Pharmaceutical composition containing anti-CCR8 antibody and application thereof

Abstract

The present disclosure relates to a pharmaceutical composition comprising an anti-CCR8 antibody and uses thereof. Specifically, the disclosure relates to a pharmaceutical composition comprising an anti-CCR8 antibody and a buffer, the buffer being an acetate buffer, a histidine buffer, or a phosphate buffer. The pharmaceutical composition has therapeutic activity. In addition, the pharmaceutical composition also has the advantages of good stability and the like.

Description

Technical Field

[0001] The present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition comprising an anti-CCR8 antibody and uses thereof. Background Art

[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] CCR8 (chemokine CC motif receptor 8) is a seven-transmembrane GPCR protein, with the N-terminus and three loops exposed outside the cell. Its main ligand is CCL1, of which the N-terminus and loop 2 play an important role in the binding of CCL1.

[0004] The CCL1-CCR8 axis plays an important role in the occurrence and development of tumors. Tumor stem cells, cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in the tumor microenvironment secrete CCL1, driving CCR8-positive Tregs in the peripheral blood to infiltrate into the tumor tissue and inhibit the activity of Teff cells. At the same time, CCL1 and TGFβ secreted by tumor cells work together to convert CD4-positive Tconvs into Tregs. Some tumor cells also express CCR8 on their surface. Under the action of CCL1, tumor cells are resistant to apoptosis (such as T cell lymphoma), promote tumor cell proliferation (such as bladder cancer), and promote tumor cell metastasis (promote melanoma metastasis into lymph nodes). At the same time, vascular endothelial cells inside the tumor tissue also express CCR8, which promotes the formation of new blood vessels under the induction of CCL1. In addition, clinically, it has been found that the lower the CCR8 expression, the better the patient's survival. This is because patients with low CCR8 expression have less Treg infiltration in the tumor microenvironment, while the proportion and activity of Teff are relatively higher.

[0005] New research has found that CCR8 antibodies can specifically kill tumor-infiltrating Treg cells with high CCR8 expression through ADCC, eliminate Treg cells with immunosuppressive activity, and inhibit tumor growth. In addition, the combination of CCL1 and CCR8, in addition to inducing Treg to enrich in the tumor microenvironment, will also enhance the tumor immunosuppressive ability by upregulating CCR8 expression in Treg, FOXP3, IL-10 and other immunosuppressive factors.

[0006] Patents for CCR8 antibodies have been published, such as WO2018181425, WO2015048801 and CN110835371. Currently, several companies have developed antibodies against CCR8 and entered the clinical trial stage, but these CCR8 antibodies mainly bind to the N-terminus of CCR8 and have no cross-reactivity with monkey CCR8 or very weak cross-reactivity with monkey CCR8. Summary of the invention

[0007] The present disclosure provides a pharmaceutical composition of an anti-CCR8 antibody. The pharmaceutical composition has therapeutic activity. In addition, the pharmaceutical composition also has advantages such as good stability.

[0008] In some embodiments, the disclosure provides a pharmaceutical composition, wherein the anti-CCR8 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0009] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 14, 15 and 16, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 17, 18 and 19, respectively;

[0010] The buffer is an acetate buffer, a histidine buffer or a phosphate buffer.

[0011] In some embodiments, as described above in the pharmaceutical composition, the buffer is acetic acid-sodium acetate buffer or histidine-histidine hydrochloride buffer.

[0012] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the buffer is acetic acid-sodium acetate buffer.

[0013] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the pH of the pharmaceutical composition is 5.0 to 6.5. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 6.0. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 5.5. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 5.7. In some embodiments, the pH of the pharmaceutical composition is 5.3 to 5.7. In some embodiments, the pH of the pharmaceutical composition is 5.5 to 6.0. In some embodiments, the pH of the pharmaceutical composition is about 5.5.

[0014] In some embodiments, the pH of the pharmaceutical composition is about 5.6. In some embodiments, the pH of the pharmaceutical composition is about 5.7. When a point value is mentioned in this disclosure, it should be understood that the point value includes a range of error. This range of error is due to factors such as laboratory environment, personnel operation, instrumentation, methodology, and measurement error. Taking pH as an example, when the measured value is about 5.5, it should be understood that it includes a range of error. As an example, when an industrial pH meter is used to measure a preparation, "about 5.5" means 5.5±0.2 (i.e., a pH of 5.3 to 5.7).

[0015] In some embodiments, the pH of the pharmaceutical composition is 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, or 6.5, or any range between these points. In some embodiments, the pH of the pharmaceutical composition is 5.5. In some embodiments, the pH of the pharmaceutical composition is 5.6. In some embodiments, the pH of the pharmaceutical composition is 5.7.

[0016] Typically, the pH of the pharmaceutical composition obtained by replacing the buffer is almost consistent with the buffer pH. At the same time, it is well known to those skilled in the art that in the process of pharmaceutical preparation, there may sometimes be a pH drift, but the drift of the pH of the pharmaceutical preparation is generally very small (within the range of ± 0.3). In some embodiments, the drift of the pH of the pharmaceutical preparation is within the range of ± 0.2. In some embodiments, the drift of the pH of the pharmaceutical preparation is within the range of ± 0.1. As an example, "pH is 5.5 drift" means that the drift of pH is within the range of 5.5 ± 0.3 (i.e., pH is 5.2 to 5.8), or the drift of pH is within the range of 5.5 ± 0.2 (i.e., pH is 5.3 to 5.7), or the drift of pH is within the range of 5.5 ± 0.1 (i.e., pH is 5.4 to 5.6).

[0017] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the concentration of the anti-CCR8 antibody is 1 mg / mL to 280 mg / mL.

[0018] In some embodiments, the concentration of the anti-CCR8 antibody is 50 mg / mL to 260 mg / mL. In some embodiments, the concentration of the anti-CCR8 antibody is 50 mg / mL to 100 mg / mL. In some embodiments, the concentration of the anti-CCR8 antibody is 50 mg / mL to 80 mg / mL.

[0019] In some embodiments, the anti-CCR8 antibody is at a concentration of 100 mg / mL to 260 mg / mL.

[0020] In some embodiments, the concentration of the anti-CCR8 antibody is 160 mg / mL to 240 mg / mL. In some embodiments, the concentration of the anti-CCR8 antibody is 180 mg / mL to 220 mg / mL. In some embodiments, the concentration of the anti-CCR8 antibody is about 200 mg / mL.

[0021] In some embodiments, the anti-CCR8 antibody is at a concentration of 200 mg / mL to 260 mg / mL.

[0022] In some embodiments, the concentration of the anti-CCR8 antibody is 230 mg / mL to 260 mg / mL. In some embodiments, the concentration of the anti-CCR8 antibody is about 250 mg / mL.

[0023] In some embodiments, the concentration of the anti-CCR8 antibody is 60 mg / mL to 200 mg / mL. In some embodiments, the concentration of the anti-CCR8 antibody is 80 mg / mL to 120 mg / mL. In some embodiments, the concentration of the anti-CCR8 antibody is about 100 mg / mL.

[0024] In some embodiments, the concentration of the anti-CCR8 antibody is 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL, 280 mg / mL, 290 mg / mL, 300 mg / mL, 10 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL or 280 mg / mL, or any range between these points.

[0025] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the pharmaceutical composition comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from polysorbates (e.g., polysorbate 80, polysorbate 20), poloxamer (e.g., poloxamer 188), Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, nutmeg In some embodiments, the surfactant is polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate 80 or poloxamer 188. In some embodiments, the surfactant is polysorbate 80.

[0026] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the surfactant concentration is 0.01 mg / mL to 1.0 mg / mL. In some embodiments, the surfactant concentration is 0.1 mg / mL to 0.8 mg / mL. In some embodiments, the surfactant concentration is 0.2 mg / mL to 0.8 mg / mL. In some embodiments, the surfactant concentration is 0.2 mg / mL to 0.6 mg / mL. In some embodiments, the surfactant concentration is 0.3 mg / mL to 0.5 mg / mL. In some embodiments, the surfactant concentration is 0.32 mg / mL to 0.48 mg / mL. In some embodiments, the surfactant concentration is 0.36 mg / mL to 0.44 mg / mL. In some embodiments, the surfactant concentration is about 0.4 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.32 mg / mL, 0.36 mg / mL, 0.4 mg / mL, 0.44 mg / mL, 0.48 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1.0 mg / mL, or any range between these point values. In some embodiments, the surfactant concentration is 0.4 mg / mL.

[0027] In some embodiments, the surfactant is 0.01 mg / mL to 1.0 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.1 mg / mL to 0.8 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.2 mg / mL to 0.6 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.3 mg / mL to 0.5 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.32 mg / mL to 0.48 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.36 mg / mL to 0.44 mg / mL of polysorbate 80. In some embodiments, the surfactant is about 0.4 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.4 mg / mL of polysorbate 80.

[0028] In some embodiments, the surfactant is poloxamer 188 at 0.4 mg / mL.

[0029] In some embodiments, the pharmaceutical composition as described in any one of the above comprises sugar. In some embodiments, the sugar is selected from the conventional composition (CH2O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. The sugar can be selected from sucrose, trehalose, glucose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc.

[0030] In some embodiments, the sugar is sucrose, trehalose, mannitol, or sorbitol. In some embodiments, the sugar is sucrose or trehalose. In some embodiments, the sugar is sucrose.

[0031] In some embodiments, the concentration of the sugar is 10 mg / mL to 100 mg / mL. In some embodiments, the concentration of the sugar is 40 mg / mL to 90 mg / mL.

[0032] In some embodiments, the concentration of the sugar is 50 mg / mL to 90 mg / mL. In some embodiments, the concentration of the sugar is 70 mg / mL to 90 mg / mL. In some embodiments, the concentration of the sugar is about 80 mg / mL.

[0033] In some embodiments, the concentration of the sugar is 40 mg / mL to 80 mg / mL. In some embodiments, the concentration of the sugar is 45 mg / mL to 55 mg / mL. In some embodiments, the concentration of the sugar is about 50 mg / mL.

[0034] In some embodiments, the concentration of the sugar is non-limiting and includes 10mg / mL, 20mg / mL, 30mg / mL, 35mg / mL, 40mg / mL, 45mg / mL, 50mg / mL, 55mg / mL, 60mg / mL, 65mg / mL, 70mg / mL, 75mg / mL, 80mg / mL, 85mg / mL, 90mg / mL, 95mg / mL or 100mg / mL, and any range between these point values. In some embodiments, the concentration of the sugar is 50mg / mL. In some embodiments, the concentration of the sugar is 80mg / mL.

[0035] In some embodiments, the sugar is 10mg / mL to 100mg / mL of sucrose. In some embodiments, the sugar is 40mg / mL to 90mg / mL of sucrose. In some embodiments, the sugar is 50mg / mL to 90mg / mL of sucrose. In some embodiments, the sugar is 70mg / mL to 90mg / mL of sucrose. In some embodiments, the sugar is about 80mg / mL of sucrose. In some embodiments, the sugar is 80mg / mL of sucrose.

[0036] In some embodiments, the sugar is 10mg / mL to 100mg / mL of sucrose. In some embodiments, the sugar is 40mg / mL to 90mg / mL of sucrose. In some embodiments, the sugar is 40mg / mL to 80mg / mL of sucrose. In some embodiments, the sugar is 45mg / mL to 55mg / mL of sucrose. In some embodiments, the sugar is about 50mg / mL of sucrose. In some embodiments, the sugar is 50mg / mL of sucrose.

[0037] In some embodiments, the pharmaceutical composition as described in any one of the above, the concentration of the buffer is 5mM to 100mM. In some embodiments, the concentration of the buffer is 5mM to 50mM. In some embodiments, the concentration of the buffer is 10mM to 50mM. In some embodiments, the concentration of the buffer is 5mM to 30mM. In some embodiments, the concentration of the buffer is 10mM to 30mM. In some embodiments, the concentration of the buffer is 15mM to 25mM. In some embodiments, the concentration of the buffer is 5mM to 20mM. In some embodiments, the concentration of the buffer is 8mM to 20mM. In some embodiments, the concentration of the buffer is about 20mM. In some embodiments, the concentration of the buffer is 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 25mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM, and any range between these point values. In some embodiments, the concentration of the buffer is 20mM.

[0038] In some embodiments, the buffer is 5mM to 100mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is 10mM to 50mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is 10mM to 30mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is 15mM to 25mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is about 20mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is 20mM acetic acid-sodium acetate buffer.

[0039] In some embodiments, the buffer is about 20 mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is about 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0040] In some embodiments, the buffer is 20 mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0041] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the pharmaceutical composition further comprises an excipient. In some embodiments, the excipient is proline, arginine hydrochloride, sodium chloride, disodium ethylenediaminetetraacetic acid (EDTA-2Na), glycine, methionine, histidine, phenylalanine, glutamic acid, aspartic acid or calcium chloride. In some embodiments, the excipient is proline.

[0042] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the concentration of the excipient is 10mM to 120mM. In some embodiments, the concentration of the excipient is 40mM to 100mM. In some embodiments, the concentration of the excipient is 40mM to 80mM. In some embodiments, the concentration of the excipient is 45mM to 55mM. In some embodiments, the concentration of the excipient is about 50mM. In some embodiments, the concentration of the excipient is 10mM, 20mM, 30mM, 40mM, 45mM, 50mM, 55mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM or 120mM, and any range between these point values.

[0043] In some embodiments, the pharmaceutical composition of any of the above, wherein the excipient is 10mM to 120mM proline. In some embodiments, the excipient is 40mM to 100mM proline. In some embodiments, the excipient is 40mM to 80mM proline. In some embodiments, the excipient is 45mM to 55mM proline. In some embodiments, the excipient is about 50mM proline. In some embodiments, the excipient is 50mM proline.

[0044] In some embodiments, the excipient is about 50 mM sodium chloride. In some embodiments, the excipient is about 100 mM arginine hydrochloride.

[0045] In some embodiments, the auxiliary material is 50 mM sodium chloride. In some embodiments, the auxiliary material is 100 mM arginine hydrochloride.

[0046] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-CCR8 antibody is a murine antibody, a chimeric antibody or a humanized antibody.

[0047] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-CCR8 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 27, and a light chain variable region as shown in SEQ ID NO: 31.

[0048] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-CCR8 antibody comprises the heavy chain sequence shown in SEQ ID NO: 46, and the light chain sequence shown in SEQ ID NO: 47.

[0049] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0050] (a) 1 mg / mL to 280 mg / mL of the anti-CCR8 antibody,

[0051] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0052] (c) 10 mg / mL to 100 mg / mL of sugar, and

[0053] (d) 5 mM to 100 mM buffer, the pH of the pharmaceutical composition is 5.0 to 6.5.

[0054] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0055] (a) 100 mg / mL to 260 mg / mL of the anti-CCR8 antibody,

[0056] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0057] (c) 40 mg / mL to 90 mg / mL of sucrose, and

[0058] (d) 10 mM to 50 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0059] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0060] (a) 160 mg / mL to 240 mg / mL of the anti-CCR8 antibody,

[0061] (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80,

[0062] (c) 50 mg / mL to 90 mg / mL of sucrose, and

[0063] (d) 10 mM to 30 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 5.7.

[0064] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0065] (a) 180 mg / mL to 220 mg / mL of an anti-CCR8 antibody,

[0066] (b) 0.3 mg / mL to 0.5 mg / mL of polysorbate 80,

[0067] (c) 70 mg / mL to 90 mg / mL of sucrose, and

[0068] (d) 15 mM to 25 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.3 to 5.7.

[0069] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0070] (a) about 200 mg / mL of an anti-CCR8 antibody,

[0071] (b) about 0.4 mg / mL of polysorbate 80,

[0072] (c) about 80 mg / mL of sucrose, and

[0073] (d) about 20 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is about 5.5.

[0074] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0075] (a) 200 mg / mL of anti-CCR8 antibody,

[0076] (b) 0.4 mg / mL of polysorbate 80,

[0077] (c) 80 mg / mL sucrose, and

[0078] (d) 20 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.5.

[0079] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0080] (a) 1 mg / mL to 280 mg / mL of the anti-CCR8 antibody,

[0081] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0082] (c) 10 mg / mL to 100 mg / mL of sugar,

[0083] (d) 10 mM to 120 mM of an excipient, and

[0084] (e) 5 mM to 100 mM buffer, the pH of the pharmaceutical composition is 5.0 to 6.5.

[0085] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0086] (a) 100 mg / mL to 260 mg / mL of the anti-CCR8 antibody,

[0087] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0088] (c) 40 mg / mL to 90 mg / mL of sucrose,

[0089] (d) 40 mM to 100 mM proline, and

[0090] (e) 10 mM to 50 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0091] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0092] (a) 200 mg / mL to 260 mg / mL of the anti-CCR8 antibody,

[0093] (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80,

[0094] (c) 40 mg / mL to 80 mg / mL of sucrose,

[0095] (d) 40 mM to 80 mM proline, and

[0096] (e) 10 mM to 30 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 5.7.

[0097] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0098] (a) 230 mg / mL to 260 mg / mL of an anti-CCR8 antibody,

[0099] (b) 0.3 mg / mL to 0.5 mg / mL of polysorbate 80,

[0100] (c) 45 mg / mL to 55 mg / mL of sucrose,

[0101] (d) 45 mM to 55 mM proline, and

[0102] (e) 15 mM to 25 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.3 to 5.7.

[0103] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0104] (a) about 250 mg / mL of an anti-CCR8 antibody,

[0105] (b) about 0.4 mg / mL of polysorbate 80,

[0106] (c) about 50 mg / mL of sucrose,

[0107] (d) about 50 mM proline, and

[0108] (e) about 20 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is about 5.5.

[0109] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0110] (a) 250 mg / mL of anti-CCR8 antibody,

[0111] (b) 0.4 mg / mL of polysorbate 80,

[0112] (c) 50 mg / mL sucrose,

[0113] (d) 50 mM proline, and

[0114] (e) 20 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.5.

[0115] In some embodiments, the pharmaceutical composition as described in any one of the above items is a liquid preparation. In some embodiments, the solvent of the liquid preparation is water.

[0116] The present disclosure also provides a lyophilized preparation, characterized in that the lyophilized preparation can form any of the above pharmaceutical compositions after reconstitution.

[0117] The present disclosure also provides a lyophilized preparation, which is a lyophilized preparation of the pharmaceutical composition as described in any one of the above items.

[0118] The present disclosure also provides a method for preparing a lyophilized preparation, comprising the step of freeze-drying the pharmaceutical composition as described in any one of the above items. In some embodiments, the freeze-drying as described in any one of the above items sequentially comprises the steps of pre-freezing, primary drying and secondary drying.

[0119] The present disclosure also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition as described in any one of the above items.

[0120] The present disclosure also provides a reconstituted solution, characterized in that the reconstituted solution is prepared by reconstituted the lyophilized preparation as described in any one of the above items.

[0121] The present disclosure also provides a reconstituted solution, which is a reconstituted preparation of the lyophilized preparation as described in any one of the above items.

[0122] In some embodiments, the components and contents of the reconstituted solution as described in any of the above items are the same as the components and contents of the aforementioned pharmaceutical composition.

[0123] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is a subcutaneous injection preparation, an intravenous injection preparation, an intraperitoneal injection preparation, or an intramuscular injection preparation. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is an intravenous injection preparation. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is a subcutaneous injection preparation.

[0124] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is suitable for subcutaneous injection, intravenous injection, intraperitoneal injection or intramuscular injection. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is suitable for intravenous injection. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is suitable for subcutaneous injection.

[0125] In some embodiments, the pharmaceutical composition, reconstituted solution or lyophilized preparation as described in any of the above items is used to prepare a drug for subcutaneous injection, intravenous injection, intraperitoneal injection or intramuscular injection. In some embodiments, the pharmaceutical composition, reconstituted solution or lyophilized preparation as described in any of the above items is used to prepare a drug for intravenous injection. In some embodiments, the pharmaceutical composition, reconstituted solution or lyophilized preparation as described in any of the above items is used to prepare a drug for subcutaneous injection.

[0126] The present disclosure also provides a medicine kit, which comprises at least one container, each container independently comprising the pharmaceutical composition as described in any one of the above items, the lyophilized preparation as described in any one of the above items, or the reconstituted solution as described in any one of the above items.

[0127] In some embodiments, the present disclosure also provides a method for diagnosing, treating, and alleviating a condition of a subject, comprising administering to the subject an effective amount of the pharmaceutical composition as described in any of the above, the lyophilized preparation as described in any of the above, the reconstituted solution as described in any of the above, or the medicine kit as described in any of the above.

[0128] In some embodiments, the present disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition as described in any one of the above, the lyophilized preparation as described in any one of the above, the reconstituted solution as described in any one of the above, or the medicine kit as described in any one of the above.

[0129] In some embodiments, the present disclosure also provides a pharmaceutical composition as described in any of the above, a lyophilized preparation as described in any of the above, a reconstituted solution as described in any of the above, or a medicine kit as described in any of the above, for use in treating or preventing a disease.

[0130] In one aspect, the present disclosure also provides use of the pharmaceutical composition as described in any of the preceding items, the lyophilized preparation as described in any of the preceding items, the reconstituted solution as described in any of the preceding items, or the medicine kit as described in any of the preceding items in the preparation of a medicament for preventing or treating a disease.

[0131] In some embodiments, the disease as described in any of the preceding items is cancer or tumor, inflammatory disease and viral infection.

[0132] In some embodiments, the disease as described in any of the preceding items is a hematological malignancy or a solid tumor. In some embodiments, the cancer or tumor is selected from prostate cancer, bladder cancer, ovarian cancer, lung cancer, liver cancer, stomach cancer, colon cancer, kidney cancer, cutaneous fibrosarcoma, osteosarcoma, cervical cancer, esophageal cancer, rectal cancer, head and neck squamous cell carcinoma, breast cancer, multiple myeloma, lymphoma and melanoma.

[0133] In some embodiments, the aforementioned disease is a disease associated with CCR8; in some embodiments, the aforementioned disease is a disease expressing CCR8.

[0134] In one aspect, the present disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition as described in any of the preceding items, a lyophilized preparation as described in any of the preceding items, a reconstituted solution as described in any of the preceding items, or a drug kit as described in any of the preceding items. The aforementioned disease is a disease expressing CCR8. The disease is cancer or a tumor, an inflammatory disease, and a viral infection. The cancer or tumor is selected from prostate cancer, bladder cancer, ovarian cancer, lung cancer, liver cancer, gastric cancer, colon cancer, kidney cancer, cutaneous fibrosarcoma, osteosarcoma, cervical cancer, esophageal cancer, rectal cancer, head and neck squamous cell carcinoma, breast cancer, multiple myeloma, lymphoma, and melanoma. DETAILED DESCRIPTION

[0135] the term

[0136] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0137] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0138] The term "antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. "Native antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric proteins of about 150,000 daltons, consisting of two light chains and two heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or a light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0139] The term "CCR8" refers to the full-length CCR8 protein, and human CCR8 has the amino acid sequence of SEQ ID NO: 1. Amino acid sequences of CCR8 molecules from non-human species (eg, mouse, monkey, rabbit, dog, pig, etc.) are available from public sources.

[0140] The term "variable region" or "variable domain" refers to the domain in an antigen-binding molecule that binds to an antigen. VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs). Among them, the term "complementarity determining region" or "CDR" refers to the region within the variable domain that mainly contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen binding specificity.

[0141] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "Kabat" numbering rules (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering rules, "ABM" numbering rules, "contact" numbering rules (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering rules (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art. The numbering rules of the present disclosure are shown in Table 1 below.

[0142] Table 1. Relationships between CDR numbering systems

[0143] CDR IMGT Kabat AbM Chothia Contact HCDR1 27-38 31-35 26-35 26-32 30-35 HCDR2 56-65 50-65 50-58 52-56 47-58 HCDR3 105-117 95-102 95-102 95-102 93-101 LCDR1 27-38 24-34 24-34 24-34 30-36 LCDR2 56-65 50-56 50-56 50-56 46-55 LCDR3 105-117 89-97 89-97 89-97 89-96

[0144] Unless otherwise specified, the variable region and CDR sequences in the disclosed embodiments are all subject to the "Kabat" numbering convention. Although in a specific embodiment, the Kabat numbering convention is used to define amino acid residues, the corresponding technical solutions of other numbering systems will be regarded as equivalent technical solutions.

[0145] The term "antibody fragment" refers to a molecule different from an intact antibody, which comprises a portion of an intact antibody that retains the antigen binding ability of the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g., scFv); and multispecific antibodies formed by antibody fragments.

[0146] Fab refers to a protein composed of VH and CH1 (Fab heavy chain) and VL and CL (Fab light chain) of immunoglobulin.

[0147] Fv refers to the antigen binding domain composed of VH and VL of immunoglobulin.

[0148] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of the human IgG heavy chain is defined as extending from the amino acid residue at the Cys226 position or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise indicated, the numbering convention for the Fc region is the EU index.

[0149] "Pharmaceutical composition" means a mixture containing one or more antibodies described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity. In this disclosure, "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0150] An "effective amount" includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0151] "Pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods.

[0152] It will be appreciated by those skilled in the art that when reference value ranges, cutoffs or specific values ​​are used, "about" can be expressed within 1 or more than 1 standard deviation. Alternatively, "about" can represent a range that differs by up to 20% (i.e., ± 20%). Since many numerical values ​​used herein are determined experimentally, it will be appreciated by those skilled in the art that such determinations can differ between different experiments and are generally different between different experiments. Due to this inherent difference, it is believed that the values ​​used herein should not be overly restricted. Therefore, the term "about" is used to cover a difference of ± 20% or less, ± 10% or less, ± 5% or less, ± 1% or less, ± 0.5% or less, or ± 0.1% or less from a specified value.

[0153] "Buffer" refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that control pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0154] "Histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, histidine-histidine sulfate and the like, preferably histidine-histidine hydrochloride buffer. Histidine-histidine hydrochloride buffer can be prepared from histidine and hydrochloric acid, or from histidine and histidine hydrochloride.

[0155] "Citrate buffer" is a buffer including citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

[0156] "Succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-succinic acid sodium salt, succinic acid-succinic acid potassium salt, succinic acid-succinic acid calcium salt, etc. A preferred succinate buffer is succinic acid-succinic acid sodium salt. Exemplarily, the succinic acid-succinic acid sodium salt can be prepared from succinic acid and sodium hydroxide, or from succinic acid and succinic acid sodium salt.

[0157] "Phosphate buffer" is a buffer including phosphate ions. Examples of phosphate buffers include citric acid-disodium hydrogen phosphate, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. A preferred phosphate buffer is citric acid-disodium hydrogen phosphate.

[0158] "Acetate buffer" is a buffer including acetate ions. Examples of acetate buffers include acetate-sodium acetate, histidine-histidine acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, etc. A preferred acetate buffer is acetate-sodium acetate.

[0159] "Poloxamer" is an α-hydrogen-ω-hydroxy poly(ethylene oxide) a-poly(propylene oxide) b-poly(ethylene oxide) a block copolymer. Propylene oxide and propylene glycol react to form polyoxypropylene glycol, and then ethylene oxide is added to form a block copolymer. Wherein, a is the number of ethylene oxide units, and b is the number of propylene oxide units. Examples of poloxamers include, but are not limited to, poloxamer 188 (P188 or PF68). Specifically, poloxamer 188, in the copolymer, the ethylene oxide units (a) are 75 to 85, the propylene oxide units (b) are 25 to 30, the ethylene oxide (EO) content is 79.9% to 83.7%, and the average molecular weight is 7680 to 9510.

[0160] "Lyophilized preparation" refers to a pharmaceutical composition in liquid or solution form or a preparation or pharmaceutical composition obtained after a liquid or solution preparation has been subjected to a vacuum freeze-drying step.

[0161] The pharmaceutical composition disclosed herein can achieve a stable effect: the antibody therein substantially retains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are currently a variety of analytical techniques for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0162] A stable formulation is one in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 1 month, at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In addition, a stable liquid formulation includes a liquid formulation that exhibits desired characteristics after being stored at a temperature of 25°C for a period of 1 month, 3 months, or 6 months. It also includes a temperature of 40°C that exhibits desired characteristics after being stored for a period of 4 weeks, 1 month, 3 months, or 6 months. A typical example of stability: measured by SEC-HPLC, usually no more than about 10%, preferably no more than about 5%, of the antibody aggregates or degrades. By visual analysis, the formulation is a light yellow, nearly colorless, clear liquid or a colorless, clear liquid, or clear to slightly milky white. The concentration, pH, weight, and molecular osmotic pressure of the formulation have no more than ±10% variation, preferably no more than ±5% variation. The formulation generally forms no more than about 10%, preferably no more than about 5% aggregation.

[0163] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured after visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines protein tertiary structure, and by FTIR spectroscopy, which determines protein secondary structure.

[0164] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0165] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.

[0166] "Treatment" and "treatment" (and grammatical variations thereof) refer to clinical interventions applied to the treated individual and may be performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving prognosis. In some embodiments, the antigen binding molecules of the present disclosure are used to delay the formation of the disease or slow the progression of the disease.

[0167] The details of one or more embodiments of the present disclosure are proposed in the above description. Although any method and material similar or identical to that described herein can be used to implement or test the present disclosure, the preferred method and material are described below. Through the description and claims, other features, objects and advantages of the present disclosure will be apparent. In the description and claims, unless otherwise clearly indicated in the context, the singular form includes the case of plural references. Unless otherwise defined, all technical and scientific terms used herein have the general meaning understood by ordinary technicians in the field to which the present disclosure belongs. All patents and publications cited in the description are incorporated by reference. The following examples are proposed to more fully illustrate the preferred embodiments of the present disclosure. These embodiments should not be construed in any way as limiting the scope of the present disclosure, and the scope of the present disclosure is limited by the claims.

[0168] Example - Preparation of anti-CCR8 antibodies

[0169] PCT / CN2023 / 091525 (application date: 2023.04.28; priority patent application number: CN202210473502.0) is incorporated into this disclosure in its entirety by reference.

[0170] Example 1. Preparation of CCR8 fusion protein and stably transfected cells

[0171] 1.1 Related protein sequences

[0172] Untagged human CCR8 gene, human CCR4 gene, and monkey CCR8 gene are transfected into CHOK1 and / or HEK293 cells to form CHOK1 or HEK293 cell lines expressing CCR8 or human CCR4 proteins of different species on the cell surface for subsequent antibody screening and identification. The Gqi5 gene and human CCR8 gene are transfected into CHOK1 cells to form CHOK1-hCCR8-Gq cells, which are used for calcium flow inhibition assay detection. The human CCR8 gene and Luc-GFP gene are transfected into HEK293 cells to form HEK293-hCCR8 / Luc-GFP cells, which are used for PBMC ADCC killing assay detection. At the same time, the N-terminal sequence and Fc tag of the human or monkey CCR8 protein are cloned into mammalian cell expression vectors, respectively, and after expression and purification in 293E cells, the Fc fusion protein is obtained for use in the experiments of the subsequent embodiments. The amino acid sequences of the relevant proteins are as follows:

[0173] The sequence of human CCR8 protein expressed on the surface of CHOK1 / HEK293 cells is as follows:

[0174] MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASEDGVLQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIISFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL

[0175] SEQ ID NO: 1

[0176] The amino acid sequence of simian CCR8 protein expressed on the surface of CHOK1 cells is as follows:

[0177] MDYTLDPSMTTMTDYYYPDSLSSPCDGELIQRNDKLLLAVFYCLLFVFSLLGNSLVILVLVVCKKLRNITDIYLLNLALSDLLFVFSFPFQTYYQLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYAIKVRTIRMGTTLSLVVWLTAIMATIPLLVFYQVASEDGVLQCYSFYNQQTLKWKIFTNFEMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLNYATHVTEIISFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSHIFIYLGRQMPRESCEKSSSCQQHSFRSSSIDYIL

[0178] SEQ ID NO: 2

[0179] The amino acid sequence of human CCR4 protein expressed on the surface of CHOK1 cells is as follows:

[0180] MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYSLVFVFGLLGNSVVVLVLFKYKRLRSMTDVYLLNLAISDLLFVFSLPFWGYYAADQWVFGLGLCKMISWMYLVGFYSGIFFVMLMSIDRYLAIVHAVFSLRARTLTYGVITSLATWSVAVFASLPGFLFSTCYTERNHTYCKTKYSLNSTTWKVLSSLEINILGLVIPLGIMLFCYSMIIRTLQHCKNEKKNKAVKMIFAVVVLFLGFWTPYNIVLFLETLVELEVLQDCTFERYLDYAIQATETLAFVHCCLNPIIYFFLGEKFRKYILQLFKTCRGLFVLCQYCGLLQIYSADTPSSSYTQSTMDHDLHDAL

[0181] SEQ ID NO: 3

[0182] The protein sequence of Gqi5 expressed on the surface of CHOK1-hCCR8 cells is as follows:

[0183] MARSLTWRCCPWCLTEDEKAAARVDQEINRILLEQKKQDRGELKLLLLGPGESGKSTFIKQMRIIHGAGYSEEERKGFRPLVYQNIFVSMRAMIEAMERLQIPFSRPESKHHASLVMSQDPYKVTTFEKRYAAAMQWLWRDAGIRAYYERRREFHLLDSAVYYLSHLERITEEGYVPTAQDVLRSRMPTTGINEYCFSVQKTNLRIVDVGGQKSERKKWIHCFENVIALIYLASLSEYDQCLEENNQENRMKESLALFGTILELPWFKSTSVILFLNKTDILEEKIPTSHLATYFPSFQGPKQDAEAAKRFILDMYTRMYTGCVDGPEGSKKGARSRRLFSHYTCATDTQNIRKVFKDVRDSVLARYLDECGLF

[0184] SEQ ID NO: 4

[0185] The gene sequence of Luc-GFP expressed on the surface of HEK293-hCCR8 cells is as follows:

[0186]

[0187] SEQ ID NO: 5

[0188] The sequence of human CCR8 N-terminal-human Fc (hereinafter referred to as hCCR8-hFc) protein is as follows:

[0189]

[0190] The sequence of human CCR8 N-terminal-mouse Fc (hereinafter referred to as hCCR8-mFc) protein is as follows:

[0191]

[0192] Notes:

[0193] The protein sequence of monkey CCR8 N-terminal-human Fc (hereinafter referred to as cynoCCR8-hFc) is as follows:

[0194]

[0195] Notes:

[0196] The protein sequence of monkey CCR8 N-terminal-mouse Fc (hereinafter referred to as cynoCCR8-mFc) is as follows:

[0197]

[0198]

[0199] Notes:

[0200] The single underline above is the signal peptide, the double underline is the N-terminus of the extracellular region, the wavy line is the connecting peptide, and the dot-dash line is the corresponding Fc fragment.

[0201] 1.2 Purification of related proteins

[0202] Purification of Fc-containing proteins, chimeric antibodies and hybridoma antibodies.

[0203] The cell expression supernatant samples were centrifuged at high speed to remove impurities. The recombinant protein containing Fc and the chimeric antibody expression supernatant were purified with a Protein A column, and the hybridoma expression supernatant was purified with a Protein G column. The supernatant was loaded onto the column at a certain flow rate. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with 100mM acetic acid at pH 3.0 and neutralized with 1M Tris-HCl at pH 8.0. The eluted sample was concentrated and replaced with PBS and then aliquoted for standby use.

[0204] Example 2. Preparation of mouse anti-human CCR8 monoclonal antibody

[0205] SJL mice were immunized with HEK293 and CHOK1 cell lines overexpressing human CCR8 and human or monkey CCR8 N-terminus and human Fc fusion proteins. After 5 immunizations, blood was collected to determine the titer of the antibody in the serum, and mice with high antibody titers in the serum and titers approaching a plateau were selected for spleen cell fusion. The fused hybridoma cells were plated in a 96-well cell culture plate and cultured in a 37°C, 5% CO2 incubator. The cell culture supernatant was tested by Mirrorball. The screened positive clones were amplified, frozen, and subcloned two to three times until a single cell clone was obtained. The selected hybridoma clones were further prepared and purified using a serum-free cell culture method. The obtained hybridoma antibodies were tested for binding to human and monkey CCR8 overexpressing cell lines using FACS (see Test Examples 1 and 2 of this disclosure for methods), and hybridoma cell lines with good binding activity and blocking activity were selected.

[0206] The monoclonal hybridoma cell lines mAbCP11 and mAb28 were selected to clone the sequences of monoclonal antibodies. The process is as follows: hybridoma cells in the logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, Cat#15596-018), and reverse transcribed into cDNA. After PCR amplification using cDNA as a template, it was sent to a sequencing company for sequencing. The variable region amino acid sequence of the antibody corresponding to the obtained DNA sequence is as follows:

[0207] Amino acid sequence of the heavy chain variable region of mAbCP11:

[0208] QVQVKESGGPGLVAPSQSLSITCTVSGFSLT RYGIN WVRQPPGKGLEWLG VIWTGGDTNYNSALKS RLSITKDKSKSQVFLKMNSLQTDDTARYYCAR EGVVNYYGSRYSFFDV WGTGTTVTVSS

[0209] SEQ ID NO: 10

[0210] The amino acid sequence of the light chain variable region of mAb CP11:

[0211] QVVLSQSPAILSSSPGEKVTMTC RASSSVSYMH WYQQKPGSSPKPWIY ATSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYC QQWSSNPYT FGGGTKVEIK

[0212] SEQ ID NO: 11

[0213] Amino acid sequence of mAb28 heavy chain variable region:

[0214] EVQLVESGGGLVQPKGSLKLSCAASGFSFN PYAMN WVRQAPGKGLEWVA RIRSKSNNYAAYYADSVKD RFTISRDDSETMLYLQMNNLKTEDTGIYYCVR GREAPQAGYAMDY WGQGTSVTVSS

[0215] SEQ ID NO: 12

[0216] The amino acid sequence of the light chain variable region of mAb28:

[0217] DIVVTQAAPSVPVTPGESVSISC RSSKSLLHSNGNTYLY WLLQRPGQSPQLLIY RMSNLAS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC MQHLEYPFT FGAGTKLELK

[0218] SEQ ID NO: 13

[0219] Table 2. Antibody CDR sequences

[0220]

[0221] Note: The CDRs in Table 2 are identified according to the Kabat numbering system.

[0222] The variable region sequences of the above mAbCP11 and mAb28 candidate molecules were amplified by PCR for VH / VK sequences, and then homologously recombined with the expression vector pTT5 (with a signal peptide and hIgG1 / hkappa constant region gene (CH1-Fc / CL) fragment). Exemplarily, the human heavy chain IgG1 constant region sequence is shown in SEQ ID NO: 44, and the human light chain κ constant region sequence is shown in SEQ ID NO: 45, and the recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pTT5 / VL-CL-pTT5 is constructed to obtain the chimeric antibodies ChCP11 and Ch28.

[0223] Example 3: Humanized design of anti-human CCR8 monoclonal antibody

[0224] The humanization of mouse monoclonal antibodies is carried out according to the methods disclosed in many documents in the field. In short, based on the typical structure of the mouse antibody VH / VL CDR, the homologous sequences of the light chain variable region (VL) and the heavy chain variable region (VH) are searched from the human germline database, the CDR region of the mouse antibody is transplanted to the human template, and some amino acids of VL and VH are replaced, and the constant region of the mouse antibody is replaced with the human constant region to obtain the final humanized molecule.

[0225] 1. Humanization of antibody mAbCP11

[0226] The humanized antibody of the mouse antibody mAbCP11 selects FR1, FR2, FR3 of IGHV2-26*01 or IGHV4-30-4*01, and FR4 of IGHJ6*01 as the heavy chain framework region template; FR1, FR2, FR3 of IGKV6-21*01 or IGKV1-39*01 or IGKV3-20*02 and FR4 of IGKJ4*01 are selected as the light chain framework region template. Optionally, the amino acid residues at positions 1, 27, 28, 30, 44, 49, 71, 73 and / or 78 (numbered according to the Kabat numbering system) on the FR region of the heavy chain variable region of the humanized antibody are substituted; and / or the amino acid residues at positions 43, 45, 46, 47, 49, 58, 60 and / or 71 on the FR region of the light chain variable region of the humanized antibody are substituted.

[0227] Table 3. Mutations of humanized antibody hCP11

[0228]

[0229] Note: The sites in the above table are numbered according to the Kabat numbering system; Graft represents the mouse antibody CDR implanted into the human germline FR region sequence, and F71Y means that the F at position 71 is mutated back to Y according to the Kabat numbering system.

[0230] Different humanized heavy chain variable regions and light chain variable regions were obtained by back mutation design in Table 3, and different antibody molecules were obtained by combining variable regions according to Table 4:

[0231] Table 4. Combinations of heavy and light chain variable regions of hCP11 humanized antibody

[0232]

[0233]

[0234] The specific sequences of the variable regions after humanization and mutation are as follows:

[0235] >hCP11VH1 (SEQ ID NO: 26):

[0236] EVTLKESGPVLVKPTETLTLTCTVSGFSLTRYGINWIRQPPGKALEWLAVIWTGGDTNYNSALKSRLTISKDKSKSQVVLTMTNMDPVDTATYYCAREGVVNYYGSRYSFFDVWGQGTTVTVSS

[0237] >hCP11VH2(SEQ ID NO:27):

[0238] EVTLKESGPVLVKPTETLTLTCTVSGFSLTRYGINWIRQPPGKGLEWLGVIWTGGDTNYNSALKSRLTISKDKSKSQVVLTMTNMDPVDTATYYCAREGVVNYYGSRYSFFDVWGQGTTVTVSS

[0239] >hCP11VH3(SEQ ID NO:28):

[0240] EVQLQESGPGLVKPSQTLSLTCTVSGFSITRYGINWIRQPPGKGLEWIGVIWTGGDTNYNSALKSRVTISKDKSKNQVSLKLSSVTAADTAVYYCAREGVVNYYGSRYSFFDVWGQGTTVTVSS

[0241] >hCP11VL1(SEQ ID NO:29):

[0242] EIVLTQSPDFQSVTPKEKVTITCRASSSVSYMHWYQQKPDQSPKPWIYATSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQWSSNPYTFGGGTKVEIK

[0243] >hCP11VL2(SEQ ID NO:30):

[0244] EIVLTQSPDFQSVTPKEKVTITCRASSSVSYMHWYQQKPDQSPKPWIYATSNLASGVPARFSGSGSGTDYTLTINSLEAEDAATYYCQQWSSNPYTFGGGTKVEIK

[0245] >hCP11VL3(SEQ ID NO:31):

[0246] DIQMTQSPSSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKSPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPYTFGGGTKVEIK

[0247] >hCP11VL4 (SEQ ID NO: 32):

[0248] EIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQSPKPWIYATSNLASGVPARFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSSNPYTFGGGTKVEIK

[0249] 2. Humanization of mAb28

[0250] The heavy chain template of the humanized mouse antibody mAb28 selected the FR1, FR2, FR3, and FR4 regions of IGHV3-72*01, IGHV3-66*01, IGHV3-23*01, or IGHV7-4-1*01, and IGHJ6*01; the light chain template selected the FR1, FR2, FR3, and FR4 regions of IGKV2-28*01, IGKV3-11*01, or IGKV1-39*01. Optionally, amino acid residues at positions 27, 29, 31, 48, 69, 71, 75 and / or 93 (numbered according to the Kabat numbering system) on the heavy chain variable region of the humanized antibody are substituted; and / or amino acid residues at positions 4, 27c, 29, 36, 43, 45, 51, 60, 93 and / or 100 on the light chain variable region of the humanized antibody are substituted.

[0251] Table 5. Mutations of humanized antibody h28

[0252]

[0253] Note: The loci in the table are numbered according to the Kabat numbering system; Grafted represents the mouse antibody CDR implanted into the human germline FR region sequence, and V75E represents the mutation of position 75 V back to E according to the Kabat numbering system.

[0254] The above mutations include mutations selected from L27cA, G29R, M51K and E93Q in the CDR of the light chain variable region of the h28 antibody. The HCDR1 of the heavy chain variable region of the h28 antibody is mutated to P31I to obtain the following CDR combination:

[0255] Table 6. Humanized antibody h28 mutation sequence

[0256]

[0257]

[0258] Different humanized heavy chain variable regions and light chain variable regions were obtained by using the mutation design in Table 5, and different antibody molecules were obtained by combining the variable regions according to Table 7.

[0259] Table 7. Combinations of heavy and light chain variable regions of h28 humanized antibody

[0260] h28VH1 h28VH2 h28VH3 h28VH4 h28VL1 h28-1 h28-4 h28-7 h28-10 h28VL2 h28-2 h28-5 h28-8 h28-11 h28VL3 h28-3 h28-6 h28-9 h28-12

[0261] The specific sequences after humanization and mutation are as follows:

[0262] >h28VH1 (SEQ ID NO: 37)

[0263] EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMNWVRQAPGKGLEWVGRIRSKSNNYAAYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARGREAPQAGYAMDYWGQGTTVTVSS

[0264] >h28VH2 (SEQ ID NO: 38)

[0265] EVQLVESGGGLVQPGGSLRLSCAASGFTFSPYAMNWVRQAPGKGLEWVSRIRSKSNNYAAYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRGREAPQAGYAMDYWGQGTTVTVSS

[0266] >h28VH3 (SEQ ID NO: 39)

[0267] EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYAMNWVRQAPGKGLEWVSRIRSKSNNYAAYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGREAPQAGYAMDYWGQGTTVTVSS

[0268] >h28VH4 (SEQ ID NO: 40)

[0269] EVQLVQSGSELKKPGASVKVSCKASGFTFTPYAMNWVRQAPGQGLEWVGRIRSKSNNYAAYYADSVKDRFVISRDTSESTAYLQISSLKAEDTAVYYCVRGREAPQAGYAMDYWGQGTTVTVSS

[0270] >h28VL1 (SEQ ID NO: 41)

[0271] DIVVTQSPLSLPVTPGEPASISCRSSKSLAHSNRNTYLYWLLQKPGQSPQLLIYRKSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLQYPFTFGAGTKLEIK

[0272] >h28VL2 (SEQ ID NO: 42)

[0273] EIVLTQSPATLSLSPGERATLSCRSSKSLAHSNRNTYLYWLQQKPGQSPQLLIYRKSNLASGIPSRFSGSGSGTDFTLTISSLEPEDFAVYYCMQHLQYPFTFGAGTKLEIK

[0274] >h28VL3 (SEQ ID NO: 43)

[0275] DIQMTQSPSSSLSASVGDRVTITCRSSKSLAHSNRNTYLYWLQQKPGKSPQLLIYRKSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQHLQYPFTFGAGTKLEIK

[0276] Example 4. Preparation of humanized antibodies

[0277] 4.1 Sequences of humanized antibodies

[0278] The expression vectors of antibody light chain and heavy chain were constructed respectively, and the humanized antibody light / heavy chains were cross-paired and combined respectively. After transfection into 293E cells, the culture supernatant was collected and purified to obtain the humanized full-length antibody. The humanized antibody heavy chain constant region can be selected from IgG1, IgG2, IgG3, and IgG4 constant regions. For example, the human heavy chain IgG1 constant region is used, and the humanized antibody light chain constant region can be selected from human κ and λ chain constant regions. The exemplary antibody constant region sequences are as follows:

[0279] Amino acid sequence of the human IgG1 heavy chain constant region

[0280] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0281] SEQ ID NO: 44

[0282] The amino acid sequence of the human light chain kappa constant region is:

[0283] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0284] SEQ ID NO: 45

[0285] Exemplarily, the humanized antibody heavy chain variable region of the aforementioned mAbCP11 or mAb28 was fused with the human heavy chain IgG1 constant region (sequence as shown in SEQ ID NO: 44) to form the antibody full-length heavy chain, and the humanized antibody light chain variable region was fused with the human light chain κ constant region (sequence as shown in SEQ ID NO: 45) to form the antibody full-length light chain, to obtain mAbCP11 and mAb28 humanized antibodies, which were used for subsequent experimental testing, and finally the humanized antibody with the best activity was selected, and its complete sequence is as follows:

[0286] Antibody hCP11-7:

[0287] hCP11-7 heavy chain sequence:

[0288] EVTLKESGPVLVKPTETLTLTCTVSGFSLT RYGIN WIRQPPGKGLEWLG VIWTGGDTNYNSALKS RLTISKDKSKSQVVLTTMTNMDPVDTATYYCAR EGVVNYYGSRYSFFDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0289] SEQ ID NO: 46 hCP11-7 light chain sequence:

[0290] DIQMTQSPSSLSASVGDRVTITC RASSSVSYMH WYQQKPGKSPKPWIY ATSNLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPYT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0291] SEQ ID NO: 47

[0292] Antibody h28-3:

[0293] h28-3 heavy chain sequence:

[0294] EVQLVESGGGLVQPGGSLRLSCAASGFTFS IYAM WVRQAPGKGLEWVG RIRSKSNNYAAYYADSVKD RFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR GREAPQAGYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0295] SEQ ID NO: 48

[0296] h28-3 light chain sequence:

[0297] DIQMTQSPSSLSASVGDRVTITC RSSKSLAHSNRNTYLY WLQQKPGKSPQLLIY RKSNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHYPFT FGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0298] SEQ ID NO: 49

[0299] Exemplarily, the humanized antibody disclosed herein is subjected to in vitro enzymatic digestion and degradation to obtain a Fab fragment. The sequence of the humanized antibody Fab fragment is as follows:

[0300] hCP11-7Fab sequence

[0301] hCP11-7-Fab heavy chain:

[0302] EVTLKESGPVLVKPTETLTLTCTVSGFSLT RYGIN WIRQPPGKGLEWLG VIWTGGDTNYNSALKS RLTISKDKSKSQVVLTTMTNMDPVDTATYYCAR EGVVNYYGSRYSFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0303] SEQ ID NO: 50

[0304] hCP11-7-Fab light chain:

[0305] DIQMTQSPSSLSASVGDRVTITC RASSSVSYMH WYQQKPGKSPKPWIY ATSNLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPYT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0306] SEQ ID NO: 51

[0307] h28-3-Fab sequence

[0308] h28-3-Fab heavy chain:

[0309] EVQLVESGGGLVQPGGSLRLSCAASGFTFS IYAM WVRQAPGKGLEWVG RIRSKSNNYAAYYADSVKD RFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR GREAPQAGYAMDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0310] SEQ ID NO: 52

[0311] h28-3-Fab light chain:

[0312] DIQMTQSPSSLSASVGDRVTITC RSSKSLAHSNRNTYLY WLQQKPGKSPQLLIY RKSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHYPFT FGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0313] SEQ ID NO: 53

[0314] Exemplarily, the present disclosure uses a mouse mIgG2a constant region fused with a humanized variable region to obtain an antibody in the form of mIgG2a for evaluation of tumor inhibition activity in mice. The sequence of the antibody in the form of mIgG2a is as follows:

[0315] hCP11-7-mIgG2a heavy chain sequence:

[0316] EVTLKESGPVLVKPTETLTLTCTVSGFSLTRYGINWIRQPPGKGLEWLGVIWTGGDTNYNSALKSRLTISKDKSKSQVVLTMTNMDPVDTATYYCAREGVVNYYGSRYSFFDV WGQGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPT IKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0317] SEQ ID NO: 54

[0318] hCP11-7-mIgG2a light chain sequence:

[0319] DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKSPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPYTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0320] SEQ ID NO: 55

[0321] h28-3-mIgG2a heavy chain sequence:

[0322] EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMNWVRQAPGKGLEWVGRIRSKSNNYAAYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARGREAPQAGYAMDYWGQGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0323] SEQ ID NO: 56

[0324] h28-3-mIgG2a light chain sequence:

[0325] DIQMTQSPSSSLSASVGDRVTITCRSSKSLAHSNRNTYLYWLQQKPGKSPQLLIYRKSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQHLQYPFTFGAGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0326] SEQ ID NO: 57

[0327] 4.2. Sequence and preparation of control antibodies

[0328] The humanized antibody 4A19 of patent WO2021194942A1 is used as the control molecule disclosed in the present invention, and its light and heavy chain amino acid sequences are as follows:

[0329] 4A19 heavy chain sequence:

[0330]

[0331]

[0332] 4A19 light chain sequence:

[0333]

[0334] The antibody 10A11 in patent WO2020138489A1 is used as the control molecule disclosed in the present invention, and its light and heavy chain amino acid sequences are as follows:

[0335] 10A11 heavy chain sequence:

[0336]

[0337] 10A11 light chain sequence:

[0338]

[0339] Example 5. Preparation of CCR8 monoclonal antibody defucosylated in vitro

[0340] CCR8 antibodies work through cellular ADCC, so the defucosyl form of the antibody is more potent than the normal IgG1 form.

[0341] The IgG1 antibody was removed by in vitro fucose removal to prepare ADCC-enhanced antibody. The specific preparation process is as follows:

[0342] Deglycosylation: Adjust the concentration of IgG1 antibody to about 10 mg / mL, adjust its pH to 7.0-7.4, add endoglycosidase Endo S with a final concentration of 50 μg / mL and Alfc enzyme solution with a final concentration of 1.5 mg / mL to the antibody solution, and incubate in a water bath at 37°C for 24 hours. Use SDS-PAGE to judge the deglycosylation effect of the antibody, then use Protein A magnetic beads to purify the antibody, and change the solution to PBS through an ultrafiltration tube (cutoff: 50 kDa).

[0343] Glycosylation transfer: Add the oxazoline substrate CT-oxa (final concentration of antibody 5 mg / mL, final concentration of oxazoline 1.67 mM) at a final concentration of 50 times the antibody concentration to the deglycosylated antibody solution, add EndoS2D184M at a final concentration of 0.1 mg / mL, and incubate in a water bath at 30°C for 1 hour. Mass spectrometry analysis was used to identify whether the prepared antibody was in the de-fucose form, and the obtained antibody was a de-fucose form antibody.

[0344] In the following test examples, the defucosylated humanized antibody is indicated by the suffix "(Defuc)", for example: the defucosylated form of hCP11-7 is hCP11-7-Defuc; the defucosylated form of h28-3 is h28-3-Defuc.

[0345] Test Example - Biological Evaluation of Anti-CCR8 Antibody Activity in Vitro and In Vivo

[0346] Test Example 1: Detecting the binding ability of antibodies to CCR8 expressed on the cell surface

[0347] In order to detect the binding of the antibody to the CCR8 protein on the cell surface, the binding activity of the antibody was detected by FACS using cells expressing CCR8 on the cell surface.

[0348] The full-length genes encoding human CCR8, human CCR4 or monkey CCR8 were cloned into mammalian cell expression vectors, and HEK293T cells were co-transfected with three plasmids, pCDH-human CCR8, pCDH-human CCR4 or pCDH-cyno CCR8, and pVSV-G and pCMV-dR8.91, respectively. CRL-11268) packaged virus. 48 hours after transfection, the virus was collected to infect CHO-K1 (ATCC, CCL-61) cells. After two weeks of pressure screening, cell subcloning was performed. After FACS detection, the cell lines human CCR8-CHO-K1, human CCR4-CHO-K1, and cynoCCR8-CHO-K1 with high expression of human CCR8, human CCR4, and monkey CCR8 were obtained respectively.

[0349] Collect cells, centrifuge at 400g, 4℃ for 5 minutes; add pre-cooled PBS containing a final concentration of 10% FBS, centrifuge at 400g, 4℃ for 5 minutes, repeat twice; distribute cells to 96-well plates, 10 5 cells / well; 100 μL of gradient diluted antibody solution was added to each well, incubated at 4°C for 60 minutes, and centrifuged at 300g to remove the supernatant; 200 μL of pre-cooled PBS containing a final concentration of 10% FBS was added to each well to resuspend the cells, 300g, 4°C centrifuged for 5 minutes, and the supernatant was removed, and repeated twice; 100 μL of 1:1000 diluted secondary antibody AlexaFluorA488 goat anti-human IgG (H+L) (Lifetechologies, Cat#A11013) was added, incubated at 4°C in the dark for 45 minutes, and the supernatant was removed by centrifugation; 200 μL of pre-cooled PBS containing a final concentration of 10% FBS was added to each well to resuspend, 400g, 4°C centrifuged for 5 minutes, and repeated twice; 100 μL of pre-cooled PBS was added to each well to resuspend the cells; detected by flow cytometer (BD, FACS CantoII); the fluorescence signal value was obtained, and the higher the signal, the stronger the binding activity of the antibody to the protein on the cell surface. The test results were analyzed using PRISM software to make a binding curve diagram, and the EC value of the antibody binding activity to the cell surface protein was obtained by fitting. 50 The binding activity of the humanized antibodies is shown in Table 8 below:

[0350] Table 8. Binding activity of humanized antibodies to CCR8 proteins on the cell surface of different species

[0351]

[0352]

[0353] Conclusion: The humanized antibodies disclosed in the present invention retain binding activity similar to that of mouse antibodies. The humanized antibody h28-3 has the strongest binding activity to human CCR8 and no binding activity to monkey CCR8. hCP11-7 has good binding activity to human and monkey CCR8. Both positive antibodies have only relatively weak binding activity to monkey CCR8.

[0354] Test Example 2: Biacore determination of affinity between antibody and CCR8 N-terminal fusion protein

[0355] The affinity of the humanized antibody to be tested for the human CCR8 N-terminal fusion protein was determined using a Biacore (GE, T200) instrument. The hCCR8-mFc fusion protein (Example 1) was captured by affinity using a CM5 biosensor chip (Cat.#BR-1005-30, Cytiva), and then a certain concentration of human CCR8 antibody molecules were passed through the chip surface for 180 seconds, followed by dissociation for 300 seconds. The Biacore T200 instrument was used to detect the reaction signal in real time to obtain the binding and dissociation curves. After each test cycle dissociation was completed, the biosensor chip was cleaned and regenerated with Glycine1.5 (Cat.#BR-1003-54, Cytiva). The data obtained from the experiment were fitted using a 1:1 or steady-state model to obtain affinity values. The affinity test results of humanized antibodies and CCR8N-mouse Fc fusion proteins are shown in Table 9.

[0356] Table 9. Affinity of humanized antibodies and CCR8 N-terminal fusion protein

[0357]

[0358] Conclusion: The humanized antibody h28-3 disclosed herein has a strong affinity to the N-terminal of human CCR8 protein, while hCP11-7 does not bind to the N-terminal fusion protein of human CCR8.

[0359] Test Example 3: Determination of the inhibitory effect of antibodies on calcium flow stimulated by ligand CCL1

[0360] The Gqi5 gene was further transfected into the human CCR8-CHO-K1 cell line obtained in Test Example 1. The full-length gene encoding Gqi5 was cloned into a mammalian cell expression vector, and HEK293T cells were co-transfected with three plasmids: pVSV-G, pCMV-dR8.91, and pCDH-Gqi5. CRL-11268) was used to package the virus. 48 hours after transfection, the virus-infected human CCR8-CHO-K1 cells were collected. After two weeks of pressure screening, the cells were subcloned. After FACS detection, the cell line human CCR8-Gq-CHO-K1 that simultaneously expressed human CCR8 and Gqi5 proteins was obtained.

[0361] After CCR8 on the surface of human CCR8-Gq-CHO-K1 cells is activated by the ligand CCL1, it will cause an increase in intracellular calcium ion levels. Further overexpression of Gqi5 protein in the CCR8-overexpressing CHOK1 cell line can improve the sensitivity of calcium flow detection. Therefore, we used the CHOK1 cell line overexpressing CCR8 and Gqi5 to detect the inhibitory effect of antibodies on calcium flow stimulated by the ligand CCL1. The detection reagent was Fluo-4 DirectTM Calcium Assay kits (Invitrogen, Cat#F10473), which was configured and used according to the instructions of the kit. The day before the experiment, the cells were seeded into 96-well plates at a density of 20,000 cells / well and cultured in a 37°C incubator overnight. The next day, the supernatant in the culture plate was removed, and 1×Fluo-4 DirectTM calciumreagent loading solution (Invitrogen, Cat#F10473) was freshly prepared at 100μL / well and incubated at 37°C for 30 minutes. After the incubation, move the 96-well plate to room temperature for 10 to 30 minutes, then add the prepared gradient dilution antibody to the 96-well plate, 25 μL / well, and incubate at room temperature for 30 minutes. TM Calcium assay buffer (Invitrogen, Cat#F10473). After the incubation, the flexstation 3 microplate reader was used for detection. 40nM CCL1 (Biolegend, Cat#582708) 25μL / well was automatically added by the machine, and the value was immediately read at EX494 / EM516nm. The inhibitory effect of each antibody on intracellular calcium flow induced by CCL1 stimulation was calculated, as shown in Table 10 below.

[0362] Table 10. Inhibitory effects of antibodies on hCCL1-stimulated calcium flux

[0363] Antibody <![CDATA[IC 50 (nM)]]> hCP11-7 2.00 h28-3 0.64 ChCP11 2.7 Ch28 2.8

[0364] Conclusion: The disclosed antibody has a strong inhibitory effect on the intracellular calcium flow induced by CCL1 stimulation.

[0365] Test Example 4: Determination of the effect of antibodies on chemotaxis blocking induced by ligand CCL1

[0366] The BaF3 recombinant cell line overexpressing CCR8 was used to test the blocking effect of the antibody on chemotaxis induced by the ligand CCL1.

[0367] The full-length gene encoding human CCR8 was cloned into a mammalian cell expression vector, and HEK293T cells were co-transfected with three plasmids: pVSV-G, pCMV-dR8.91, and pCDH-human CCR8. CRL-11268) was used to package the virus. After 48 hours of transfection, the virus-infected Ba / F3 cells (Nanjing Kebai, CBP60474) were collected and screened under pressure for two weeks, and then the cells were subcloned. The recombinant cell line human CCR8-Ba / F3 with high expression of human CCR8 protein was obtained by FACS detection.

[0368] CCL1 (R&D, Cat#272-I-050 / CF) and antibodies were prepared with RPMI1640 complete medium (GESH30809.01), and human CCR8-Ba / F3 cells were cultured with RPMI1640+10% FBS+10ng / mL mIL3 (Peprotech, Cat#213-13)+4μg / mL puromycin. On the day of the experiment, the cells were resuspended in complete medium and the cell density was adjusted to 8×10 after counting. 6 / mL. Mix the cells and the prepared antibodies in a 1:1 ratio and incubate in 37°C culture medium for 30 minutes. Then open the top cover of the Chemotaxis System (Neuron Probe Cat#106-8), remove the filter membrane, add 20ng / mL CCL1 protein to the lower chamber, 30μL per well; gently cover the filter membrane, add the cell antibody mixture on the membrane, 50μL / well; cover the top cover, place in an incubator (37°C, 5% CO2), and incubate for 2 hours. Then use a clean paper towel to absorb the liquid on the filter membrane, open the filter membrane, and add 30μL Cell Titer-Glo solution (Promega Cat#G7573) to each well of the lower chamber, and incubate at room temperature in the dark for 5 minutes. Transfer the liquid to a 96-well white-bottom plate to which 40μL Cell Titer-Glo solution has been added, and read the plate using a microplate reader (PerkinElmer, Vector3) using the chemiluminescence method. The experimental data was processed with Graphpad to calculate the IC of the antibody for the BaF3 cell chemotaxis blocking experiment 50 , see Table 11.

[0369] Table 11. Chemotaxis inhibition of human CCR8-Ba / F3 cells by antibodies

[0370] Antibody <![CDATA[IC 50 (nM)]]> hCP11-7 2.63 h28-3 0.34 Ch28 0.86

[0371] Conclusion: The antibodies disclosed herein have strong ligand blocking effects.

[0372] Test Example 5: Determination of the killing effect of antibody-induced PBMC on cells expressing CCR8

[0373] HEK293 recombinant cells overexpressing hCCR8 and Luc-GFP were used to detect antibody-induced killing. The full-length genes encoding human CCR8 and Luc-GFP were cloned into mammalian cell expression vectors, and HEK293T cells were co-transfected with four plasmids: pVSV-G, pCMV-dR8.91, pCDH-human CCR8, and pCDH-Luc-GFP ( CRL-11268) packaged virus. 48 hours after transfection, virus-infected HEK293 (ATCC, CRL-1573) cells were collected. After two weeks of pressure screening, cell subcloning was performed. Recombinant cells with different expression levels of CCR8 were obtained by FACS detection, among which HEK293-hCCR8 / Luc-GFP clone 1 was a human CCR8 high-expressing cell line; HEK293-hCCR8 / Luc-GFP clone 11 was a human CCR8 low-expressing cell line, and the CCR8 expression level of clone 11 was equivalent to the expression level of CCR8 on Treg cells in PBMC.

[0374] HEK293-hCCR8 / Luc-GFP clone 1 (CCR8 high expression) and clone 11 (CCR8 low expression) cells were plated at 5000 cells / well in a 96-well plate with a white transparent bottom. Fresh PBMC cells were centrifuged and the density was adjusted to 0.6×10 6 / mL, and then add 50μL of cell suspension to the well plate so that there are 30,000 effector cells in each well (E:T ratio=6:1). Add 50μL of RPMI1640 complete medium to the control well without effector cells. Take 10μL of the prepared antibody and add it to the 96-well plate so that the initial concentration of the antibody in the cells is 10nM, and add 10μL of RPMI1640 complete medium to the control well without antibody. The negative control is only the medium and target cells, and the control with target cells and effector cells but no antibody. The well plate was placed in a 37℃ cell culture incubator and incubated for 24 hours. Then use ONE-Glo reagent (Promega, E6120) to quantify the number of surviving target cells. After taking out the well plate, add 50μL of the prepared ONE-Glo reagent to each well and incubate at room temperature for 10 minutes, stick a white sticker on the bottom of the well plate, and then use Wallac Victor 3 microplate reader to measure the luminescence fluorescence signal value. The antibody concentration was used as the horizontal axis and the measured luminescence signal value was used as the vertical axis. GraphPad Prism9 software was used to draw the dose-effect curve and calculate the EC of each antibody. 50 The results are shown in Tables 12 and 13.

[0375] Table 12. Antibody killing effect on CCR8 high expression cell clone 1 cell line

[0376]

[0377]

[0378] Table 13. Emax% of the killing effect of antibodies on CCR8 low-expressing cell clone 11 cell line

[0379] Antibody Emax% hCP11-7 17.8 h28-3 16.2 hCP11-7-Defuc 12.0 h28-3-Defuc 7.9

[0380] Table 12 shows that the PBMCs induced by the two antibodies disclosed herein have strong ADCC killing ability against the CCR8 highly expressed cell line Clone 1. For the ADCC enhanced defucosylating antibody disclosed herein, the ADCC killing ability of the induced PBMCs against the highly expressed cell line Clone 1 was enhanced.

[0381] Table 13 shows that all antibodies or their enhanced defucosylated forms did not show significant ADCC killing effect on clone11 cells with low CCR8 expression, and their Emax values ​​were relatively low, making it difficult to fit and calculate the EC values. 50 .

[0382] In summary, the disclosed antibodies can induce PBMC to significantly kill cells with high CCR8 expression (clone 1), but have no significant killing effect on cells with low CCR8 expression (clone 11), which shows that the antibodies have obvious differentiated killing ability on cells with different CCR8 expression levels.

[0383] Test Example 6: Determination of the killing effect of antibodies on Tregs with low CCR8 expression in normal PBMCs

[0384] The fluorescence amplification ligand BATDA (bis(acetoxymethyl) 2,2':6',2"-terpyridine-6,6"-dicarboxylic acid) and target cells (HEK293-hCCR8 / Luc-GFP clone 1 or Treg cells) are co-incubated. BATDA can quickly enter the target cells and form hydrophilic TDA (2,2':6',2"-tripyridine-6,6"-dicarboxylic acid) under hydrolysis and remain in the cells. When the antibody disclosed herein causes ADCC to target cells in PBMC, the target cells are lysed to release TDA. The released TDA combines with a solution containing the lanthanide element europium (DELIFA Eu reagent) to form strong fluorescence. The stronger the fluorescence signal, the stronger the ADCC effect of the antibody.

[0385] This experiment used the DELFIA EUTDA kit ( EuTDA Cytotoxicity Reagents, PerkinElmer, Catalog No. AD0116) were used to detect the ADCC killing effect of the test antibody on different target cells (HEK293-hCCR8 / Luc-GFP clone 1 or Treg cells). The time-resolved fluorometer was read on Victor3. The lysis percentage was calculated according to the instructions of the kit.

[0386] The antibodies to be tested include h28-3-Defuc and hCP11-7-Defuc, which are prepared with RPMI1640 complete medium (GESH30809.01) to a final concentration of 10000 pM, 250 pM and 6.25 pM. C25-hIgG1-Defuc is a negative control.

[0387] The test results are as follows:

[0388] Table 14. Treg cell lysis rate %

[0389] Antibody concentration (pM) h28-3-Defuc hCP11-7-Defuc C25-hIgG1-Defuc 10000 5.19 -1.06 1.17 250 -1.28 -1.99 -0.9 6.25 0.92 -3.79 -2.34

[0390] Table 15. HEK293-hCCR8 / Luc-GFP Clone 1 Lysis Rate %

[0391] Antibody concentration (pM) h28-3-Defuc hCP11-7-Defuc C25-hIgG1-Defuc 10000 51.1 37.9 6.3 250 51.1 49.1 -0.8 6.25 39 24.2 -1.3

[0392] Conclusion: Table 14 shows that the two ADCC-enhanced defucose-form antibodies disclosed herein have no significant killing effect on Treg cells in PBMCs. The killing effect is the same as that of the unrelated antibody C25, and the lysis % is less than 10.

[0393] Table 15 shows that for HEK293-hCCR8 / Luc-GFP clone 1 with high expression of CCR8, both of the two defucosyl forms of the present invention showed significant killing effects, and the lysis % was significantly higher than that of the irrelevant antibody C25. This experiment further proves that the antibodies of the present invention can differentially kill cells with high expression of CCR8 (HEK293-hCCR8-Luc-GFP clone 1) through ADCC, but have no significant killing effect on Treg cells with low expression of CCR8 in normal PBMC.

[0394] Test Example 7: Determination of the site where the antibody binds to CCR8

[0395] In order to determine the binding site of CCR8 antibodies to CCR8, we constructed a series of recombinant cell lines by mutating the amino acids at the N-terminus of CCR8 and constructing chimeric antigens of CCR8 and CCR4, and added a V5 tag at the N-terminus for easy detection. Antibody anti-V5 tag (sinobiology, Cat. No.: 100378-MM04) was used as a positive control.

[0396] The N-terminus of CCR4 was used to replace the N-terminus of CCR8 to obtain the chimeric protein plasmid P2746. The loop 2 of CCR4 was used to replace the loop 2 of CCR8 to obtain the chimeric protein plasmid P2748. The loop 3 of CCR4 was used to replace the loop 3 of CCR8 to obtain the chimeric protein plasmid P2749. The chimeric protein gene encoding human CCR8 and human CCR4 was cloned into the mammalian cell expression vector pCDH, and the three plasmids pVSV-G, pCMV-dR8.91 and the chimeric protein plasmid were co-transfected into HEK293T cells ( CRL-11268) was used to package the virus. After 48 hours of transfection, the virus was collected and infected with CHO-K1 (ATCC, CCL-61) cells. After two weeks of pressure screening, cell subcloning was performed, and recombinant cell lines with high expression of chimeric antigens were obtained through FACS detection.

[0397] The amino acid sequence of the chimeric protein plasmid is as follows:

[0398] >P2746-CCR4 N:

[0399]

[0400]

[0401] >P2748-CCR4 Ring 2:

[0402]

[0403] >P2749-CCR4 ring 3:

[0404]

[0405] Note: The wavy underline represents the V5 tag, the single underline represents the N-terminus, and the double underline represents the extracellular regions of loop 1, loop 2, and loop 3.

[0406] Loop 2 sequence of CCR8:

[0407] YQVASEDGVLQCYSFYNQQTLKWKIFTNFKM

[0408] SEQ ID NO: 65

[0409] Loop 3 sequence of CCR8:

[0410] HSMHILDGCSISQQLTY

[0411] SEQ ID NO: 66

[0412] The same method as in Test Example 1 was used to detect the binding ability of the antibody to these recombinant cell lines using FACS. The results are shown in Table 16 below:

[0413] Table 16. Antibody binding ability to each cell line

[0414]

[0415]

[0416] Note: The data in the table are the average fluorescence intensity of the binding

[0417] Conclusion: All antibodies did not bind to the CHOK1 wild-type cell line, and the average fluorescence intensity was less than 300, while the anti-V5 tag antibody bound to all cell lines expressing the CCR8 fragment, and the average fluorescence intensity was greater than 600, indicating that the recombinant cell lines we constructed can be used for detection.

[0418] If we replace the N-terminus of CCR8 with the N-terminus of CCR4 (P2746), all the antibodies tested will not bind, indicating that the binding of the antibodies tested to CCR8 requires the N-terminus of CCR8.

[0419] At the same time, if loop 2 and loop 3 of CCR4 are used to replace loop 2 (P2748) and loop 3 (P2749) of CCR8 respectively, both positive antibodies can bind to P2748 and P2749 cell lines, indicating that the binding of the two positive antibodies to CCR8 does not require the participation of loop 2 and loop 3, and mainly binds to the N-terminus of CCR8.

[0420] When loop 2 of CCR8 was replaced by loop 2 of CCR4 (P2748), hCP11-7 had no binding at all. When loop 3 of CCR8 was replaced by loop 3 of CCR4 (P2749), the binding of hCP11-7 was also significantly reduced, indicating that the binding of hCP11-7 to CCR8 requires the participation of loop 2 and loop 3 of CCR8. Therefore, HCP11-7 mainly binds to the N-terminus of CCR8 and the spatial conformation formed by loop 2 and loop 3.

[0421] At the same time, loop 2 and loop 3 of human and monkey CCR8 are highly conserved in amino acid sequence, while the N-terminal amino acid sequence similarity of human and monkey CCR8 is not high. hCP11-7 binds to the spatial conformation of CCR8 N-terminus, loop 2, and loop 3, so it has good monkey CCR8 cross-activity, while the two positive antibodies mainly bind to the N-terminus of CCR8, so the binding activity with monkey CCR8 is very weak or non-existent.

[0422] Test Example 8: Evaluation of the efficacy of antibodies on the B-hCCR8 mouse MC38 cell tumor model

[0423] In this experiment, human CCR8 transgenic mice (B-hCCR8 female mice, purchased from Biocytogen Laboratory Animal Co., Ltd.) of the C57BL / 6N strain were inoculated with MC38 mouse colon cancer cells. 3 The mice were randomly divided into groups and given antibody treatment. The effect of the drugs on tumor growth in tumor-bearing mice was evaluated by comparing the size of the tumor volume after treatment with different antibodies. Because CCR8 antibodies mainly work by removing Tregs with high CCR8 expression in tumor TILs through ADCC, the antibodies used in this experiment were all in the form of mIgG2a.

[0424] MC38 cells were subcutaneously inoculated in the right rib of B-hCCR8 mice at a density of 5×10 5 / 100μL / mouse; the average tumor volume is about ~118mm 3 At 14:00 pm, 56 mice were selected according to the tumor volume and randomly divided into 7 groups, 8 mice in each group. Intraperitoneal administration (ip) began on the day of grouping (D0), and the frequency of administration was twice a week (BIW). The tumor volume and mouse body weight were measured twice a week. During the entire treatment process, the body weight of each group was no abnormality compared with the blank group.

[0425] The formula for calculating tumor volume (V) is: V = 1 / 2 × length × width 2

[0426] Relative tumor proliferation rate T / C% = (T-T0) / (C-C0) × 100%

[0427] Tumor inhibition rate TGI% = 1-T / C%

[0428] C0 and T0 are the tumor volumes of the blank control group and the experimental group at the beginning of the experiment, respectively. C and T are the tumor volumes of the blank control group and the experimental group at the end of the experiment, respectively.

[0429] The tumor inhibition results are shown in Table 17.

[0430] Table 17. Antitumor effects of drugs on MC38 tumor-bearing mice

[0431]

[0432] Conclusion: The disclosed antibody has a good tumor inhibition effect.

[0433] Preparation Example - Anti-CCR8 Antibody Preparation

[0434] SEC Size Exclusion Chromatography:

[0435] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecule.

[0436] SEC% (SEC monomer content percentage) = A monomer / A total × 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas). ΔSEC% = SEC% of the preparation before the stability experiment - SEC% of the preparation after the stability experiment.

[0437] Instrument for SEC determination: Agilent HPLC 1260;

[0438] Column: Waters, SEC 3.5μm 7.8×300mm Column

[0439] NR-CE capillary gel electrophoresis:

[0440] A method of electrophoresis in which the gel is moved into a capillary tube as a supporting medium and the samples are separated according to their molecular weight at a certain voltage.

[0441] NR-CE% (NR-CE main peak content percentage) = A main peak / A total × 100% (A main peak is the peak area of ​​the main peak in the sample, A total is the sum of all peak areas). ΔNR-CE% = NR-CE% of the preparation before the stability experiment - NR-CE% of the preparation after the stability experiment.

[0442] CE measurement instrument: Beckman capillary electrophoresis instrument, model PA800 plus.

[0443] Viscosity determination:

[0444] By continuously rotating the rotor in the fluid, the rotational torque sensor measures the degree of twisting of the spring, namely the torque, which is proportional to the resistance formed by the viscous drag of the rotor immersed in the sample. The torque is therefore also proportional to the viscosity of the liquid, thereby measuring the viscosity of the protein solution.

[0445] protein

[0446] The protein used in the following formulation examples is the anti-CCR8 antibody hCP11-7 (hereinafter referred to as "protein" or "CCR8 antibody protein") as described above.

[0447] The instrument used to measure the concentration of the fusion protein is a UV-visible spectrophotometer, model: Nano Drop oneC, with an optical path of 1 mm.

[0448] Formulation Example 1. pH and buffer system screening

[0449] A formulation of 200 mg / mL CCR8 antibody protein, 80 mg / mL sucrose, and 0.4 mg / mL polysorbate 80 (PS80) was prepared using the pH and buffer system shown in Table 18. A forced degradation study (40°C for 4 weeks) was performed on the samples, and the effects of different pH and buffer systems on protein stability were investigated using appearance, SEC, and NR-CE as evaluation indicators.

[0450] The results are shown in Table 18. Appearance, SEC and NR-CE data showed that after 4 weeks at 40°C, the purity of the preparations between the pH 5.0-6.0 groups was slightly different.

[0451] Table 18. pH and buffer system screening results

[0452] Group Buffer system Placement conditions Appearance △SEC% △NR-CE% 1 20mM AA pH 5.0 40℃ W4 Clarity -3.0 -5.2 2 20mM AA pH 5.5 40℃ W4 Clarity -2.9 -4.1 4 20mM His-HCl pH 5.5 40℃ W4 Clarity -2.8 -5.3 5 20mM His-HCl pH 6.0 40℃ W4 Clarity -3.5 -6.1 10 20mM PB pH 6.5 40℃ W4 Clarity -5.9 -9.0

[0453] Note: AA stands for acetic acid-sodium acetate; His-HCl stands for histidine-histidine hydrochloride; PB stands for disodium hydrogen phosphate-sodium dihydrogen phosphate; 40℃ W4: 40℃ for 4 weeks, the same below.

[0454] Formulation Example 2. Surfactant Screening

[0455] 200 mg / mL protein, 80 mg / mL sucrose, and the surfactants shown in Table 19 were prepared using 20 mM AA buffer (pH 5.0). The samples were subjected to forced degradation studies (25°C, 300 rpm shaking for 7 days; 40°C for 4 weeks), and the effects of different surfactants on protein stability were investigated using appearance and SEC as evaluation indicators.

[0456] The results are shown in Table 19. The forced degradation appearance and SEC data showed no significant differences among the surfactant groups.

[0457] Table 19. Surfactant screening results

[0458]

[0459]

[0460] Note: PF68 stands for Poloxamer 188; D7 stands for 7 days, the same below.

[0461] Formulation Example 3. Protein Concentration Screening

[0462] 20 mM AA buffer (pH 5.0) was used to prepare 80 mg / mL sucrose, 0.4 mg / mL PS80, and formulations with the protein concentrations shown in Table 20. The samples were subjected to a forced degradation study (40°C for 4 weeks), and SEC and NR-CE were used as evaluation indicators to investigate the effect of different protein concentrations on protein stability.

[0463] The results are shown in Table 20. SEC and NR-CE data showed that after 4 weeks at 40°C, the change value of the purity item increased with the increase of protein concentration, but the difference between the groups was acceptable.

[0464] Table 20. Protein concentration screening results

[0465] Group Protein concentration (mg / mL) Placement conditions ΔSEC% ΔNR-CE% 1 200 40℃ W4 -3.0 -5.2 2 150 40℃ W4 -2.4 -4.5 3 100 40℃ W4 -2.0 -3.5

[0466] Formulation Example 4. Screening of different ionic strengths

[0467] A preparation of 200 mg / mL protein, 80 mg / mL sucrose, and 0.4 mg / mL PS80 was prepared using the buffer system shown in Table 21. The samples were subjected to a forced degradation study (40°C for 4 weeks), and the effects of different ionic strengths on protein stability were investigated using appearance, SEC, and NR-CE as evaluation indicators.

[0468] The results are shown in Table 21. Appearance data showed that there was no difference in appearance between the 20mM and 50mM ionic strength preparation groups. SEC and NR-CE data showed that after 4 weeks at 40°C, the purity of the preparation at 20mM ionic strength was slightly better than that at 50mM ionic strength.

[0469] Table 21. Screening results at different ionic strengths

[0470] Group Buffer system Placement conditions Appearance △SEC% △NR-CE% 1 20mM AA pH 5.0 40℃ W4 Clarity -3.0 -5.2 2 50mM AA pH 5.0 40℃ W4 Clarity -3.4 -8.0

[0471] Formulation Example 5. Screening of viscosity-reducing excipients for high-concentration protein formulations

[0472] A preparation containing 250 mg / mL of CCR8 antibody protein was prepared using 20 mM AA buffer (pH 5.0) and the viscosity reducers shown in Table 22. The samples were subjected to viscosity measurement to investigate the effects of different viscosity reducers on protein viscosity.

[0473] The results are shown in Table 22. The viscosity data showed that 50 mM Pro had the best viscosity reduction effect on protein.

[0474] Table 22. Screening results of protein viscosity reducers

[0475]

[0476]

[0477] Note: NaCl represents sodium chloride; Arg·HCl represents arginine hydrochloride; Pro represents proline.

[0478] Formulation Example 6. Stability of High Concentration Protein Formulations

[0479] A preparation of 250 mg / mL CCR8 antibody protein, 50 mg / mL sucrose, 0.4 mg / mL PS80, and 50 mM Pro was prepared using 20 mM AA buffer (pH 5.0). The samples were subjected to a forced degradation study (40°C for 4 weeks) and the stability of the high-concentration preparation after the addition of a viscosity reducer was investigated using appearance, SEC, and NR-CE as evaluation indicators.

[0480] The results are shown in Table 23. Appearance data showed that the protein preparation was still clear and transparent after being placed at 40°C for 4 weeks. SEC and NR-CE data showed that after being placed at 40°C for 4 weeks, the protein purity item changed slightly and was within an acceptable range (<10%), so the high-concentration protein preparation with the addition of 50 mM Pro viscosity reducer was relatively stable.

[0481] Table 23. Viscosity reducer stability results

[0482] Group Viscosity reducer Placement conditions Appearance △SEC% △NR-CE% 1 50mM Pro 40℃ W4 Clarity -3.0 -6.1 .

Claims

1. A pharmaceutical composition comprising an anti-CCR8 antibody and a buffer, wherein the anti-CCR8 antibody comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 14, 15 and 16, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 17, 18 and 19, respectively; The buffer is an acetate buffer, a histidine buffer or a phosphate buffer; Preferably, the buffer is acetic acid-sodium acetate buffer or histidine-histidine hydrochloride buffer; More preferably, the buffer is acetic acid-sodium acetate buffer.

2. The pharmaceutical composition according to claim 1, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; preferably, the pH of the pharmaceutical composition is 5.0 to 6.0; further preferably, the pH of the pharmaceutical composition is 5.0 to 5.7; more preferably, the pH of the pharmaceutical composition is 5.3 to 5.7; most preferably, the pH is about 5.

5.

3. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the anti-CCR8 antibody is 1 mg / mL to 280 mg / mL; Preferably, the concentration of the anti-CCR8 antibody is 100 mg / mL to 260 mg / mL; Further preferably, the concentration of the anti-CCR8 antibody is 160 mg / mL to 240 mg / mL; More preferably, the concentration of the anti-CCR8 antibody is 180 mg / mL to 220 mg / mL; Most preferably, it is about 200 mg / mL.

4. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the anti-CCR8 antibody is 1 mg / mL to 280 mg / mL; Preferably, the concentration of the anti-CCR8 antibody is 100 mg / mL to 260 mg / mL; Further preferably, the concentration of the anti-CCR8 antibody is 200 mg / mL to 260 mg / mL; More preferably, the concentration of the anti-CCR8 antibody is 230 mg / mL to 260 mg / mL; Most preferably, it is about 250 mg / mL.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition comprises a surfactant; preferably, the surfactant is polysorbate or poloxamer; more preferably, the surfactant is polysorbate 80 or poloxamer 188; most preferably, the surfactant is polysorbate 80.

6. The pharmaceutical composition according to claim 5, wherein the concentration of the surfactant is 0.01 mg / mL to 1.0 mg / mL; preferably, the concentration of the surfactant is 0.1 mg / mL to 0.8 mg / mL; further preferably, the concentration of the surfactant is 0.2 mg / mL to 0.6 mg / mL; more preferably, the concentration of the surfactant is 0.3 mg / mL to 0.5 mg / mL; most preferably, it is about 0.4 mg / mL.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition comprises sugar; preferably, the sugar is sucrose, trehalose, mannitol or sorbitol; more preferably, the sugar is sucrose.

8. The pharmaceutical composition according to claim 7, wherein the concentration of the sugar is 10 mg / mL to 100 mg / mL; preferably, the concentration of the sugar is 40 mg / mL to 90 mg / mL; further preferably, the concentration of the sugar is 50 mg / mL to 90 mg / mL; more preferably, the concentration of the sugar is 70 mg / mL to 90 mg / mL; most preferably, it is about 80 mg / mL.

9. The pharmaceutical composition according to claim 7, wherein the concentration of the sugar is 10 mg / mL to 100 mg / mL; preferably, the concentration of the sugar is 40 mg / mL to 90 mg / mL; further preferably, the concentration of the sugar is 40 mg / mL to 80 mg / mL; more preferably, the concentration of the sugar is 45 mg / mL to 55 mg / mL; most preferably, the concentration of the sugar is about 50 mg / mL.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the concentration of the buffer is 5 mM to 100 mM; preferably, the concentration of the buffer is 10 mM to 50 mM; further preferably, the concentration of the buffer is 10 mM to 30 mM; more preferably, the concentration of the buffer is 15 mM to 25 mM; most preferably, it is about 20 mM.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutical composition further comprises an excipient; preferably, the excipient is proline, arginine hydrochloride, sodium chloride, disodium edetate, glycine, methionine, histidine, phenylalanine, glutamic acid, aspartic acid or calcium chloride; more preferably, the excipient is proline.

12. The pharmaceutical composition according to claim 11, wherein the concentration of the excipient is 10 mM to 120 mM; preferably, the concentration of the excipient is 40 mM to 100 mM; further preferably, the concentration of the excipient is 40 mM to 80 mM; more preferably, the concentration of the excipient is 45 mM to 55 mM; most preferably, it is about 50 mM.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the anti-CCR8 antibody is a murine antibody, a chimeric antibody or a humanized antibody; Preferably, the anti-CCR8 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 27, and a light chain variable region as shown in SEQ ID NO: 31; More preferably, the anti-CCR8 antibody comprises the heavy chain sequence shown in SEQ ID NO:46, and the light chain sequence shown in SEQ ID NO:

47.

14. A pharmaceutical composition according to any one of claims 1 to 13, comprising the following components: (a) 1 mg / mL to 280 mg / mL of the anti-CCR8 antibody, (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant, (c) 10 mg / mL to 100 mg / mL of sugar, and (d) 5 mM to 100 mM buffer, the pH of the pharmaceutical composition is 5.0 to 6.5; Preferably, the pharmaceutical composition comprises the following components: (a) 100 mg / mL to 260 mg / mL of the anti-CCR8 antibody, (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80, (c) 40 mg / mL to 90 mg / mL of sucrose, and (d) 10 mM to 50 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 5.0 to 6.0; Further preferably, the pharmaceutical composition comprises the following components: (a) 160 mg / mL to 240 mg / mL of the anti-CCR8 antibody, (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80, (c) 50 mg / mL to 90 mg / mL of sucrose, and (d) 10 mM to 30 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 5.0 to 5.7; More preferably, the pharmaceutical composition comprises the following components: (a) 180 mg / mL to 220 mg / mL of an anti-CCR8 antibody, (b) 0.3 mg / mL to 0.5 mg / mL of polysorbate 80, (c) 70 mg / mL to 90 mg / mL of sucrose, and (d) 15 mM to 25 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 5.3 to 5.7; Most preferably, the pharmaceutical composition comprises the following components: (a) about 200 mg / mL of an anti-CCR8 antibody, (b) about 0.4 mg / mL of polysorbate 80, (c) about 80 mg / mL of sucrose, and (d) about 20 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is about 5.

5.

15. A pharmaceutical composition according to any one of claims 1 to 13, comprising the following components: (a) 1 mg / mL to 280 mg / mL of the anti-CCR8 antibody, (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant, (c) 10 mg / mL to 100 mg / mL of sugar, (d) 10 mM to 120 mM of an excipient, and (e) 5 mM to 100 mM buffer, the pH of the pharmaceutical composition is 5.0 to 6.5; Preferably, the pharmaceutical composition comprises the following components: (a) 100 mg / mL to 260 mg / mL of the anti-CCR8 antibody, (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80, (c) 40 mg / mL to 90 mg / mL of sucrose, (d) 40 mM to 100 mM proline, and (e) 10 mM to 50 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 5.0 to 6.0; Further preferably, the pharmaceutical composition comprises the following components: (a) 200 mg / mL to 260 mg / mL of the anti-CCR8 antibody, (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80, (c) 40 mg / mL to 80 mg / mL of sucrose, (d) 40 mM to 80 mM proline, and (e) 10 mM to 30 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 5.0 to 5.7; More preferably, the pharmaceutical composition comprises the following components: (a) 230 mg / mL to 260 mg / mL of an anti-CCR8 antibody, (b) 0.3 mg / mL to 0.5 mg / mL of polysorbate 80, (c) 45 mg / mL to 55 mg / mL of sucrose, (d) 45 mM to 55 mM proline, and (e) 15 mM to 25 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 5.3 to 5.7; Most preferably, the pharmaceutical composition comprises the following components: (a) about 250 mg / mL of an anti-CCR8 antibody, (b) about 0.4 mg / mL of polysorbate 80, (c) about 50 mg / mL of sucrose, (d) about 50 mM proline, and (e) about 20 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is about 5.

5.

16. A method for preparing a lyophilized preparation, comprising the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 15.

17. A lyophilized preparation obtained by the method of claim 16.

18. The pharmaceutical composition according to any one of claims 1 to 15, which is a subcutaneous injection preparation, an intravenous injection preparation, an intraperitoneal injection preparation or an intramuscular injection preparation; preferably a subcutaneous injection preparation.

19. A method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 15, or the lyophilized preparation of claim 17; Preferably, the disease is selected from cancer or tumor, inflammatory disease and viral infection; More preferably, the cancer or tumor is selected from prostate cancer, bladder cancer, ovarian cancer, lung cancer, liver cancer, stomach cancer, colon cancer, kidney cancer, cutaneous fibrosarcoma, osteosarcoma, cervical cancer, esophageal cancer, rectal cancer, head and neck squamous cell carcinoma, breast cancer, multiple myeloma, lymphoma and melanoma.

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