is assessed by tumor volume, calculated using the formula described in example 3-7-1.

The tumor growth inhibition rate (TGI - Tumor Growth Inhibition) is calculated using the following formula:

TGI (%) = (1 - (Mean tumor volume in the study group at the time of measurement - Average tumor volume in the study group at the time of initial administration) / (Average tumor volume in the control group at the time of measurement average tumor volume in the control group at the time of initial administration introduction)) x 100

As a result, the effectiveness of the drug TGI = 60% or more on the 13th day after administration, which was established for mNS-mFa55 in doses of 0.1 mg/kg or more and for SW1208-mFa55 in doses of 1 mg/kg or more (Fig. 10 and 11).

Example 3-7-5. Drug administration to evaluate Treg cells in the tumor and confirm systemic effects in the spleen.

On day 7 after transplantation, a control anti-mouse CTLA4 antibody (mNS-mFa55) is administered via the tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg and 100 mg/kg, and a switching anti-CTLA4 antibody ( SW1208-mFa55) is administered via the tail vein at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg and 500 mg/kg. In table Figure 13 shows details of drug treatment to evaluate Treg cells in the tumor and confirm systemic effects in the spleen.

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Table 13

Testing intratumoral and systemic effects in the FM3A cell transplant model (mNS-mFa55 and SW1208-mFa55)

Group Animals Medicine Dose Introduction Date of introduction 1 3 His buffer - To the tail vein 7th day after transplantation 2 3 mNS-mFa55 0.1 mg/kg To the tail vein 7th day after transplantation 3 3 mNS-mFa55 1 mg/kg To the tail vein 7th day after transplantation 4 3 mNS-mFa55 10 mg/kg To the tail vein 7th day after transplantation 5 3 mNS-mFa55 100 mg/kg To the tail vein 7th day after transplantation 6 3 SW1208mFa55 OD mg/kg To the tail vein 7th day after transplantation 7 3 SW1208mFa55 1 mg/kg To the tail vein 7th day after transplantation 8 3 SW1208mFa55 10 mg/kg To the tail vein 7th day after transplantation 9 3 SW1208mFa55 100 mg/kg To the tail vein 7th day after transplantation 10 3 SW1208mFa55 500 mg/kg To the tail vein 7th day after transplantation

Example 3-7-6. Tumor and spleen resection in FM3A cell-transplanted mouse model.

On day 6 after antibody injection, mice were euthanized under anesthesia and the tumor and spleen were resected. A cell suspension is prepared from the resected spleen using RPMI-1640 medium (SIGMA) containing 10%.

FBS (SIGMA), and then hemolyzed using a mouse erythrocyte lysis kit (R&D) to obtain spleen cells. Resected tumors are crushed using a Tumor Dissociation Kt, mouse kit (Miltenyi). Both spleen cells and crushed tumors react with the following antibodies, and the fractions of immune cells present are analyzed by FACS analysis: anti-CD45 antibody (BD, clone: 30-F11), anti-CD3 antibody (BD, clone: 145-2C11), anti-CD4 antibody (BD, clone: RM4-5), anti-PDxP3 antibody (eBioscience, clone: FJK-16s)), anti-ICOS antibody (eBioscience, clone: 7E17G9), anti -KLRG1 antibody (Biolegend, clone: 2F1/KLRG1). FACS analysis is performed using a BD LSR Fortessa X-20 flow cytometer (BD).

Example 3-7-7. Evaluation of Treg tumor in the FM3A cell transplant model.

Changes in Treg effector cells (CD4+ FoxP3 ⁺ KLRG1') in the tumor after administration of a control anti-mouse CTLA4 antibody (mNS-mFa55) or a switch anti-CTLA4 antibody (SW1208-mFa55) were assessed. As a result, both mNS-mFa55 and SW1208-mFa55 reduced the proportion of effector Treg cells to less than 0.2% CD45-positive cells at doses of 1 mg/kg or more (Fig. 12). Example 37-8. Evaluation of systemic effects on the spleen in the FM3A cell transplant model.

Changes in activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) in the spleen after administration of mNS-mFa55 or SW1208-mFa55 were assessed using FACS analysis. As a result, the ratio of activated helper T cells to CD45-positive cells in the spleen increased significantly with mNS-mFa55 administration, but there was no significant increase in the ratio of activated helper T cells to CD45-positive cells in the spleen even with increasing doses of SW1208-mFa55 ( Fig. 13). It was confirmed that although the switch antibody showed the same potency as the control antibody, it did not induce a response in tissues other than the tumor, and the concept of activity only locally in the tumor was proven in vivo in mice.

Example 4. Production of modified CTLA4 antibodies and evaluation of their activity.

The anti-CTLA4 switching antibodies obtained in Example 3 were further modified and evaluated.

Example 4-1. Enhancing CTLA4 binding ability by altering ATP binding enhancement.

Based on the results of the structural analysis performed in Example 2-1, it was shown that CDR2 of the antibody heavy chain interacts with the phosphate group of AMP. It is believed that when the small molecule is ATP, the γ-phosphate group may cause steric hindrance to the heavy chain CDR2. The amino acids in this region are then replaced to study the increase in ATP binding capacity.

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Specifically, 04H1389-G1m/04L1086-lam1 and 04H1382-G1m/04L1086-lam1 were obtained by introducing R53Q and G55H changes into the heavy chain variable regions of 04H1207-G1m/04L1086-lam1 and 04H1208G1m/04L1086-lam1 obtained in Example 3. In addition, 04H1389-G1m/04L1305-k0MT was obtained by replacing the light chain of 04H1389-G1m/04L1086-lam1 with the human κ chain sequence. In table 14 lists the SEQ ID NOs of the heavy chain variable regions, light chain variable regions, heavy chain constant regions, light chain constant regions and hypervariable regions of these antibodies.

Table 14

Amino acid sequences of heavy chains, light chains and their hypervariable regions (designated SEQ ID NO:)

Antibody Variable region Constant region Hypervariable region (HVR) (M) Heavy chain Light chain Heavy chain Light chain H1 H2 NZ L1 L2 L3 04H1389-G1m/04L1086-lam1 136 97 82 87 107 110 102 122 117 133 04Н1382-G1 m/04L1086-I am 1 135 97 82 87 107 109 102 122 117 133 04 Η1389-G1 m/04L13O5-kOMT 136 95 82 96 107 110 102 122 117 133

Biacore T200 is used to assess the binding of the resulting variants to ATP and to human CTLA4. The ATP binding assay was performed at 37°C using 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl ₂ , 0.05% Tween20 as running buffer. First, Sure Protein A (GE Healthcare) is immobilized on a SMZ S Series sensor chip (GE Healthcare), and the antibodies are captured on the chip by reacting with an antibody solution prepared in running buffer. Next, the ability to bind to the antibody is assessed by interaction with an ATP solution prepared in a running buffer. The chips were regenerated with 25 mM NaOH and 10 mM glycine-HCl (pH 1.5) and assayed by antibody recapture. To bind the amount of each antibody to ATP, the amount of ATP bound per unit amount of antibody is calculated by adjusting the amount of binding when ATP is injected at a concentration of 100 nM with the amount of antibody captured on the chip surface. Binding to human CTLA4 was measured using Biacore T200 according to the method described in Example 3-3. In table Figure 15 shows the results of these measurements.

Table 15 Human ATP and CTLA4 binding assay

Antibody ATP binding Binding to human CTLA4 Kd for human CTL A4 (M) Without ATP ATP 1 µM ATP 10 µM ATP 100 µM Without ATP ATP 10 µM ATP 100 µM 04H1207-G1m/04L10864am 1 0.00013 0.010 0.037 0.111 0.166 N.A. 4.0E-07 7.6E-08 04H1208-G1m/04L1086-lam1 0.00018 0.003 0.022 0.084 0.139 N.A. 5.1E-07 9.1E-08 04H1389-G1m/04L1086-lam1 0.00243 0.022 0.138 0.189 0.193 *5.1E-06 4.3E-08 2.1E-O8 04H1382 G1m/04L1086lam1 0.00204 0.008 0.090 0.152 0.159 N.A. 4.6E-08 1.4E-08 04H1389-G1 m/04L1305-kOMT 0.00190 0.030 0.120 0.154 0.151 *3.2E-06 1.9E-08 5.4E-09

NA is an extremely small value for determining _KD .

*The value of K _D is determined using a stationary model.

Antibodies 04H1389-G1m/04L1086-lam1 and 04H1382-G1m/04L1086-lam1 have increased binding ability to ATP compared to parental antibodies 04H1207-G1m/04L1086-lam1 and 04H1208-G1m/04L1086-lam1, for which R was not introduced 53Q/G55H . The binding ability of 04H1389-G1m/04L1086-lam1 and 04H1382-G1m/04L1086-lam1 to human CTLA4 increases approximately 10-fold in the presence of 10 μM ATP compared to the parent antibodies 04H1207-G1m/ ^04L1086 -lam1 and 04H1208-G1m/04 L1086-lam1 , ^for which R53Q/G55H has not been introduced. Comparison of the amount bound shows that the ability to bind to human CTLA4 was increased at lower ATP concentrations. It has also been shown that 04H1389-G1m/04L1305-k0MT, in which the 04H1389-G1m/04L1086-lam1 light chain is replaced by a human κ chain sequence, has equivalent ATP-binding capacity and ATP-dependent ability to bind human CTLA4 as 04H13 89- G1 m/04L 1086-lam1.

Example 4-2. Preparation of control anti-human CTLA4 antibody and anti-CTLA4 switch antibody and evaluation of their binding capacity.

A control antibody against human CTLA4 (MDX10D1H-mFa55/MDX10D1Lmk1, abbreviation: hNS-mFa55) and a switch antibody against CTLA4 (04H1389-mFa55/04L1305mk1, abbreviation: SW1389-mFa55) were generated. The hNS-mFa55 antibody uses the MDX10D1H heavy chain variable region (SEQ ID NO: 26) and the MDX10D1L light chain variable region (SEQ ID NO: 27), and the mFa55 mouse heavy chain constant region (SEQ ID

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NO: 18) and wild-type mouse light chain mk1 constant region (SEQ ID NO: 19). A mouse heavy chain constant region is also used, to which modifications have been added to enhance binding to the Fcy receptor. The antibody is expressed and purified in a manner known to those skilled in the art.

The SW1389-mFa55 antibody uses the heavy chain variable region 04H1389 (SEQ ID NO: 29) and the light chain variable region 04L1305 (SEQ ID NO: 30), and the mouse heavy chain constant region mFa55 (SEQ ID NO: 18) is used as the constant regions ) and wild-type mouse light chain mkl constant region (SEQ ID NO: 19). A mouse heavy chain constant region is also used, to which modifications have been added to enhance binding to the Fcy receptor. The antibody was expressed and purified in a manner known to those skilled in the art.

The binding of the designed hNS-mFa55 and SW1389-mFa55 to human CTLA4 was assessed. The running buffer is 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl ₂ , 0.05% Tween 20 with the addition of ATP to the required concentrations, the analysis is carried out at 37°C. First, rabbit anti-mouse IgG (Thermo Fisher Scientific) is immobilized on S-Series CM5 sensor chips (GE Healthcare), and the antibody is captured on the chip by reacting with an antibody solution prepared in an ATP-free running buffer. The binding ability of the anti-human CTLA4 antibody in the presence or absence of ATP is then assessed by reacting with a solution of human CTLA4 prepared in running buffer with ATP added to the desired concentration, or a solution of human CTLA4 prepared in running buffer without ATP. The chips were regenerated with 25 mM NaOH and 10 mM glycine-HCl (pH 1.5) and assayed by antibody recapture. The dissociation constant of each anti-CTLA4 antibody was calculated using Biacore T200 Evaluation Software 2.0. Specifically, the binding rate constant ka (l/mol/s) and the dissociation rate constant kd (1/s) are calculated by globally fitting the sensorograms obtained from measurements using the 1:1 Langmuir binding model, and the dissociation constant KD (mol/l ) are calculated from these values. In table Figure 16 shows the results of these analyses.

Table 16

Analysis of binding of mouse constant region variants to human CTLA4

Antibody K _D for human CTLA4 (M) ATP 1 µM ATP 10 µM ATP 100 µM MDX10DlH-mFa55/MDxl0DlL-mkl 3.2E-08 3.8E-08 3.6E-08 04H1389-mFa55/04L1305-mkl 5DE-08 1.8E-08 8.9E-09

Both antibodies generated using mouse constant regions were confirmed to bind to human CTLA4. SW1389-mFa55 was also shown to bind to human CTLA4 in an ATP-dependent manner, similar to 04H1389-G1m/04L1305-k0MT prepared using the same human variable region and constant region shown in Table. 15.

Example 4-3. Efficacy of each anti-CTLA4 switch antibody and anti-CTLA4 nonswitch antibody in a syngeneic tumor cell transplantation model using a human CTLA4 knockout, human CD3 transgenic mouse, increase/decrease of Treg cells in the tumor, and changes in a marker of systemic response in the spleen.

Example 4-3-1. Cell line.

Hepa1-6/hGPC3 cells were used. This cell line is obtained by purchasing the Hepa1-6 mouse liver cancer cell line from the ATCC collection, which constitutively expresses the human Glypican 3 gene (hGPC3, SEQ ID NO: 181) by transfection and cloning. Hepa1-6/hGPC3 cells are maintained and passaged in D-MEM (high glucose) medium (SIGMA) containing 10% FBS (SIGMA) and 600 μg/ml GENETICIN (Gibco).

Example 4-3-2. Preparation of a mouse model with a transplanted syngeneic tumor line.

A human CTLA4 KI, a human CD3 EDG-replaced mouse (hCTLA4 KI hCD3 EDG-replaced mouse), which is a hybrid of a human CTLA4 knockout mouse (Blood, 2005, 106(9): 3127-3133), and a mouse obtained by the authors of the present invention, are used. with human CD3 EDG replacement (Sci Rep 2017, 7: 45839). Hepa1-6/hGPC3 cells were transplanted subcutaneously into hCTLA4 KI hCD3 EDG replacement mice, and the model was determined to engraft when the average volume of transplanted tumors reached approximately 200 mm ³ to approximately 400 mm ³ .

The volume of the transplanted tumor is calculated using the formula:

Tumor volume=larger diameterxsmaller diameterxsmaller diameter/2.

Example 4-3-3. Obtaining medications for administration.

Anti-CTLA4 switching antibody (SW1389-mFa55) and control anti-human CTLA4 antibody (hNS-mFa55) obtained in Example 4-2 were prepared as drugs for administration to the Hepa16/hGPC3 cell transplantation model at concentrations of 0.01, 0 ,1, 1, 5, 10 and 20 mg/ml and 0.01, 0.1, 1 and 3 mg/ml, respectively, using His buffer (150 mM NaCl/20 mM HisHCl buffer, pH 6.0) .

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Example 4-3-4. Administration of a drug to measure the antitumor effect.

On day 7 after transplantation, mice were administered SW1389-mFa55 at doses of 0.1, 1, 10, and 100 mg/kg, and hNS-mFa55 was administered at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg and 30 mg/kg via the tail vein. In table 17 shows details of drug treatment when measuring the antitumor effect.

Table17

Measuring the antitumor effect in the Hepa1-6/hGPC3 cell transplant model

Group Animals Medicine Dose Introduction Date of introduction 1 4 His buffer - To the tail vein 7th day after transplantation 2 4 hNS-mFa55 0.1 mg/kg To the tail vein 7th day after transplantation 3 4 hNS-mFa55 1 mg/kg To the tail vein 7th day after transplantation 4 4 hNS-mFa55 10 mg/kg To the tail vein 7th day after transplantation 5 4 hNS-mFa55 30 mg/kg To the tail vein 7th day after transplantation 6 4 SW1389mFa55 OD mg/kg To the tail vein 7th day after transplantation 7 4 SW1389mFa55 1 mg/kg To the tail vein 7th day after transplantation 8 4 SW1389mFa55 10 mg/kg To the tail vein 7th day after transplantation 9 4 SW1389mFa55 100 mg/kg To the tail vein 7th day after transplantation

Example 4-3-5. Evaluation of antitumor effect.

The antitumor effect is assessed by tumor volume, calculated using the formula described in example 4-3-2. JMP 11.2.1 (SAS Institute Inc.) was used for statistical analysis.

Tumor growth inhibition (TGI - tumor growth inhibition) is calculated using the formula:

TGI (%) = (1 - (Average tumor volume in the study group at the time of measurement - Average tumor volume in the study group before antibody administration) / (Average tumor volume in the control group at the time of measurement - average tumor volume in the control group before antibody administration )) x 100

As a result, both hNS-mFa55 and SW1389-mFa55 showed drug efficacy TGI=60% or more on day 18 post-administration at doses of 1 mg/kg or more (FIGS. 14 and 15).

Example 4-3-6. Drug administration to evaluate Treg cells in the tumor and confirm systemic effects in the spleen.

On the 7th day after transplantation, SW1389-mFa55 was administered to mice at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg and 500 mg/kg via the tail vein, and hNS-mFa55 was administered i.d. doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg and 30 mg/kg via the tail vein. In table Figure 18 shows details of drug use to evaluate Treg cells in the tumor and confirm systemic effects in the spleen.

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Table 18

Validation of intratumoral and systemic effects in the Hepal______________________6/hGPC3 (hNS-mFa55 and SW1389-mFa55) transplanted cell model_____________________

Group Animals Medicine Dose Introduction Date of introduction 1 3 His buffer - To the tail vein 7th day after transplantation 2 3 hNS-mFa55 0.1 mg/kg To the tail vein 7th day after transplantation 3 3 hNS-mFa55 1 mg/kg To the tail vein 7th day after transplantation 4 3 hNS-mFa55 10 mg/kg To the tail vein 7th day after transplantation 5 3 hNS-mFa55 30 mg/kg To the tail vein 7th day after transplantation 6 3 SW1389mFa55 OD mg/kg To the tail vein 7th day after transplantation 7 3 SW1389mFa55 1 mg/kg To the tail vein 7th day after transplantation 8 3 SW1389mFa55 10 mg/kg To the tail vein 7th day after transplantation 9 3 SW1389mFa55 100 mg/kg To the tail vein 7th day after transplantation 10 3 SW1389mFa55 500 mg/kg To the tail vein 7th day after transplantation

Example 4-3-7. Tumor and spleen resection in Hepa1-6/hGPC3 cell-transplanted mouse models.

On day 6 after antibody administration, mice were euthanized under anesthesia, and the tumor and spleen were resected. From the obtained spleens, a cell suspension is prepared using RPMI-1640 medium (SIGMA) containing 10% FBS (SIGMA), and then hemolyzed using a mouse erythrocyte lysis kit (R&D) to obtain spleen cells. The resulting tumors are crushed using the Tumor Dissociation Kt, mouse kit (Miltenyi). Both spleen cells and crushed tumors react with the following antibodies, and the fractions of immune cells present are analyzed by FACS analysis: anti-CD45 antibody (BD, clone: 30-F11), anti-CD3 antibody (BD, clone: UCHT1), anti-CD4 antibody (BD, clone: RM4-5), anti-PDxP3 antibody (eBioscience, clone: FJK-16s), anti-ICOS antibody (eBioscience, clone: 7E17G9), anti-CCR7 antibody (Biolegend, clone: 4B12). FACS analysis is performed using a BD LSR Fortessa X-20 flow cytometer (BD).

Example 4-3-8. Evaluation of tumor Treg in a Hepa1-6/hGPC3 cell transplant model.

Changes in Treg effector cells (CD4+ FoxP3 ⁺ CCR7 ^low KLRG1') in the tumor after administration of SW1389-mFa55 or hNS-mFa55 were assessed. As a result, both hNS-mFa55 and SW1389-mFa55 reduced the proportion of effector Treg cells to less than 0.2% CD45-positive cells at doses of 1 mg/kg or more (Fig. 16).

Example 4-3-9. Evaluation of the systemic effect in the spleen in the Hepa1-6/hGPC3 cell transplantation model.

Changes in activated helper T cells (CD4 Foxp3 ^- ICOS ⁺ ) in the spleen after administration of hNS-mFa55 or SW1389-mFa55 were assessed by FACS. As a result, the ratio of activated helper T cells to CD45 positive cells in the spleen increased significantly with hNS-mFa55 administration, but no significant increase in activated helper T cells in the spleen was observed with SW1389-mFa55 administration, even with increasing administration dose (Fig. 17 ). This confirms that the switching antibody is as effective as the control antibody but does not induce a response in non-tumor tissues. Thus, the concept of exerting activity only locally in the tumor was proven in vivo in mice with human CTLA4 KI.

Example 4-4. Preparation of control anti-CTLA4 antibody and switch anti-CTLA4 antibody for toxicity testing in cynomolgus monkeys.

Prepare control anti-CTLA4 antibody (MDX10D1H-Kn125/MDX10D1Lk0MT//MDX10D1H-H1076/MDX10D1L-k0MT, abbreviation: NS-ART1) and switch anti-CTLA4 antibody (04H1389-Kn125/04L1305-k0MT//04H1389- H1076)/04L1305 -k0MT, abbreviation: SW1389ART1). The NS-ART1 antibody uses the heavy chain variable region MDX10D1H (SEQ ID NO: 26) and the light chain variable region MDX10D1L (SEQ ID NO: 27), and the human heavy chain constant region Kn125 (SEQ ID NO: 31) is used as the constant regions ) and the human heavy chain constant region H1076 (SEQ ID NO: 32), described in the prior patent literature WO 2013/002362, and the human light chain constant region k0MT (SEQ ID NO: 33). A human heavy chain constant region is also used, to which modifications have been added to enhance binding to the Fcy receptor. The antibody is expressed and purified in a manner known to those skilled in the art.

The SW1389-ART1 antibody uses the heavy chain variable region 04H1389 (SEQ ID NO: 29) and the light chain variable region 04L1305 (SEQ ID NO: 30), and the human heavy chain constant region Kn125 (SEQ ID NO: 31) is used as the constant regions ), human heavy chain constant region H1076 (SEQ ID NO: 32) and human light chain constant region k0MT (SEQ ID NO: 33). A human heavy chain constant region is also used, to which modifications have been added to enhance binding to the Fcy receptor. The antibody is expressed and purified in a manner known to those skilled in the art.

Heterodimerized antibodies (antibodies containing two different heavy chain polypeptides and/or two different light chain polypeptides) in the present invention are denoted by the following rule: (first heavy chain variable region)-(first heavy chain constant region)/(first light chain variable region) -(first light chain constant region)//(second heavy chain variable region)-(second heavy chain constant region)/(second light chain variable region)-(second light chain constant region). For example, if the name of an antibody is 04H1389Kn125/04L1305-k0MT//04H1389-H1076/04L1305-k0MT, this means that in this antibody the first variable region of the heavy chain is 04H1389, the first constant region of the heavy chain is Kn125, the first variable region of the light chain is 04L1305, the first light chain constant region is k0MT, the second heavy chain variable region is 04H1389, the second heavy chain constant region is H1076, the second light chain variable region is 04L1305, and the second light chain constant region is k0MT.

Example 4-5. Conducting a toxicity test in a cynomolgus monkey.

To evaluate and compare toxicity, including systemic response, NS-ART1 antibody or SW1389-ART1 antibody prepared in Example 4-4 was administered to male cynomolgus monkeys (3 each) at a dose of 60 mg/kg once a week for a total of 5 once. Administration is carried out slowly intravenously using a syringe pump, and general condition is monitored, body weight is measured, blood/blood chemistry is performed, bone marrow is examined, pathological examination is performed, and plasma drug concentrations are measured.

The appearance of antibodies to the drug is observed during the period of administration of both antibodies, but exposure is maintained until the end of the administration period. Administration of the NS-ART1 antibody results in changes similar to systemic autoimmune diseases, such as the appearance of autoantibodies, inflammatory changes (vasculitis, inflammatory cell infiltration), anemic changes and T-cell activation, while the above changes were not observed with the administration of the SW1389-antibody. ART1. These results indicate that the SW1389-ART1 antibody has reduced toxicity in vivo.

Example 5. Production of modified CTLA4 antibodies and evaluation of their activity.

The anti-CTLA4 switching antibodies obtained in Example 4 were further modified and evaluated.

Example 5-1. Antibody optimization through complex modifications and scaffold replacements.

Amino acid changes are specifically introduced into CDR 04H1389-G1m/04L1086-lam1 and the changes are examined with improved profiles. Targeted administration and assessment of amino acid changes are carried out using the method described in Example 3-3. Variations have been created that incorporate a combination of the changes found in the present invention and replace the frameworks. In table 19 lists the SEQ ID NOs of the heavy chain variable regions, light chain variable regions, heavy chain constant regions, light chain constant regions and hypervariable regions of these antibodies. Light chains 04L1594-lam1, 04L1581-lam1, 04L1610 ^- lam1, 04L1612-lam1 and 04L1610 ^- lam1 in table. 19 are introduced with changes in CDRs and frameworks compared to the light chain of the parent antibody 04L1086-lam1, and have frameworks and constant region germline human λ chain sequences. In addition, 04L1615-k0MT, 04L1616-k0MT, and 04L1617-k0MT have changes introduced in the 04L1086-lam1 CDR and have the framework and constant region of human κ chain germline sequences. The heavy chain variable region 04H1389v373 was introduced with changes in the CDR of the heavy chain variable region 04H1389 of the parent antibody. Heavy chain variable regions 04H1637, 04H1643, 04H1654, 04H1656, 04H1642 and 04H1735 have changes introduced in CDR 04H1389, as well as substitutions of framework sequences with other germline sequences.

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Table 19

Amino acid sequences of heavy chains, light chains and their hypervariable regions _________________________(indicated by SEQ ID NO:)__________________________

Antibody Variable region Constant region Hnpervarnatable region (HVR) (M) Heavy chain Light chain Heavy chain Light chain H1 H2 nz L1 L2 L3 04Н 1 389v373-G 1 m/04L1086-lam 1 137 97 82 87 107 111 102 122 117 133 04Н1637-G1m/O4L1086-lam1 138 97 82 87 107 111 102 122 117 133 04H1637-G1m/04L1594-ram1 04H1637-G1m/04L1 581 -laml 138 138 144 145 82 82 87 87 107 107 111 111 102 102 124 126 125 127 133 133 04H1637-G1 m/04L 16104am i 138 146 82 87 107 111 102 128 117 133 04H1643-G1m/04L1610-lam1 139 146 82 87 107 111 102 128 117 133 04H1654-Glm/04L1610-tam1 140 146 82 87 107 112 102 128 117 133 04H1656-Gtm/04Lt610-laml 141 146 82 87 107 111 152 128 117 133 04H1654-G1m/04L1612-lam1 140 147 82 87 107 112 102 129 117 133 04H1656-Gtm/04L1612-lam 1 141 147 82 87 107 111 152 129 117 133 04H1654-G1m/04Ll 606-lam 1 140 148 82 87 107 112 102 124 117 133 04H1656-G1m/04LI 606Ham1 141 148 82 87 107 111 152 124 117 133 04H1389-G1 m/04Ll 615-kOMT 136 149 82 96 107 110 102 130 117 133

The binding activity of the created variants to human CTLA4 is assessed by the method described in example 3-3 (Table 20).

Table 20

Human CTLA4 binding assay

Antibody Kd for human CTLA4 (Μ) Without ATP ATP 1 µM ATP 10 µM MDX10D1H-G1 m/MDX WD1 L-kOMT 4.8E-08 5.0E-08 4.9E-08 04Н1389-G1 m/04L1086-lam 1 *5.7E“06 1.8E-07 3.7E-08 04H1389v373-Glm/04L1086-lam1 * 5.3E-06 1.4E-07 2.8E-08 04H1637-G1 m/04L1086-lam 1 *5.3E-06 1.6E-07 3.0E-08 04H1637-G 1 m/04L1594-lam 1 *4.3E-06 1.4E-07 2.8E-08 04H1637-G 1 m/04L1581 -lam 1 *3.2E-06 8.9E-08 1.7E-08 04H1637-G 1 m/04L1610-lam 1 *Z.ZE-06 1.0E-07 1.8E-08 04H1643-G1 m/04L1610-lam 1 *2.8E-06 8.9E-08 1.4E-08 04H1654-G1 m/04L1610-lam 1 * 5.3E-06 1.2E-07 1.EE-08 04H1656-G 1 m/04L1610-lam 1 *2.2E-06 9.8E-08 1.EE-08 04H1654-G 1 m/04L1612-lam 1 *4.8E-06 2.0E-07 Z.ZE-08 04H1656-G 1 m/04L1612-lam 1 *7.6E-06 1.6E-07 3.2E-08 04H1654-G 1 m/04Ll 606-lam 1 *6.8E-06 1.4E-07 2.5E-08 04H1656-G 1 m/04L1606-lam 1 *2.3E-O6 9.5E-08 TEE-08 04H138 9-G1 m/04L 1305-kOMT *2.4E-06 8.2E-08 1.5E-08 04H138 9-G 1 m/04L 1615-k0MT *2.0E-06 7.2E-08 1.5E-08

* The value of K _D is determined using a stationary model.

KD values in the table marked * are calculated using a steady-state model. All variants obtained using 04H1389-G1m/04L1086-lam1 as the parent antibody were shown to bind to human CTLA4 in an ATP-dependent manner and have a stronger binding capacity than the parent antibody with a KD of 3.7x10 ^-8 M under conditions when ATP is present in an amount of 10 µM. All of these antibodies were shown to have stronger binding capacity than the existing anti-human CTLA4 antibody MDX10D1H-G1m/MDX10D1L-k0MT when ATP was present at 10 μM.

The ability of the resulting variants to bind to human CTLA4 in the presence of ADP or AMP was assessed using Biacore T200 and compared with the ability to bind in the presence of ATP. The binding ability to human CTLA4 in the presence of ADP or AMP is measured using the method described in Example 3-3, as for the binding ability in the presence of ATP, but using ADP or AMP instead of ATP (Table 21).

Table 21

Assessment of dependence on ATP, ADP and AMP

Antibody Kinetic parameters for human CTLA4. ATP 10 µM ADP 10 µM AMP 10 µM KofSsec *) LgYasek ^-1 ) Y<M) Ysek ^-1 ) £r(M) k'y(M ^_1 sec ^_1 ) foices ¹ ) Y<M) MDX10D1H-G1 m/MDX10D1 L-kOMT 2.9E+05 1.ZE-02 4.6E-08 3.6E+05 1.ZE-02 3.6E-08 3.4E+05 1.ZE-02 3.9E-0S 04H1389-G1 m/04L1086-laml 1.0E+05 3.2E-03 3.2E-08 1.4E+05 6.1E-03 4.4E-08 1.4E-U5 1.8E-02 1.ZE-07 04H1637-G1 m/04L161 Q-laml 1.2E+05 1.9E-03 1.5E-08 1.6E+05 3.5E-03 2.2E-08 1.5E+05 1.0E-02 7ΌΕ-08 04H1654-G1 m/04L1610-laml 7.1E+04 1.4E-03 1.9E-08 8.8E+04 2.8E-03 3.2E-08 8.9E+04 8.9E-03 1ΌΕ-07 04H1656-G1 m/04Ll 610-laml 1.3E+05 1.9E-03 1.5E-08 1.7E+05 4.3E-03 2.5E-08 1.7E+05 1.4E-02 8.2E-08 04H1654-G1 m/04L1612-laml 7.4E+04 2.7E-03 3.6E-08 8.9E+04 5.0E-03 5.6E-08 9.4 E+04 1.6E-02 1.7E-07 04H1656-G1m/04L1612-lam1 1.1E+05 2.9E-03 2.6E-08 1.5E+05 6.4E-03 4.2E-08 1.5E+05 2.0E-02 1.ZE-07 04H1654-G1 m/04L1606-laml 6.5E+04 1.ZE-OZ 2.1E-08 8.0E+04 2.8E-03 3.5E-08 8.1E1-04 9.0E-03 1.1E-07 04H1656-G1 m/04L1606-laml 1.2E+05 2.2E-03 1.9E-08 T6E+05 4.8E-03 3.0E-08 1.6E+05 1.6E-02 1.0E-07 04H1389-G1 m/04L1305-kOMT 1.1E+05 1.9E-03 1.7E-08 1.6E+05 3.8E-03 2.5E-08 1.5E+05 1.2E-02 7.9E-08 04H1389-G1 m/04L1615-kOMT 1.3E+05 1.5E-03 1.1E-08 1.8E+05 Z.OE-OZ 1.7E-08 1.7E+O5 9.7E-03 5.5E-08

The existing anti-human CTLA4 antibody MDX10D1H-G1m/MDX10D1L-k0MT shows similar kinetic parameters regardless of the type and presence of small molecules, whereas all ATP-dependent anti-CTLA4 antibodies bind to human CTLA4 not only in the presence of ATP, but also in the presence of ADP or AMP, and the binding capacity in the presence of these small molecules was higher than the binding capacity in the absence of small molecules shown in Table. 20. Thus, these antibodies have been shown to be antibodies that bind to CTLA4 in an ATP-, ADP-, and AMP-dependent manner. These antibodies have the highest binding capacity in the presence of ATP, followed by the highest binding capacity in the presence of ADP and the lowest binding capacity in the presence of AMP. In all cases, the binding capacity in the presence of ADP was approximately 3 times stronger than the binding capacity in the presence of AMP, and the binding capacity in the presence of ATP was approximately 5 times stronger than the binding capacity in the presence of AMP. The value of ka is the same in the presence of either small molecule, but there is a difference in the value of kd. Since dissociation in the presence of ADP occurs faster than in the presence of ATP, and further dissociation in the presence of AMP occurs faster than in the presence of ADP, the difference in KD value depending on the type of small molecule is shown to be due to the difference in the rate of dissociation.

The binding ability of some variants to mouse CTLA4 and cynomolgus CTLA4 is then assessed. Binding activity to human CTLA4, mouse CTLA4 and cynomolgus CTLA4 was assessed using Biacore T200 according to the method described in Example 3-3 (Table 22). Cynomolgus CTLA4 was obtained by the following method.

The CyCTLA4-His-BAP gene (SEQ ID NO: 50), which is a fusion of a His tag and a BAP tag at the C-terminus of the extracellular region of cynomolgus macaque CTLA4, was synthesized and inserted into an animal expression plasmid. The resulting plasmid is introduced by lipofection into the FreeStyle 293-F cell line, obtained from human embryonic kidney cells (Invitrogen), these cells are seeded in a flask after suspension in the FreeStyle 293 expression medium (Invitrogen) at a cell density of 1.33.x 10 ⁶ cells/ml. Three hours after transfection of the plasmid, biotin is added to a final concentration of 100 μM, the cells are cultured in a CO ₂ incubator (37 ° C, 8% CO ₂ , 125 rpm) for 4 days and the antigen is purified by a method known to specialists in the field , from the culture supernatant of each sample. The absorbance of purified antigen solutions at 280 nm is measured using a spectrophotometer. From the measured values obtained, the concentration of the purified antigen is calculated using the extinction coefficient calculated by the PACE method (Protein Science, 1995, 4, 2411-2423).

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Table 22

Human, mouse, cynomolgus macaque CTLA4 binding assay

Kd for human CTLA4 (Μ) Kd for mouse CTLA4 (M) Kd for CTLA4 monkey (M) Antibody ATP 1 µM ATP 10 µM ATP 100 µM ATP 1 µM ATP 10 µM ATP 100 µM ATP 1 µM ATP 10 µM ATP 100 µM MDX10D1H-G1 m/MDX10D1 L-kOMT 4.7E-08 5.3E-08 5.4E-08 N.T. N.T. N.T. 1 1.6E-O7 1 1.2E-O7 1 1.2E-O7 04H1389~G1m/04L1305-kOMT 7.2E-08 1.4E-08 4.6E-09 2.1 E-07 3.2E-08 1.2E-08 I 1.4E-07 1 2.6E-08 1 1.2E-08 04H1654-G1m/04L1610-lam1 1.1E-07 1.5E-08 4.4E-09 1.8E-07 2.7E-08 1.0E-08 I 1.8E-O7 1 2.8E-08 1 8.1E-09 04H1656-G1m/04L1610-lam1 7.9E-08 1.6E-08 6.2E-09 2.3E-07 3.7E-08 1.4E-08 I 1.2E-O7 1 2.6E-08 1 1.0E-08 04Ш 654-G1 m/04L1612-lam 1 1.7E-07 Z.OE-O8 1.1E-08 3.9E-07 4.8E-08 1.8E-08 I 3.1E-07 1 5.4E-08 1 2.1E-08 04H1656-G1m/04L1612-laml 9.5E-08 2.5E-08 7.8E-09 3.2E-07 5.4E-08 2.1E-08 1 1.6E-O7 1 4.1E-08 1 1.6E-08 O4H1389-G1m/Q4L1615-k0MT 5.8E-08 1.2E-08 5.0E-09 1.6E-07 2.6E-08 1.0E-O8 I 1.1E-07 1 2.1E-08 1 9.3E-09

N.T. - They didn’t investigate.

All six small molecule-dependent CTLA4 antibodies studied were found to bind not only to human CTLA4, but also to mouse CTLA4 and cynomolgus CTLA4 in an ATP-dependent manner.

Example 5-2. Preparation of modified anti-CTLA4 switch antibodies and negative control antibodies.

Generate modified anti-CTLA4 switching antibodies (04H1654-mFa55m2P1/04L1610 ml0r//04H1656-mFa55m2N1/04L1610^-ml0r, abbreviation: SW1610^-mFa55;04H1654-mFa55m2P1/04L1612ml0r//04H1656-mFa55m2N1/04L1612-m10r, abbreviation: SW1612-mFa55; and 04H1389-mFa55/04L1615mk1, abbreviation: SW1615-mFa55) and negative control antibody (IC17Hdk-mFa55/IC17L-mk1, abbreviation: KLH-mFa55).

The SW1615-mFa55 antibody uses the heavy chain variable region 04H1389 (SEQ ID NO: 29) and the light chain variable region 04L1615 (SEQ ID NO: 34), and the mouse mFa55 heavy chain constant region (SEQ ID NO: 18) is used as the constant regions ) and wild-type mouse light chain mk1 constant region (SEQ ID NO: 19). A mouse heavy chain constant region to which modifications have been added to enhance binding to the Fcy receptor is also used. The antibody is expressed and purified in a manner known to those skilled in the art.

In the antibody SW1610 ^- mFa55, as constant regions, one heavy chain variable region 04H1654 (SEQ ID NO: 35) is linked to the mouse heavy chain constant region mFa55m2P1 (SEQ ID NO: 36), the other heavy chain variable region 04H1656 (SEQ ID NO: 37 ) is linked to the mouse heavy chain constant region mFa55m2N1 (SEQ ID NO: 38), and for the light chain variable region 04L1610 (SEQ ID NO: 39) to the wild-type mouse light chain constant region mFa55m2N1 (SEQ ID NO: 39). (SEQ ID NO: 22). The antibody is expressed and purified in a manner known to those skilled in the art.

In the antibody SW1612-mFa55, as constant regions, one heavy chain variable region 04H1654 (SEQ ID NO: 35) is linked to the mouse heavy chain constant region mFa55m2Pl (SEQ ID NO: 36), another heavy chain variable region 04H1656 (SEQ ID NO: 37 ) is linked to the mouse heavy chain constant region mFa55m2N1 (SEQ ID NO: 38), and for the light chain variable region 04L1612 (SEQ ID NO: 40) to the wild-type mouse light chain constant region mFa55m2N1 (SEQ ID NO: 22). The antibody is expressed and purified in a manner known to those skilled in the art.

In the negative control antibody, as constant regions, the heavy chain variable region IC17Hdk (SEQ ID NO: 51) is linked to the mouse heavy chain constant region mFa55 (SEQ ID NO: 18), and for the light chain variable region IC17L (SEQ ID NO: 52) using the wild-type mouse light chain mk1 constant region (SEQ ID NO: 19). The antibody is expressed and purified in a manner known to those skilled in the art.

Example 5-3. Evaluation of the binding ability of antibodies having a mouse constant region to human CTLA4.

The ability of anti-CTLA4 antibodies having a mouse constant region to bind to antigen was assessed by the method described in Example 4-2 (Table 23). All of these mouse constant region antibodies were found to have the same ATP-dependent ability to bind to human CTLA4 as the antibodies shown in Table 1. 22, having the same variable region, but with a human constant region.

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Table 23

Analysis of binding of mouse constant region variants to human CTLA4

Kd for human CTLA4 (Μ) Antibody ΑΤΦ 1 µM ΑΤΦ 10 µM ΑΤΦ 100 µM MDX10D1 H-mFa55/MDX10D1 L-mk1 3.2Ε-08 3.8Ε-08 3.6E-08 04Н1654-mFa55m2P1 /04L1610-ml0r 5.8Ε-08 1.7Ε-08 8.5E-09 04H1656-mFa55m2N1/04L1610-ml0r 4.7Ε-08 1.ZE-08 7.4E-09 04H1654-mFa55m2P1 /04L1612-ml0r 9.4Ε-08 2.9E-08 1.2E-08 04H1656-mFa55m2N1/04L1612-rrlOr 6.7Ε-08 2.0E-08 1.0E-08 04H1389-rriFa55/04L1615-mk1 4.5Ε-08 1.5E-08 8.7E-09

Example 5-4. Efficacy of anti-CTLA4 switching antibodies in a syngeneic tumor cell transplant model using human CTLA4 knockin, human CD3 transgenic mouse, with increased/decreased Treg cells in the tumor and altered systemic response marker in the spleen.

Example 5-4-1. Cell line.

Hepa1-6/hGPC3 cells were used. This mouse liver cancer cell line Hepa1-6 was acquired from the ATCC collection and has constitutive expression of the human Glypican 3 (hGPC3) gene as a result of transfection and cloning. Hepa1-6/hGPC3 cells are maintained and passaged in D-MEM (high glucose) medium (SIGMA) containing 10% FBS (Sigma) and 0.6 mg/ml G418 (Nacalai Tesque).

Example 5-4-2. Generation of a mouse model with a transplanted syngeneic tumor.

Human CTLA4 KI, human CD3 EDG knockout mice (hCTLA4 KI hCD3 EDG mice) are used, which are a hybrid of a human CTLA4 knockout mouse (Blood, 2005, 106(9):3127-3133) and a mouse produced by the present authors invention, with replacement by human CD3 EDG (Sci Rep, 2017, 7: 45839). Hepa1-6/hGPC3 cells are transplanted subcutaneously into hCTLA4 KI hCD3 EDG replacement mice, and the model is determined to be established when the average volume of transplanted tumors reaches from about 200 mm to about 400 mm ³ .

The volume of the transplanted tumor is calculated using the following formula:

Tumor volume=larger diameterxsmaller diameterxsmaller diameter/2.

Example 5-4-3. Obtaining the drug for administration.

The drugs administered to Hepa16/hGPC3 cell transplant models are the anti-CTLA4 switch antibodies (SW1610 ^- mFa55, SW1612-mFa55, SW1615-mFa55) prepared in Example 5-2. Drugs for administration are prepared using His buffer (20 mM His-HCl, 150 mM NaCl, pH 6.0) so that they are at concentrations of 0.03, 0.1 and 0.3 mg/ml.

Example 5-4-4. Administration of a drug to measure the antitumor effect.

On day 8 after transplantation, mice were injected via the tail vein with three samples of anti-CTLA4 switching antibodies at doses of 0.3, 1, and 3 mg/kg, respectively. In table 24 shows details of drug treatment when measuring the antitumor effect.

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Table 24

Measuring the antitumor effect in a Hepa1-6/hGPC3 cell transplant model _____________________(anti-CTLA4 switching antibodies)_____________________

Group Animals Medicine Dose Introduction Date of introduction 1 5 His buffer - To the tail vein 8th day after transplantation 2 5 SW1615mFa55 0.3 mg/kg To the tail vein 8th day after transplantation 3 5 SW1615mFa55 1 mg/kg To the tail vein 8th day after transplantation 4 5 SW1615mFa55 3 mg/kg To the tail vein 8th day after transplantation 5 5 SW1615mFa55 o.z mg/kg To the tail vein 8th day after transplantation 6 5 SW1615mFa55 1 mg/kg To the tail vein 8th day after transplantation 7 5 SW1615mFa55 3 mg/kg To the tail vein 8th day after transplantation 8 5 SW1615mFa55 0.3 mg/kg To the tail vein 8th day after transplantation 9 5 SW1615mFa55 1 mg/kg To the tail vein 8th day after transplantation 10 5 SW1615mFa55 3 mg/kg To the tail vein 8th day after transplantation

Example 5-4-5. Evaluation of antitumor effect.

The antitumor effect is assessed by tumor volume, calculated using the formula described in example 5-4-2.

The rate of tumor growth inhibition (TGI - Tumor Growth Inhibition) is calculated using the following formula:

TGI (%) = (1 - (Mean tumor volume in the study group at the time of measurement - Average tumor volume in the study group at the time of initial administration) / (Average tumor volume in the control group at the time of measurement average tumor volume in the control group at the time of initial administration introduction)) x 100

As a result, the effectiveness of the drug SW1610 ^- mFa55 and SW1612-mFa55 according to the TGI criterion is 60% or more on the 16th day after administration at doses of 1 mg/kg or more, and SW1615-mFa55 at doses of 3 mg/kg or more (Fig. 18-20).

Example 5-4-6. Drug administration to evaluate Treg cells in the tumor and confirm systemic effect in the spleen.

On the ^10th day after transplantation, mice are administered through the tail vein SW1610 ^- mFa55 in doses of 50 mg/kg, 100 mg/kg and 200 mg/kg, SW1612-mFa55 is administered in doses of 50 mg/kg, 100 mg/kg and 200 mg /kg, and SW1615mFa55 was administered at doses of 50 mg/kg, 100 mg/kg, 200 mg/kg and 400 mg/kg. In addition, in the control group, the negative control antibody IC17Hdk-mFa55/IC17L-mkl (abbreviation: KLH-mFa55) was administered via the tail vein at a dose of 400 mg/kg. In table Figure 25 shows details of drug treatment to evaluate Treg cells in the tumor and confirm systemic effects in the spleen.

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Table 25

Evaluation of intratumoral and systemic effects in mouse models transplanted with Hepa1-6/hGPC3 cells (switching anti-CTLA4 antibodies)

Group Animals Medicine Dose Introduction Date of introduction 1 3 KLH-mFa55 400 mg/kg To the tail vein 10th day after transplantation 2 3 SW1610mFa55 50 mg/kg To the tail vein 10th day after transplantation 3 3 SW1610mFa55 100 mg/kg To the tail vein 10th day after transplantation 4 3 SW1610mFa55 200 mg/kg To the tail vein 10th day after transplantation 5 3 SW1612mFa55 50 mg/kg To the tail vein 10th day after transplantation 6 3 SW1612mFa55 100 mg/kg To the tail vein 10th day after transplantation 7 3 SW1612mFa55 200 mg/kg To the tail vein 10th day after transplantation 8 3 SW1615mFa55 50 mg/kg To the tail vein 10th day after transplantation 9 3 SW1615mFa55 100 mg/kg To the tail vein 10th day after transplantation 10 3 SW1615mFa55 200 mg/kg To the tail vein 10th day after transplantation AND 3 SW1615mFa55 400 mg/kg To the tail vein 10th day after transplantation

Example 5-4-7. Resection of tumors and spleens in model mice transplanted with Hepa1-6/hGPC3 cells.

On day 6 after antibody administration, mice were euthanized under anesthesia, and tumors and spleens were separated. From the obtained spleens, a cell suspension was prepared using RPMI1640 medium (SIGMA) containing 10% FBS (SIGMA), and then hemolyzed using a mouse erythrocyte lysis kit (R&D) to obtain spleen cells. The resulting tumors are crushed using a mouse tumor dissociation kit (Miltenyi). Both spleen cells and crushed tumors react with the following antibodies and the fractions of immune cells present are analyzed by FACS: anti-CD45 (BD, clone: 30-F11), anti-CD3 (BD, clone: UCHT1), anti-CD4 antibody (BD, clone: RM4-5), anti-FoxP3 antibody (eBioscience, clone: FJK-16s), anti-ICOS antibody (eBioscience, clone: 7E17G9), anti-CCR7 antibody (Biolegend, clone : 4B12), antibody against KLRG1 (Biolegend, clone: 2F1/KLRG1). FACS analysis is performed using a BD LSR Fortessa X-20 (BD).

Example 5-4-8. Assessment of tumor Tregs in a Hepa1-6/hGPC3 cell transplantation model.

Assess changes in Treg effector cells (CD4+ FoxP3 ⁺ CCR7 ^low KLRG1') in tumors after administration of anti-CTLA4 switching antibodies. As a result, SW1610 ^- mFa55, SW1612-mFa55 and SW1615mFa55 reduced the proportion of effector Treg cells to less than 0.2% CD45-positive cells at all administered doses (Fig. 21).

Example 5-4-9. Evaluation of the systemic effect in the spleen in a model with transplanted Hepa1-6/hGPC3 cells.

Changes in activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) in the spleen after administration of anti-CTLA4-switching antibodies are assessed using the FACS method. As a result, the ratio of activated helper T cells to CD45-positive cells in the spleen does not increase significantly at the 50 mg/kg evaluated doses for SW1610 ^- mFa55 and SW1612-mFa55 and at 200 mg/kg or less evaluated doses for SW1615-mFa55. Dunnett's test was performed on the KLH-mFa55 treated group using JMP 11.2.1 (SAS Institute Inc.) to determine the significance test (FIG. 22). It has been confirmed that although all switching antibodies are effective, they do not induce a response in tissues other than the tumor and that they only have the ability to be active locally in the tumor.

Example 6: Creation of an Fc region variant capable of enhancing ADCC/ADCP activity.

To obtain antibodies with enhanced ADCC and ADCP activity, which are cytotoxic effector functions, the production of Fc region variants with increased ability to bind to the activating FcYR receptors FcYRIIIa and FcYRIIa is being explored.

Example 6-1. Generation and evaluation of variants with increased binding to FcyR.

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Get heterodimerized antibody 04H1637-Kn125/04L1610^-lam1//04H1637-H1076/04L1610 laml having the heavy chain constant regions Kn125 and H1076 with increased FcyR binding ability described in WO 2013/002362 and having 04H1637 as the heavy chain variable region and 04L1610^-lam1 as a light chain. In particular, the antibody heavy chain gene 04H1637-Kn125 (SEQ ID NO: 162) was created, which contains 04H1637 (SEQ ID NO: 138) as one heavy chain variable region and has L234Y/L235Q/G236W/S239M/H268D/D270E/ S298A introduced into G1d (SEQ ID NO: 158) with Gly and Lys removed at the C-terminus of the human IgG1 heavy chain constant region, and also has Y349C/T366W changes in the CH3 region that promote heterodimerization. Similarly, the antibody heavy chain gene 04H1637-H1076 (SEQ ID NO: 163) was generated, which contains 04H1637 (SEQ ID NO: 138) as another heavy chain variable region and has D27OE/K326D/A33OM/K334E introduced into the G1d constant region human IgG1 heavy chain (SEQ ID NO: 158), and also has changes D356C/T366S/L368A/Y407V in the CH3 region, which promote heterodimerization. Using 04L1610^-lam1 (SEQ ID NO: 161) as the light chain of the antibody, obtaining the heterodimer 04H1637-Kn125/04L1610^-lam1//04Н1637-Н1076/04L1610 lam1 in a manner known to those skilled in the art. Antibody heavy chain genes 04H1637-Kp462 (SEQ ID NO: 164), 04H1637-H1441 (SEQ ID NO: 165), 04H1637-H1445 (SEQ ID NO: 166), 04H1637-Kn461 (SEQ ID NO: 167) and 04H1637- H1443 (SEQ ID NO: 168), which, in addition to L235Q, G236W, S239M, H268D, D270E, S298A, K326D and K334E, have changes made to the CH2 region of L234F and ADZOC described in WO 2013/002362 as changes, which alter binding to FcyR; G236A, I332E and I332D, reported in Mol. Cancer Ther., 2008, 7, 2517-2527 and WO 2004/029207; and T250V and T307P, specified in WO 2013/118858, as changes to improve stability, in combination. In addition, the antibody heavy chain gene O4H1654-KT462 (SEQ ID NO: 182) was obtained, in which Gly and Lys at the C-terminus of human IgG1 were removed (IGHG1*03), and it has the same changes as Kn462 in the CH2 region , an E356K change that promotes heterodimerization as described in WO 2006/106905 in the CH3 region and also contains 04H1654 (SEQ ID NO: 140) as the heavy chain variable region. In a similar way, the antibody heavy chain gene 04H1656-HT441 (SEQ ID NO: 170) was created, in which Gly and Lys at the C-terminus of human IgG1 (IGHG1*03) are removed, and it has the same changes as H1441 in the CH2 region, change K439E , which promotes heterodimerization as described in WO 2006/106905 in the CH3 region, and also contains 04H1656 (SEQ ID NO:141) as the heavy chain variable region. The genes 04H1656-HT445 (SEQ ID NO: 171), 04H1654-KT461 (SEQ ID NO: 183) and 04H1656HT443 (SEQ ID NO: 173) were created in a similar way. In addition, a combination of changes to improve the blood kinetics of antibodies described in Mabs, 2017, 9, 844-853 was investigated. In particular, the gene 04H1654-KT473 (SEQ ID NO: 184) was created, in which N434A/Y436T/Q438R/S440E was introduced into the CH3 region of 04H1654-KT462 (SEQ ID NO: 182), which is a combination of changes that enhance binding to Human FcRn under acidic conditions and changes that reduce binding to rheumatoid factor. Similarly, a gene was created for 04H1656-HT482 (SEQ ID NO: 185), which has N434A/Y436T/Q438R/S440E introduced in 04H1656-HT445 (SEQ ID NO: 171). Similarly, the antibody heavy chain 04H1654-KT481 (SEQ ID NO: 186) and the antibody heavy chain 04H1656-HT498 (SEQ ID NO: 187) were obtained by introducing the same changes into 04H1654-KT461 and 04H1656-HT443, respectively. By combining these heavy chains and using 04L1610^-lam1 or 04L1612-lam1 (SEQ ID NO: 188) as the light chain, the desired heterodimerized antibodies were obtained.

The extracellular domains of FcyR are obtained by the following method. First, the FcyR extracellular domain genes are synthesized in a manner known to those skilled in the art. Also, the sequences of each FcyR are obtained based on information registered in NCBI. In particular, FcyRI was obtained based on the NCBI sequence with accession number NM_000566.3, FcYRIIa was obtained based on the NCBI sequence with accession number NM_001136219.1, FcYRIIb was obtained based on the NCBI sequence with accession number NM_004001.3, FcYRIIIa was obtained based on the NCBI sequence with accession number access NM_004001.3. derived from the sequence accession number NCBINM_001127593.1, and a His tag was added to the C-terminus. The polymorphic site of FcYRIIa was obtained with reference to J. Exp. Med., 1990, 172, 19-25, and the FcYRIIIa polymorphic site was prepared with reference to J. Clin. Invest., 1997, 100, 1059-1070. Expression vectors are produced by inserting the resulting gene fragments into expression vectors in animal cells. The resulting expression vectors were transiently introduced into FreeStyle293 cells, derived from human embryonic kidney cancer cells (Invitrogen), to express proteins of interest. Once the culture supernatants are obtained, they are passed through a 0.22 μm pore filter and purified in principle through the following four steps. The first step is cation exchange column chromatography (SP Sepharose FF), the second step is His-tag affinity column chromatography (HisTrap HP), the third step is gel filtration column chromatography (Superdex200), and the fourth step is aseptic filtration. However, for FcyRI, the first step was anion exchange column chromatography using Q-Sepharose FF. The purified protein concentration is calculated by measuring the absorbance at 280 nm using a spectrophotometer and using the coefficient

- 84 045931 extinction factor calculated from the obtained value using a method such as PACE (Protein Science, 1995, 4, 2411-2423). Human FcRn is obtained by the method described in WO 2010/107110.

The interaction between the resulting antibodies and human FcyR was analyzed using a Biacore T200 method. 50 mM Na-phosphate, 150 mM NaCl, 0.05% Tween20 (pH 7.4) are used as a running buffer; the analysis is carried out at 25°C. Series S SA chips (GE Healthcare) are used as sensor chips, on which the CaptureSelect Human Fablambda Kinetics biotin conjugate (Thermo Fisher Scientific) is immobilized. The antibodies under study are captured by these chips, and conditions are created for each FcyR, diluted in the working buffer, to interact with them. The chips were regenerated with 10 mM glycine-HCl (pH 1.5) and 25 mM NaOH, and the assay was performed by antibody recapture. Dissociation constants KD (mol/L) for FcyR and each antibody were calculated using Biacore T200 Evaluation Software 2.0 using the 1:1 Langmuir binding model for FcYRIa and FcYRIIIa and the steady state affinity model for FcYRIIa. For FcYRIIb, the amount of FcYRIIb binding per unit amount of antibody is calculated by adjusting the amount of FcYRIIb binding obtained from the sensogram constructed by measuring with the amount of antibody captured on the chip surface.

The interaction between the resulting antibodies and human FcRn was analyzed using a Biacore T200 method. 50 mM Na-phosphate, 150 mM NaCl, 0.05% Tween20 (pH 7.4) are used as a running buffer; the analysis is carried out at 25°C. Series S SA chips (GE Healthcare) are used as sensor chips, on which the CaptureSelect Human Fablambda Kinetics biotin conjugate (Thermo Fisher Scientific) is immobilized. The antibodies under study are captured by these chips, and conditions are created for FcRn, diluted in a running buffer, to interact with them. The chips were regenerated with 10 mM glycine-HCl (pH 1.5) and 25 mM NaOH, and the assay was performed by antibody recapture. The dissociation constant for FcRn and each antibody was calculated using a steady state model using Biacore T200 2.0 evaluation software. Table 26 shows the measurement results.

Table 26

Analysis of binding of Fc region variants to human FcyRs and FcRn receptors

Antibody Zaman ish in the house CH2_in the constant region of the heavy chain Kp or KT Substitutions in the CH2 domain in the constant region of the heavy chain H1 or HT Co for hFcRn <M) Relative in magnitude between Glm and hFcRn IQ, for hFcyR (M) Binding quantity Relative KD value between Glm and hFcyR Relative binding amount hFcyR hFcyR Ite R hFcyR Ite H hFcyR tlte F hFcyR Illa V hFcyR lib hFo-jrr la hFoyR Ite R hFcyR Ga H HFcyR Illa F hFcyR Ute V hFcyR lib 04Н163?-G1m/04L1610-lani1 1.ZE-06 1.0 4.9E-1I 2.0E-06 1.5ENE6 1.4E-06 246-07 0.008 10 10 1.0 1.0 1.0 10 Q4H1 637-Kn 125/04(.1 $1 0-1 "t1 // 04Н1 637—HI076/04L1610-larr1 Zo !gS Sg «la SM *·> «ch and « « og?oe/kz2es»/Azzom/K334E N.T. NT 4.CE-1I 1-2E-08 4.ιε-07 3.4E-09 1 96-09 0007 1.2 1.6 3.6 406.3 125.6 0.9 04Н1 637-Kh462/04L1 61 0-Ι«ί>1// 04H1637-HI44l/04L1610-lam1 1-2 MF/L235O/G23eWZ S23SM/T25OV/H2S8DZ D2706/S298A/T307P/ K326D G236AZT25OV/D27OEZ S29SA/T3O7P/K325DZ K3346 N.T. NT 1.5E-10 2.2E-07 7.0E-08 1.7E-09 5.2E-C9 0.013 03 9.2 20.9 79.5 45.8 1.6 04H1 637-Kn462/04L1 61 0-laml // 04H1637-N1445/04iv10-1lt1 L234F/L235Q/G236WZ S239M/T25OV/H268D/ D27O6/S298A/T307P7 K326D > ζβ ¢3 W g" m " and " yoi 2 * with tn g with N.T. NT 7.66-11 1.16-07 3.9E-08 3.86-09 1.8E-09 0030 0.6 187 3 7.5 382.2 130 7 3.7 04H1 63?-Kn461 /041.1 51 ol ₉ ri1 // 04H1 637-HI443/04L1610-lam1 L2 34F/L235Q/G236WZ $ШМ/Т25Ф7/НШС>/ D 270E/S2S8A7T307P/ K32FD/1332E G236A/T250V/D270E/ S298A/T3O7P/K326OZ 1E32E/K3346 N.T. NT 4.26-11 1.9E-C7 1.36-07 2.26-09 1.16-09 0025 12 10.7 11.1 616 5 225 7 3 1 04H1854-KT462/O4L1610-lam1// 04H1 658-HT445/04Ll610-lam1 L2 34F/L235Q/G238WZ S239M/T250V/H258D/ ΒΪ7Ο£Ζ$298Α/Τ3ΰ7Ρ/ K326D o~o 5s! “W® O O! fO « « 0 ω < 1.5E-O6 0.9 ZOE-11 1.6E-07 1 IE-07 33E-O9 2.2E-09 0025 1.6 13.0 13.9 414.7 109.6 3 1 04H1 654- KT461/04L1610-laml // 04H1S58 -HT448/04LI6l0-teml L234F/L235Q/G236W/ S2 39M/T2 5OV/H2 88D/ D27M/S2S8A/T307P/ K326D/J332E G236A/T250V/D270EZ S298A/T3O7P/K326OZ Ι332Ε/Κ3346 1.5E-O6 0.9 3 26-I 2.4E-07 2.OE-O7 26E-09 1.56-09 0025 1.5 8 4 7.3 525.5 156 1 3.1 04H1 854-KT473/04L1610-larn1// 04H1 65i-HT482/04LI610-lam1 L234F/L235Q/G236WZ S239M/T25OV/H28SD/ D270E/S298A/T307PZ KYA26G. SSuj he y 3 §&o N 1- 2 4 < ϋ « « e « o SCH-Y " 5.8E-07 2 3 4 6E-1I 1.5E-07 8.16-08 3.8E-09 2.IE-09 0026 1.1 13.9 18.0 3634 1146 32 04H1654-KT481/04L1610-lam1// 04H1 85U—HT498/04L1610-tem1 L234F/L235O/G236WZ S239M/T25OV/H268D/ D 2706/8298А/Т307Р/ K326D/1332E G236A/T2S0V/D2706Z S298A/T3O7PZK326DZ 1E32E/K.3346 57E-O7 2.3 3.3 E-11 2.6E-C7 2.3E-O7 28E-O9 1.6E-09 0022 1.$ 77 6.5 49S.2 152.0 2.7 04H1654-KT462/O4Ll612-lam1/Z 04H1S5C-HT445/04L1612-lam1 Li 34F/L235Q/G236W/ S239M/T25OV/H268O/ D270E/5293A/T307PZ K378D G236A/T250V/D270EZ 8298A/T3O7P/K326OZ A33OK/I332D/K334E 1 66-OS 0 3 4.16-11 1.46-0? 9.16-08 3.46-09 2.36-09 0.028 1.2 14.2 16.1 408.0 105.7 3.4 04Hl854-KT46l/O4Ll612-teml// D4H1658-HT443/04L1612-lam1 L2 S4F/L235O/G236WZ S23SM/T25OV/H268D/ D270E/S298A/T307P/ K3?fin/13.19F G236AZT250V/D270EZ S298A/T3O7P/K.326DZ I332E/K334E 1 6E-O6 0 8 3.9E-11 2.5E-07 2.IE-07 2.6E-O9 1.BE-09 0.025 1.3 8 2 7.1 536 8 1588 3.1 Q4H1654-KT473/04L1612-lani1// 04H1 656-HT482ZO4L101 2-larril L234F/L235O/G236WZ S239M/T25OV/H268D/ D270E/S298AZT307P/ K326D G236A/T250V/D270EZ S298A/T3O7P/K326DZ A330K/I332D/K334E 6 2E-O7 2.1 4.BE-11 1.5E-07 9.1E-O8 39E-O9 2.2E-09 0026 1.0 138 1S2 354.6 1100 32 04H1654-KT4ei/O4Liei2-lam1// 04H165t-HT49e/O4L1612-lam1 L2 34F/L235Q/G236WZ S239M/T250V/H268D/ D270E/S29BA/T307PZ K326D/J332E G236A/T250V/D270EZ S298A/T3O7P/K326DZ I332E/K334E 6.OE-O7 2 2 4.3E-11 2.7E-C7 2.3E-07 296-09 1.76-09 0022 eleven 7 6 6.4 479 2 145.2 5 6

N.T.: Not explored.

The KD values for hFcRn (M) and KD for hFcYR receptors (M) in the table indicate the dissociation constants for hFcRn and each FcyR, respectively, and the amount of binding indicates the amount of FcYRIIb bound per unit amount of antibody when FcYRIIb was allowed to interact at 1000 nM. The terms relative value for KD between G1m and hFcRn and relative value for KD between G1m and hFcYR receptors are values obtained by dividing the KD value for 04H1637-G1m/04L1610'lam1 for hFcRn and for each FcyR by the KD value for each variant, respectively . The term relative binding amount means the value obtained by dividing the amount of binding of each variant to FcYRIIb by

- 85 045931 binding quantity 04H1637-G1m/04L1610-lam1. Amino acid sequences of the heavy chain of antibody 04H1637-G1m and the light chain of antibody 04L1610^-lam1 are shown in SEQID NO: 160 and 161, respectively. It was shown that all the obtained heterodimerized antibodies have increased binding to FcYRIIa and FcYRIIIa compared to 04H1637-G1m/04L1610^-lam1, which has the constant region of native human IgG1. In addition, it was shown that 04H1637-Kn462/04L1610^-lam1//04H1637H1441/04L1610^-lam1, 04H1637-Kn462/04L1610^-lam1//04H1637-H1445/04L1610^-lam1 and 04H1637Kn461/04L1610^-laml//04H1637-H1443/04L1610^-lam1 have increased binding to FcYRIIa compared to 04H1637-Kn125/04L1610^-lam1//04H1637-H1076/04L1610^-lam1, which has a variant of the Fc region described in WO 2013/002362. It has been shown that the binding capacity of 04H1637Kn462/04L1610^-lam1//04H1637-H1445/04L1610^-lam1 with FcYRIIIa is equal to that of 04H1637-Kn125/04L1610 lam1//04H1637-H1076/04L1610^-lam1, a binding capacity 04H1637-Kn461/04L1610 lam1//04H1637-H1443/04L1610^-lam1 with FcYRIIIa is higher compared to 04H1637-Kn125/04L1610 lam1//04H1637-H1076/04L1610^-lam1. It is shown that in a similar way 04H1654-KT462/04L1610 laml//04H1656-HT445/04L1610^-lam1 and 04H1654-KT462/04L1612-lam1//04H1656-HT445/04L1610^-lam1, having different changes for heterodimerization of the constant region and the CH3 region in IGHG1*03, have comparable FcyR binding profiles to 04H1637-Kn462/04L1610^-lam1//04H1637H1445/04L1610^-lam1, and also shown that 04H1654-KT461/04L1610^-lam1//04H1656-HT443/04L1610^-lam1 and 04Н1654-KT461/04L1612-lam1//04H1656-HT443/04L1610^-lam1 have comparable FcyR to FcyR binding profiles to 04H1637-Kn461/04L1610^-lam1//04H1637-H1443/04L1610^-lam1. In addition, 04H1654KT473/04L1610^-lam1//04H1656-HT482/04L1610^-lam1, 04H1654-KT481/04L1610^-lam1//04H1656HT498/04L1610^-lam1, 04H1654-KT473/04L1612-lam1//04H1656-HT482/04L1612-lam1 and 04H1654KT481/04L1612-laml//04H1656-HT498/04L1612-lam1 with modifications to improve blood kinetics have improved FcRn binding ability human and comparable ability binding to FcyR compared to antibodies before changes were made to improve blood kinetics.

The 04H1656-HT451 gene (SEQ ID NO: 272) was also created, in which N434A/Y436T/Q438R/S440E is introduced into the 04H1656-HT441 sequence, which is a combination of changes that increase binding to human FcRn under acidic conditions, and changes that reduce binding to rheumatoid factor. The amino acid sequence of the heavy chain of antibody HT451 is shown in SEQ ID NO: 276. The heterodimerized antibody is prepared by combining O4H1654-KT473 and 04H1656-HT451 and using 04L1610 ^- lam1 as the antibody light chain. In table 27 shows the results of an analysis of the interaction of the resulting antibodies with human FcRn and with human FcyR.

Table 27

Analysis of binding of Fc region variants to FcyR and FcRn

Antibody $ flj pq s i

Kp for hFcyR (M)

I*

Relative KD value between Gkn and hFcyR

Sil

04Н1656-G1 m/04L1610-lam 1

04H1654-KT462/04L1610-I am 1 // 04H1656-HT441/04L1610-lam1

04H1654-KT473/04L1610-lam 1 //

04H1656-HT451 /04L1610-lam 1

But

HFcyR la hFcyR Pa R hFcyR Pa Η hFcyR Pa F

HFcyR Sha V

HFcyR Kb hFcyR la

HFcyR Ila R hFcyR Pa H hFcyR Illa F hFcyR Sha V hFcyR p

1.7E-O6

9.2E-11

1.6E-06

1.ZE-06

1.0E-06

9.7E-07

0.003

1.0

1.0

1.0

1.0

1.0

1.0

2ΌΕ-Ο6

1.SE-07

9.9E-08

1.7E-08

4.4E-09

0.014

0.8

9.0

13.5

58.1

218.3

5.4E-07

3.2

1.8E-07

9.2E-08

2.0E-08

5.1E-09

0.012

0.6

8.9

14.5

50.8

190.3

1.5 * The value of K _D is determined using a stationary model.

It was established that the resulting heterodimerized antibodies O4H1654-KT462/O4L161O lam1//04H1656-HT441/04L1610^-lam1 and 04H1654-KT473/04L1610^-laml//04H1656-HT451/04L1610^-lam1 have enhanced binding to activating FcyRs, namely FcYRIIIa and FcYRIIIa, compared to 04H1656-G1m/04L1610^-lam1, which has the constant region of native human IgG1. In addition, binding to FcYRIIb, which is FcyR inhibitory, was maintained at the same level as 04H1656-G1m/04L1610^-lam1 in both of these antibodies. Shown to be 04H1654-KT473/04L1610 lam1//04H1656-HT451/04L1610^-lam1, in which the N434A/Y436T/Q438R/S440E fragment was introduced into 04H1654-KT462/04L1610^-lam1//04H1656-HT441/04L1610^-lam1 exhibits increased ability to bind to human FcRn compared to before the modification.

Enhanced FcyR binding variants were then generated using various heterodimerization changes described in Nat. Biotechnol., 1998, 16, 677-681, their binding activity to human FcRn and FcyR was assessed. Antibody heavy chain genes 04H1389-Ks462 (SEQ ID NO: 191) and 04H1389-Km462 (SEQ ID NO: 199) are obtained, which contain 04H1389 (SEQ ID NO: 136) as the heavy chain variable region and the heavy chain constant region, which has the same changes as KT462, introduced into the CH2 region of the constant region with Gly and Lys removed at the C-terminus of human IgG1 (IGHG1*03), and, in addition, contains T366W in the CH3 region, introduced as a change to

- 86 045931 heterodimerization for 04H1389-Ks462, and y349C/T366W introduced as a heterodimerization change for 04H1389-Km462. In addition, the heavy chain genes of the antibodies 04H1389-Hs445 (SEQ ID NO: 192) and 04H1389-Hm445 (SEQ ID NO: 200) were obtained, which have the same changes as HT445 introduced into the CH2 region, and, in addition, have in the CH3 region T366S/L368A/Y407V, introduced as changes for heterodimerization of 04H1389-Hs445. Similarly, 04H1389-Ks461 (SEQ ID NO: 193), 04ffi389-Km461 (SEQ ID NO: 201), 04H1389-Hs443 (SEQ ID NO: 194) and 04H1389-Hm443 (SEQ ID NO: 202) are obtained, which have those the same changes as KT461 and HT443 in the CH2 region. In addition, antibody heavy chain genes 04H1389-Ks473 (SEQ ID NO: 195), 04H1389-Hs482 (SEQ ID NO: 196), 04H1389-Ks481 (SEQ ID NO: 197), 04H1389-Hs498 (SEQ ID NO: 198) are obtained ), 04H1389-Km473 (SEQ ID NO: 203), 04H1389-Hm482 (SEQ ID NO: 204), 04H1389-Km481 (SEQ ID NO: 205) and 04H1389-Hm498 (SEQ ID NO: 206), which have N434A changes /Y436T/Q438R/S440E, which improve blood kinetics, introduced into the above heavy chain constant regions Ks462, Hs445, Ks461, Hs443, Km462, Hm445, Km461 and Hm443, and contain 04H389 as a variable region. 04L1615-k0MT (SEQ ID NO: 190) is used as the light chain and the hetero dimers of interest are obtained. For comparison, a homodimer 04H1389-G1m/04L16150k0MT containing 04H1389-G1m (SEQ ID NO: 189) was obtained. The interaction between the designed antibodies and human FcyR receptors was analyzed using Biacore T200. 50 mM Na-phosphate, 150 mM NaCl, 0.05% Tween20 (pH 7.4) are used as a running buffer; the analysis is carried out at 25°C. For the sensor chip, Series S SA chips (GE Healthcare) were used, onto which the CaptureSelect Human Fab-kappa Kinetics biotin conjugate (Thermo Fisher Scientific) was immobilized. The antibodies of interest are captured on these chips, and each FcyR, diluted in running buffer, is allowed to interact with them. The chips were regenerated with 10 mM glycine-HCl (pH 1.5) and 25 mM NaOH and assayed by antibody recapture. Dissociation constants KD (mol/L) for the FcyR of each antibody were calculated using Biacore T200 Evaluation Software 2.0 and a 1:1 Langmuir binding model for FcYRIa and FcYRIIIa, and a steady-state affinity model for FcYRIIa. For FcYRIIb, the amount of FcYRIIb binding per unit amount of antibody is calculated by adjusting the amount of FcYRIIb binding obtained from the sensogram generated from the measurement with the amount of antibody captured on the chip surface. To measure binding to FcRn, 50 mM Na-phosphate, 150 mM NaCl, 0.05% Tween20 (pH 6.0) are used as a running buffer, and the dissociation constant KD (mol/l) is calculated using the steady state model (Table 28 ). As for the dissociation constants for FcYRIIIa in Table. 28, the values marked with * are the values calculated using the stationary affinity model.

Table 28

Analysis of binding of Fc region variants to human FcyR and FcRn receptors

Antibody Code hFcRn CM) Relative KD value between Glm and hFcRn Kd giving hFcyR (M) Binding quantity Relative KD value between Glm and hFcyR Relative binding amount hFcy Ria hFcyR Ila R hFcyR Pa H hFcyR Sha F hFcyR Sha V hFcyR lib hFcyRla hFcyR Ila R hFcyR Pa H hFcyR Sha V hFcyR pn 04Н1 389-G1m/04L1615-kOMT 1.3E-06 1.0 4.5E-11 1.5E-06 1.IE-06 N.T. 9.1E-07 0.011 1.0 1.0 1.0 1.0 1.0 O4H1389-Ks462/O4l_1615-kOMT// 04H1389-Hs445/04L1615-kOMT 1.6E-06 0.8 4.7E-11 1.6E-07 6.7E-08 2.IE-09 1.1E-09 0.030 1.0 9.1 15.8 829.9 2.6 04H1389-Ks461 Z04L1615-kOMT// 04H1389-Hs443/04L1615-kOMT 1.7E-06 0.8 3.0E-11 2.5E-07 1.6E-07 1.2E-09 5.6E-10 0.024 1.5 5.9 6.7 1627.2 2.1 04H1389-Ks473/04L1615-kOMT// 04H1389-Hs482/O4L161 5-kOMT 4.5E-O7 2.9 5.1E-11 1.6E-07 7.5E-08 2.3E-09 1.2E-09 0.026 0.9 9.0 14.1 760.2 2.3 04H1389-Ks481 /04L161 5-kOMT// 04H1389-Hs498/04L 1615-kOMT 4.6E-07 2.8 2.8E-11 2.6E-O7 1.9E-07 1.3E-09 6ΌΕ-10 0.023 1.6 5.6 5.6 1500.7 2.0 04H1389-Km462/04L1615-kOMT// 04H1389-Hm445/04L1615-kOMT 1.7E-06 0.8 4.8E-11 1.6E-07 7.4E-08 2.2E-09 1.1E-09 0.029 0.9 9.5 14.4 810.6 2.6 04H1389-Km461 /04L1615-kOMT// 04H1389-Hm443/04L1615-kOMT 1.7E-06 0.7 2.5E-11 2.6E-07 1.9E-07 1.2E-09 5.6E-10 0.024 1.8 5.8 5.7 1620.3 2.1 04H1389-Km473/04L1615-kOMT// 04H1389-Hm482/04L1615-kOMT 4.6E-Q7 2.8 6.2E-11 1.8E-O7 8.7E-08 2.6E-09 1.3E-09 0.024 0.7 8.2 12.3 705.1 2.1 04H1389-Km481 /04L1615-kOMT// 04H1389-Hm498/04L1615-kOMT 4.7E-07 2.8 4.2E-11 2.9E-07 2.1E-07 1.4E-09 6.2E-10 0.021 1.1 5.1 5.0 1469.1 1.9

* The value of K _D is determined using a stationary model.

N.T. - They didn’t investigate.

All heterodimerized antibodies produced in the present invention have increased binding activity to FcYRIIa and FcYRIIIa compared to 04H1389-G1m/04L1615-k0MT. Hm482/04L1615-k0MT and 04H1389-Km481/04L1615-k0MT//04H1389-Hm498/04L1615-k0MT, which were modified to improve the kinetics of antibodies in the blood, have increased binding activity to human FcRn compared to the original antibody before the modification and regarding

- 87 045931 FcyR binding activity they had the same binding profile as the parent antibody.

In addition, the gene 04H1389-Hm441 (SEQ ID NO: 273), which contains 04H1389 (SEQ ID NO: 136) as a heavy chain variable region, has the same changes as HT441 introduced into the CH2 region of the heavy chain, and contains Y349C. /366W used for changes to heterodimerize in the CH3 region. In contrast, 04H1389-Hm451 (SEQ ID NO: 274) was produced with N434A/Y436T/Q438R/S440E introduced, which is a combination of changes that increase binding to human FcRn under acidic conditions and changes that decrease binding to rheumatoid factor. The amino acid sequences of the antibody heavy chains Hm441 and Hm451 are shown in SEQ ID NO: 277 and 278, respectively. Heterodimerized antibodies were prepared using 04H1389Km473, 04H1389-Hm451 or 04H1389-Hm482 as the antibody heavy chain and 04L1305-k0MT as the antibody light chain. In table Figure 29 shows the results of an analysis of the interaction of the obtained antibodies with human FcRn and with human FcyR receptors.

Table 29

Analysis of binding of Fc region variants to human FcRn and human FcyR receptors

Antibody Code hFcRn (M) Relative KD value between Glm and hFcRn K _D for hFcyR (M) Binding quantity Relative KD value between Glm and hFcyR Otho strongly e binding amount hFcyR la hFcyR Pa R hFcyR Ila H hFcyR Illa F hFcyR Sha V hFcyR lib hFcyRla hFcyR Ila R hFcyR Ila H hFcyR Illa F hFcyR Sha V hFcyR lib 04Н1389-G1 m/04L1305-kOMT 1.0E-06 1.0 6.9E-11 1.6E-06 1.1E-06 6.9E-07 *8.8E-07 0.011 1.0 1.0 1.0 1.0 1.0 1.0 04Н1389-Km473/04L1305-kOMT// 04H1389-Hm451 /04L13O5-kOMT 3.3E-07 3.0 1.5E-10 1.6E-07 5.3E-08 1.7E-08 3.9E-09 0.015 0.5 9.7 20.0 41.3 224.7 1.3 04H1389-Km473/04L1305-kOMT // 04H1389-Hm482/04L1305-kOMT 3.7E-07 2.7 6.1E-11 8.6E-08 3.5E-08 2.7E-09 9.5E-10 0.034 1.1 18.5 30.8 258.1 930.5 3.0

* The KD value is determined using a stationary model.

It was shown that the resulting heterodimerized antibodies 04R1389-Km473/04L1305k0MT//04H1389-Hm451/04L1305-k0MT and Ο4Η1389-Κ^473/Ο4Ε13Ο5-ΜΜΤ//Ο4Η1389-^482/Ο4Ε1 3Ο5k0MT have increased binding to activating receptors FcyR, namely FcYRIIa and FcYRIIIa, compared to 04H1389-G1m/04L1305-k0MT, which have the constant region of native human IgG1. Both of these antibodies were also shown to have increased ability to bind to human FcRn compared to 04H1389-G1m/04L1305-k0MT.

The FcyR-enhanced binding constant region variants found in the present invention are then compared to existing FcyR-binding enhanced variants. Generate antibody heavy chain genes MDX10D1H-Kn125 (SEQ ID NO: 217), MDX10D1H-H1076 (SEQ ID NO: 218), MDX10D1H-Kn462 (SEQ ID NO: 219), MDX10D1H-H1445 (SEQ ID NO: 220), MDX10D1H -Kn461 (SEQ ID NO: 221) and MDX10D1H-H1443 (SEQ ID NO: 222), which contain MDX10D1H (SEQ ID NO: 154) as the heavy chain variable region, and the heavy chain constant regions listed in table. 26. and the MDX10D1H-G1m gene (SEQ ID NO: 210), containing the CH2 region of native human IgG1. The antibody heavy chain gene MDX10D1H-GASDIE (SEQ ID NO: 215) was created, which has the changes G236A/S239D/I332E in the CH2 region, as described in Mol. Cancer Ther., 2008, 7, 2517-2527, as a variant with enhanced binding to FcYRIIa. In addition, the antibody heavy chain gene MDX10D1HGASDALIE (SEQ ID NO: 216) was generated, which has G236A/S239D/A330L/I332E in the CH2 region, as described in J. Struct. Biol., 2016, 194, 78-89 as a variant with enhanced binding to FcYRIIIa. To obtain antibodies of interest, the MDX10D1L-k0MT antibody (SEQ ID NO: 211) is used as the light chain. The binding activity of these antibodies to human FcYR is measured as described above using CaptureSelect Human Fab-kappa Kinetics biotin conjugate (Table 30).

Table 30

Analysis of binding of Fc region variants to human FcyR receptors

Antibody K _D for hFcyR (M) Binding amount Relative KD value between Glm and hFcyR Relative binding amount hFcyR Ila R hFcyR Ila H hFcyR Sha F hFcyR Sha V hFcyR lib hFcyR Pa R hFcyR Pa H hFcyR Sha V hFcyR lib MDX10D1H-G1 m/MDX 10D1 L-kOMT 1.5E-O6 1.0E-06 N.T. *9.5E-07 0.012 1.0 1.0 1.0 1.0 MDX10D1H-GASD1E/MDX1OD1 L-kOMT S.1E-08 5.5E-08 2.3E-08 1.4E-08 0.072 24.2 18.9 68.2 5.8 MDX10D1H-GASDAUE/MDX10D1 L-kOMT 7.9E-O7 9.7E-07 1.2E-08 6.9E-09 0.044 1.9 1 I 1.1 1 I 138.1 I I 3.6 MDX10D1H-Kn125/MDX10D1H-HI076/MDX1OD1L-k0M1 9.6E-07 2.7E-07 1.9E-09 1.IE-09 0.009 1.5 3.8 898.8 0.7 MDX10D1H-Kn462/MDX10D1H-HI445/MDX10D1L-k0Ml 1ΌΕ-Ο7 2.8E-08 2.7E-O9 1.2E-09 0.037 14.9 37.3 807.1 3.0 MDX10D1H-Kn461/MDX10D1H-HI443/MDX10D1L-k0Ml 1.7E-O7 8.8E-08 1.4E-09 5.7E-10 0.031 9.0I 1 11.8 1 I 1076.4 I I 2.5

* The value of K _D is determined using a stationary model. NT - Not explored.

- 88 045931

The KD value for hFcYR (M) in the table means the dissociation constant for each of the listed FcyRs, and the binding amount means the amount of FcYRIIb binding per unit amount of antibody when FcYRIIb has the opportunity to interact at 1000 nM. The relative KD value between G1m and hFc','R receptors means the value obtained by dividing the KD value of MDX10D1H-G1m/MDX10D1L-k0MT for each FcyR by the KD value of each variant, and the relative binding amount means the value obtained by dividing the binding amount of each variant with FcYRIIb on the amount of MDX10D1H-G1m/MDX10D1L-k0MT binding.

The resulting heterodimers MDX10D1H-Kn125/MDX10D1H-H1076/MDX10D1L-k0MT, MDX10D1H-Kn462/MDX10D1H-H1445/MDX10D1L-k0MT and MDX10D1H-Kn461/MDX10D1HH1443/MDX10D1L-k0 MT all have increased binding to FcYRIIIa compared to existing antibodies MDX10D1H-GASDIE/ MDX10D1L-k0MT and MDX10D1H-GASDALIE/MDX10D1Lk0MT with enhanced FcyR binding. MDX10D1HKn462/MDX10D1H-H1445/MDX10D1L-k0MT binding to FcYRIIaH was also shown to be approximately 2-fold higher compared to the existing FcYRIIa-enhanced MDX10D1H-GASDIE/MDX10D1L-k0MT antibody.

Example 6-2. In vitro assessment of ADCC activity of various antibodies with altered constant regions.

To analyze ADCC activity in vitro, the hFcYRIIIaV ADCC Reporter Bioassay, Core Kit (Promega) is used. Add 25 μl of hCTLA4-CHO cell suspension as target cells to each well of a 96-well plate at a concentration of up to 2x10 ⁶ /ml using a medium containing assay buffer (90% RPMI1640, 10% FBS). Then add 25 µl of each antibody solution diluted in assay buffer so that the final concentration is 0, 0.001, 0.01, 0.1 and 1 µg/ml. 25 µl of Jurkat cells expressing hFcYRIIIaV (included in the kit), prepared to a concentration of 6x10 ⁶ /ml with the medium, are then added as an effector cell suspension, so that the solutions are mixed to a total volume of 75 µl. The plate is left to stand at 37°C overnight in a 5% CO ₂ incubator. The plate is then left at room temperature for 15 minutes and 75 µl of Bio-Glo reagent is added to each well. The Bio-Glo reagent uses the Bio-Glo Luciferase Assay System (buffer and substrate). The luminescence of each well is then measured using a plate reader. The value obtained by dividing the luminescence value of each well by the luminescence value of the wells without antibodies is determined as the fold induction, which is used as an indicator to evaluate the ADCC of each antibody. The results obtained are presented in Fig. 23. In the figure, the fold induction is presented in relative luminescence units (RLU).

These results indicate that the ADCC activity of Fc-altered antibodies against hCTLA4-CHO cells is higher than that of wild-type human IgG1 constant region.

Example 6-3. In vitro assessment of ADCP activity of various antibodies with altered constant regions.

The hFcYRIIaH ADCP Reporter Bioassay Core Kit (Promega) is used to assay ADCC activity in vitro. Add 25 μl of hCTLA4-CHO cells as target cells to each well of a 96-well plate at a concentration of 1x10 ⁶ /ml prepared using assay buffer-based medium (with 4% RPMI1640 medium in which contains a small amount of IgG serum). Then add 25 µl of each antibody solution diluted in assay buffer so that the final concentration is 0.001, 0.01, 0.1 and 1 µg/ml. Finally, 25 μl of hFcYRIIaH-expressing Jurkat cells included in the kit are added as an effector cell suspension such that the solutions are mixed to a total volume of 75 μl. The plate is left at 37°C overnight in a 5% CO ₂ incubator. The density of the cell solution of Jurkat cells expressing hFcYRIIaH is 8.25x10 ⁵ /ml. The plate is then left at room temperature for 15 minutes and 75 µl of Bio-Glo reagent is added to each well. The Bio-Glo reagent used is the Bio-Glo Luciferase Assay System (Buffer and Substrate). The luminescence in each well is then measured using a plate reader. The value obtained by dividing the luminescence value of each well by the luminescence value of the wells without antibodies is determined as the fold induction, which is used as an index to evaluate the ADCP of each antibody. The results obtained are presented in Fig. 24. In the figure, fold induction is represented in relative luminescence units (RLU).

The results show that the ADCP activity of antibodies containing an altered Fc fragment against hCTLA4-CHO cells is higher than that of human wild-type IgG1 constant region.

Example 6-4. In vitro assessment of ADCC activity of an anti-CTLA4 switch antibody with an altered Fc fragment.

Generate anti-CTLA4 switch antibodies with altered Fc (04H1654-Kn462/04L1610 lam1//04H1656-H1445/04L1610^-lam1, abbreviation: SW1610-ART6; 04H1654-Kn462/04L1612

- 89 045931 lam1//04H1656-H1445/04L1612-lam1, abbreviation: SW1612-ART6;and 04H1389-Kn462/04L1305k0MT//04H1389-H1445/04L1305-k0MT, abbreviation: SW138 9-ART6).

In the SW1610-ART6 antibody, one heavy chain variable region 04H1654 (SEQ ID NO: 35) is linked to the human heavy chain constant region Kn462 (SEQ ID NO: 43), another heavy chain variable region 04H1656 (SEQ ID NO: 37) is linked to the human constant heavy chain the human heavy chain region H1445 (SEQ ID NO: 44) as the constant region, and for the light chain variable region 04L1610 (SEQ ID NO: 39) with the wild type human light chain constant region lam1 (SEQ ID NO: 39). ID NO: 53). The antibody is expressed and purified in a manner known to those skilled in the art.

In the SW1612-ART6 antibody, one heavy chain variable region 04H1654 (SEQ ID NO: 35) is linked to the human heavy chain constant region Kn462 (SEQ ID NO: 43), another heavy chain variable region 04H1656 (SEQ ID NO: 37) is linked to the human constant heavy chain the human heavy chain region H1445 (SEQ ID NO: 44) as the constant region, and for the light chain variable region 04L1612 (SEQ ID NO: 40) with the human wild type laml light chain constant region (SEQ ID NO: 53). The antibody is expressed and purified in a manner known to those skilled in the art.

In antibody SW1389-ART6, two heavy chain variable regions 04H1389 (SEQ ID NO: 29) are linked, respectively, to the human heavy chain constant region Kn462 (SEQ ID NO: 43) and the human heavy chain constant region H1445 (SEQ ID NO: 44) As a constant region, as well as for the light chain variable region 04L1305 (SEQ ID NO: 30), the wild-type human 1<0MT light chain constant region (SEQ ID NO: 33) is used. The antibody is expressed and purified in a manner known to those skilled in the art.

The hFcYRIIIaV ADCP Reporter Bioassay Core Kit (Promega) is used to assay ADCC activity in vitro. Add 12.5 μl of hCTLA4-CHO cells as target cells to each well of a 96-well plate at a concentration of 2x10 ⁶ /ml prepared using assay buffer-based medium (with 4% RPMI1640 medium, which contains a small amount of IgG serum). Then sequentially add ATP solutions diluted with assay buffer, so that the final concentration is 0 and 100 μM, and solutions of antibodies SW1389-ART6, SW1610-ART6 and SW1612-ART6, diluted with assay buffer to a final concentration of 0, 0.001, 0 .01, 0.1 and 1 µg/ml. Finally, 25 µl of Jurkat cells expressing hFcYRIIIaV (included in the kit) are diluted with medium to a concentration of 3x10 ⁶ /ml and added as an effector cell suspension such that the total volume in the mixture is 75 µl. The plate is left at 37°C for 6 hours in an incubator with 5% CO ₂ . The plate is then left at room temperature for 15 minutes and 75 µl of Bio-Glo reagent is added to each well. The Bio-Glo reagent used is the Bio-Glo Luciferase Assay System (Buffer and Substrate). The luminescence in each well is then measured using a plate reader. The value obtained by dividing the luminescence value of each well by the luminescence value of the wells without antibodies is determined as the fold induction, which is used as an index to evaluate the ADCP of each antibody. The results obtained are presented in Fig. 25 (SW1389-ART6), fig. 26 (SW1610-ART6) and fig. 27 (SW1612-ART6). In the figures, fold induction is presented in relative luminescence units (RLU).

From the results obtained, it was confirmed that the ADCC activity of anti-CTLA4 switching antibodies possessing the altered fragment against hCTLA4-CHO cells differs in the presence and absence of ATP, and that ATP-dependent cytotoxicity against hCTLA4-CHO cells is present.

Example 6-5. In vitro assessment of the neutralizing activity of anti-CTLA4 switch antibodies.

Generated modified anti-CTLA4 switch antibodies, human antibodies with variable regions SW1389, SW1610, SW1612 and SW1615.

For antibody SW1389, 04H1389 (SEQ ID NO: 29) is used as the heavy chain variable region and 04L1305 (SEQ ID NO: 30) is used as the light chain variable region. After binding of the variable regions to the human constant regions, the antibody is expressed and purified in a manner known to those skilled in the art.

For antibody SW1610, 04H1654 (SEQ ID NO: 35) and 04H1656 (SEQ ID NO: 37) are used as the heavy chain variable region and 04L1610 (SEQ ID NO: 39) as the light chain variable region. After binding of the variable regions to the human constant regions, the antibody is expressed and purified in a manner known to those skilled in the art.

For antibody SW1612, 04H1654 (SEQ ID NO: 35) and 04H1656 (SEQ ID NO: 37) are used as the heavy chain variable region and 04L1612 (SEQ ID NO: 40) as the light chain variable region. After binding of the variable regions to the human constant regions, the antibody is expressed and purified in a manner known to those skilled in the art.

For antibody SW1615, 04H1389 (SEQ ID NO: 29) is used as the heavy chain variable region and 04L1615 (SEQ ID NO: 34) is used as the light chain variable region. After binding the variable regions to the human constant regions, the antibody is expressed and purified by

- 90 045931 bom, known to experts in the field.

To measure in vitro neutralizing activity, CTLA-4 Blockade Bioassay (Promega) is used. To each well of a 96-well plate, 25 µl of aAPC-Raji cells attached to the kit are added as target cells, prepared at a concentration of 1x10 ⁶ / ml in the medium, and the assay buffer (10% FBS in RPMI1640) is used as the medium. . ATP solutions are then diluted with assay buffer to final concentrations of 0 and 100 μM, after which antibody solutions having variable regions SW1389, SW1610, SW1612, and SW1615 are serially diluted with assay buffer to final concentrations of 0, 0.001, 0.01, 0.1 , 1 and 10 µg/ml. Finally, 25 μl of IL2-luc2CTLA4-Jurkat cells (included in the kit), prepared to a concentration of 2x10 ⁶ /ml by adding medium, are added as a suspension of effector cells so that the solution is mixed to a total volume of 75 μl. The plate is left at 37°C for 6 hours in a 5% CO ₂ incubator. The plate is then left at room temperature for 15 minutes and 75 µl of Bio-Glo reagent is added to each well. The Bio-Glo reagent uses the Bio-Glo Luciferase Assay System (buffer and substrate). The luminescence of each well is then measured using a plate reader. The value obtained by dividing the luminescence value of each well by the luminescence value of the wells without antibodies is determined as the fold induction, which is used as an indicator to evaluate the neutralizing activity of each antibody. The results obtained are shown in Fig. 28 (SW1389), fig. 29 (SW1610), fig. 30 (SW1612) and fig. 31 (SW1615). In the figures, fold induction is presented in relative luminescence units (RLU).

From the results obtained, it was confirmed that the neutralizing activity of anti-CTLA4 switch antibodies against hCTLA4-expressing cells differs in the presence and absence of ATP, and that the neutralizing activity is ATP-dependent.

Example 6-6. In vitro evaluation of the cytotoxic activity of an anti-CTLA4 switch antibody against CTLA4-positive regulatory T cells.

Anti-CTLA4 switching antibodies with an altered Fc fragment are obtained (04H1654KT473/04L1610 ^- lam1//04H1656-HT451/04L1610 ^- lam1, abbreviation: SW1610 ^- ART5+ACT1;04H1654KT473/04L1610 ^- lam1//04H165 6-HT482/04L1610 ^- lam1, abbreviation : SW1610-ART6+ACT1; 04H1389Km473/04L1305-k0MT//04H1389-Hm451/04L1305-k0MT, abbreviation: SW1389-ART5+ACT1; 04H1389Km473/04L1305-k0MT//04H1389-H m482/04L1305-k0MT, abbreviation: SW1389-ART6 +ACT1).

For the SW1610-ART5+ACT1 antibody, O4H1654-KT473 (SEQ ID NO: 184) is used as one heavy chain, 04H1656-HT451 (SEQ ID NO: 272) is used as the other heavy chain, and 04L1610 lam1 (SEQ ID NO: 161) is used as the light chain. The antibody is expressed and purified in a manner known to those skilled in the art.

For the SW1610-ART6+ACT1 antibody, 04H1654-KT473 (SEQ ID NO: 184) is used as one heavy chain, 04H1656-HT482 (SEQ ID NO: 185) is used as the other heavy chain, and 04L1610 lam1 (SEQ ID NO: 161) is used as the light chain. The antibody is expressed and purified in a manner known to those skilled in the art.

For the SW1389-ART5+ACT1 antibody, 04H1389-Km473 (SEQ ID NO: 203) is used as one heavy chain, 04H1389-Hm451 (SEQ ID NO: 274) is used as the other heavy chain, and 04L1305k0MT (SEQ ID NO: 275) is used as a light chain. The antibody is expressed and purified in a manner known to those skilled in the art.

For the SW1389-ART6+ACT1 antibody, 04H1389-Km473 (SEQ ID NO: 203) is used as one heavy chain, 04H1389-Hm482 (SEQ ID NO: 204) is used as the other heavy chain, and 04L1305k0MT (SEQ ID NO: 275) is used as a light chain. The antibody is expressed and purified in a manner known to those skilled in the art.

The in vitro cytotoxic activity of the obtained anti-CTLA4 antibodies with an altered Fc fragment against CTLA4-positive regulatory T cells (CD3+ CD4+ CD25+ CD45RA ^- CTLA4+) is assessed. First, human PBMC (CTL Cryopreserved Human MKPC, CTL) are freeze-thawed and suspended in 50 U/ml interleukin 2 (HJI-2)/RPMI/10% FBS such that the cell density is 2x10 ⁶ cells/ml, and cultured at 37°C for 4 days in an incubator with 5% CO2. After 4 days, cells are collected and washed twice with RPMI/10% FBS and then seeded at 100 µl into each well of a 96-well U-bottom plate (8 x ¹⁰⁵ cells/well or ⁵ x 105 cells/well) and into each well. add 25 μl of KLH-G1m solution adjusted with RPMI/10% FBS to a concentration of 8 mg/ml. Then add 25 µl of each antibody solution prepared for each concentration (0, 2.4, 8, 24 and 80 µg/ml or 0, 0.8, 8, 80 and 800 µg/ml) in RPMI/10% FBS into each well of a 96-well U-bottom plate. In addition, add 50 μl of ATP solution adjusted to 0 or 400 μM in RPMI/10% FBS, the mixture is thoroughly suspended and then left at 37°C for 6 hours in a CO2 incubator (5 μl of ATP solution adjusted to 0 or 4000 µM, added every 2 hours (a total of twice)). After 6 hours, PBMCs are collected, washed twice with auto MACS Rinsing Solution (Miltenyi), reacted with the following antibodies, and the immune cell fractions present are analyzed by FACS:

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Claims (8)

viability reagent (Biolegend, Zombie Aqua), anti-CD3 antibody (BD, clone: UCHT1), anti-CD4 antibody (BD, clone: RPA-T4), anti-CD8 antibody (BD, clone: SK1), antibody to CD45RA (Biolegend, clone: HI100), antibody to CD25 (BD, clone: 2A3), antibody to CD16 (Biolegend, clone: 3G8), antibody to CD56 (Biolegend, clone: HCD56), antibody to CTLA4 (Biolegend company, clone: BNI3). FACS analysis was performed on a BD LSR Fortessa X-20 flow cytometer (BD). The proportion of CTLA4-positive regulatory T cells among viable cells is calculated at each antibody concentration, and the relative value at which the value at antibody concentration 0 is taken as 100% is determined as the survival (%) of CTLA4-positive regulatory T cells, which is used in as an indicator when assessing the cytotoxic activity of each antibody. The results obtained are presented in Fig. 32 (SW1389ART5+ACT1), fig. 33 (SW1389-ART6+ACT1), fig. 34 (SW1610-ART5+ACT1) and fig. 35 (SW1610ART6+ACT1). These results confirm that the cytotoxic activity of Fc-altered anti-CTLA4 switching antibodies against CTLA4-positive regulatory T cells differs in the presence and absence of ATP, and that there is also an ATP-dependent cytotoxic activity against CTLA4-positive regulatory T cells. Although the above inventions have been described in detail with reference to illustrations and examples to promote a clear understanding, the above description of the present invention and examples should not be construed as limiting the scope of the present invention. The description of all patent and scientific publications cited herein is expressly incorporated herein by reference. Industrial applicability. The Ahtu-CTLA-4 antibodies of the present invention and methods of using them can be used in the development, production, provision, use, and the like. pharmaceuticals with little side effects, since the antibodies of the present invention have immune cell activating activity, cytotoxic activity and/or antitumor activity, have little effect on non-tumor tissues such as normal tissues. In addition, the Fc region variant-containing polypeptides of the present invention and methods for preparing and using them can be used in the development, production, provision, use, and the like. such pharmaceuticals. CLAIM (1) the VH sequence shown in SEQ ID NO: 98 and the VL sequence shown in SEQ ID NO: 99; (1) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 101; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 113; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 114; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 115; 1. An anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody having CTLA-4 binding activity that is dependent on the concentration of an adenosine-containing compound, the antibody comprising: (a) HVR-H1 (SEQ ID NO: 223) containing an amino acid sequence SX|TMN. where X ₁ represents H, A, R or K; (b) HVR-H2 (SEQ ID NO: 224) containing the amino acid sequence SISXX2SX3YIYYAX4SVX5G, where X1 is S or T, X ₂ is R or Q, X ₃ is G or H, X ₄ is D, E or R, and X ₅ represents K or R; (c) HVR-H3 (SEQ ID NO: 225) containing the amino acid sequence YGX1REDMLWVFDY, where X1 represents K or A; (d) HVR-L1 (SEQ ID NO: 226) containing the amino acid sequence X1GX2STX3VGDYX4X5VX6, where X1 is T, D, Q or E, X ₂ is T or P, X ₃ is D or G, X ₄ is is N or T, X5 is Y or W, and X6 is S or H; (e) HVR-L2 (SEQ ID NO: 227) containing the amino acid sequence X1T X ₂ X ₃ KRX ₄ where X1 is E, F or Y, X ₂ is S or I, X ₃ is K or S and X ₄ represents S, E or K; and (e) HVR-L3 (SEQ ID NO: 228) containing the amino acid sequence X1TYAAPLGP X ₂ where X 1 is S or Q and X ₂ is M or T. (2) the VH sequence shown in SEQ ID NO: 83 and the VL sequence shown in SEQ ID NO: 97; (2) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 104; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 116; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 115; 2. Antibody according to claim 1, which contains: (3) the VH sequence shown in SEQ ID NO: 86 and the VL sequence shown in SEQ ID NO: 134; 3. The antibody of claim 1, which contains: (a) a VH that is at least 95% identical to any of the amino acid sequences of SEQ ID NOs: 83-86, 98 and 135-141; (b) VL that is at least 95% identical to any of the amino acid sequences of SEQ ID NOs: 88-95, 97, 99, 134 and 144-149; or (c) VH having any amino acid sequence from SEQ ID NOs: 83-86, 98 and 135-141, and VL having any amino acid sequence from SEQ ID NOs: 88-95, 97, 99, 134 and 144-149 . (3) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 106; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence - 92 045931 activity SEQ ID NO: 122; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; (4) the VH sequence shown in SEQ ID NO: 136 and the VL sequence shown in SEQ ID NO: 95; 4. Antibody to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), which contains: (4) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 108; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 121; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 123; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 153; (5) the VH sequence shown in SEQ ID NO: 140 and the VL sequence shown in SEQ ID NO: 146; (5) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 110; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; (6) the VH sequence shown in SEQ ID NO: 141 and the VL sequence shown in SEQ ID NO: 146; (6) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 112; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; (7) the VH sequence shown in SEQ ID NO: 140 and the VL sequence shown in SEQ ID NO: 147; (7) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; (8) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 112; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; (9) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; or (10) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 109; (c) HVR-NZ containing the amino acid sequence SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; and (e) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133. (8) the VH sequence shown in SEQ ID NO: 141 and the VL sequence shown in SEQ ID NO: 147; -