Anti-CCR8 antibodies and uses thereof

Abstract

Provided are a CCR8-targeting antibody or an antigen-binding fragment thereof, and an application thereof. The antibody targeting CCR8 or the antigen binding fragment of the antibody is specifically bound with CCR8. The invention further provides application of the antibody in blocking a signal channel for binding / inducing CCR1 and CCR8, clearing CCR8 positive Treg cells through ADCC action and enhancing anti-tumor immunity.

Description

Anti-CCR8 antibodies and uses thereof Technical Field

[0001] The present invention relates to the field of monoclonal antibodies and / or engineered antibodies. Specifically, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CCR8, and compositions comprising the same. Also provided are nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof of the present invention, vectors and host cells for expressing the antibodies or antigen-binding fragments thereof of the present invention, and therapeutic and diagnostic / detection methods and uses of the antibodies or antigen-binding fragments thereof of the present invention. Background Art

[0002] The body's immune system is a core defense system against tumor development and progression, and the inability of the immune system to recognize and eliminate malignant cells plays a significant role in the pathogenesis of cancer. In the tumor microenvironment, high expression of multiple immune checkpoints reduces immune activation and inhibits anti-tumor immunity. Monoclonal antibodies targeting the inhibitory immune checkpoints CTLA-4 and PD-1 / PD-L1 produce significant anti-tumor responses by upregulating immune activation in the tumor microenvironment. Currently, multiple monoclonal antibodies targeting CTLA-4 and PD-1 / PD-L1 have been approved for the treatment of various solid tumors, including the anti-CTLA-4 antibody ipilimumab, the anti-PD-1 antibodies nivolumab and pembrolizumab, and the anti-PD-L1 antibody atezolizumab. Compared with traditional treatments (including chemotherapy, radiotherapy and surgery), cancer immunotherapy has brought significant improvements to patients in terms of survival rate and quality of life (K Esfahani et al., A review of cancer immunotherapy: from the past, to the present, to the future, Curr Oncol. 2020 Apr; 27(Suppl 2): ​​S87-S97.).

[0003] Although immune checkpoint inhibitors have been clinically approved for the treatment of a variety of solid tumors, their response rates vary greatly across tumor types, and the overall response rate is low. The overall response rate (ORR) of immune checkpoint inhibitors in non-small cell lung cancer (NSCLC), urothelial carcinoma, and head and neck cancer is only about 20%, while it is less than 10% in solid tumors such as gastric cancer (Schoenfeld AJ and Hellmann MD. Acquired Resistance to Immune Checkpoint Inhibitors. Cancer Cell. 2020 Apr 13; 37(4): 443-455. doi: 10.1016 / j.ccell.2020.03.017.). Some patients have a persistent response to immune checkpoint inhibitors, but many patients who initially respond later develop acquired resistance, for example, more than 50% of NSCLC and gastric cancer patients develop acquired resistance (Schoenfeld AJ and Hellmann MD). Even some solid tumor patients not only do not benefit from immune checkpoint inhibitors, but unfortunately develop hyperprogressive tumors. This occurs in different proportions in patients with various solid tumors after immune checkpoint treatment. For example, 14% of patients with NSCLC will develop hyperprogression (Champiat S et al. Hyperprogressive disease: recognizing a novel pattern to improve patient management. Nat Rev Clin Oncol. 2018 Dec;15(12):748-762.doi:10.1038 / s41571-018-0111-2.). At present, there is still a need to develop new immune checkpoint inhibitors or immunomodulators to supplement or overcome the shortcomings and problems of immune checkpoint inhibitors currently used in clinical practice.

[0004] The low overall response rate, acquired resistance and hyperprogression of immune checkpoint inhibitors are related to multiple mechanisms in the internal signaling pathways of tumor cells and the tumor microenvironment (Baxter MA et al. Resistance to immune checkpoint inhibitors in advanced gastro-oesophageal cancers. Br J Cancer. 2021 Oct; 125(8): 1068-1079. doi: 10.1038 / s41416-021-01425-7.). Regulatory T cells (Treg) play an important role in immune regulation and are related to the occurrence and development of tumors. Compared with the periphery, the number of Treg in the tumor microenvironment increases and the function is enhanced; Treg inhibits anti-tumor immunity through mechanisms such as secretion of inhibitory cytokines such as IL-10, consumption of IL-2 and direct contact inhibition, which has an important relationship with the natural and acquired resistance of immune checkpoint inhibitors (Baxter MA et al.). PD-1 blockade can induce dysfunctional PD-1 + CD8 + T cell recovery, but also enhances PD-1 + Treg cell-mediated immunosuppression. If the number of Treg cells expressing PD-1 in the tumor microenvironment is greater than that of PD-1 + CD8 +T cells, PD-1 blockade may lead to hyperprogression of tumors (Kumagai S et al. The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies. Nat Immunol. 2020 Nov;21(11):1346-1358.doi:10.1038 / s41590-020-0769-3.). Preclinical animal models and clinical studies have shown that eliminating Tregs in the tumor microenvironment can promote anti-tumor immunity and increase the therapeutic effect of PD-1 / PD-L1. Clinically, it has been found that the combination of the anti-CTLA-4 antibody ipilimumab and the anti-PD-1 antibody nivolumab is more beneficial for the survival of patients with melanoma and renal cell carcinoma (Hayashi H and Nakagawa K. Combination therapy with PD-1 or PD-L1 inhibitors for cancer. Int J Clin Oncol. 2020 May; 25(5):818-830. doi:10.1007 / s10147-019-01548-1.)

[0005] Currently, antibodies with the function of clearing Tregs under clinical research include antibodies targeting targets such as CTLA-4, CD25, co-stimulatory factor OX40, and GITR, and have made some progress in clinical practice (Togashi Y et al. Regulatory T cells in cancer immunosuppression-implications for anticancer therapy. Nat Rev Clin Oncol. 2019 Jun; 16(6): 356-371. doi: 10.1038 / s41571-019-0175-7.). However, these targets are highly expressed on both peripheral Tregs and effector T cells, so the simultaneous clearance of peripheral Tregs and effector T cells reduces their clinical efficacy and increases toxicity, thus limiting their clinical application. In recent years, it has been found that the chemokine receptor CCR8 is highly expressed on Treg cells in human tumors (including breast cancer, non-small cell lung cancer, colorectal cancer, melanoma, hepatocellular carcinoma and pancreatic ductal adenocarcinoma), but is not expressed or is lowly expressed on Treg and effector T cells in peripheral and normal tissues (Plitas G et al. Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer. Immunity. 2016 Nov 15; 45(5): 1122-1134. doi: 10.1016 / j.immuni.2016.10.032, and De Simone M et al. Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells. Immunity. 2016 Nov 15;45(5):1135-1147.doi:10.1016 / j.immuni.2016.10.021.). High CCR8 expression is associated with poor prognosis in breast cancer, non-small cell lung cancer, and colorectal cancer. Compared with CCR8-negative Treg cells, CCR8-positive Treg cells in the tumor microenvironment have a stronger ability to inhibit the proliferation of effector T cells (Gang, Yi et al. Identification and functional analysis of heterogeneous FOXP3 +Treg cell subpopulations in human pancreatic ductal adenocarcinoma[J].Science Bulletin,2018.Doi:10.1016 / j.scib.2018.05.028). In animal models, it was found that CCR8 antibodies can specifically eliminate Tregs in the tumor microenvironment, inhibit tumor growth, and have a synergistic effect with PD-1 antibodies (US patent application number US20190071508 and Van Damme H et al. Therapeutic depletion of CCR8+tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy.J Immunother Cancer.2021 Feb;9(2):e001749.doi:10.1136 / jitc-2020-001749.). The development of antibodies specific for human CCR8 may increase the therapeutic effect of immune checkpoints in clinical practice and has important application value.

[0006] SUMMARY OF THE INVENTION

[0007] The present invention uses cells overexpressing human CCR8 and mice immunized with CCR8 nucleic acid to screen for anti-human CCR8-specific antibodies through hybridoma technology. These anti-CCR8 antibodies have high affinity for both human and monkey CCR8, blocking the binding and induced signaling pathways of CCL1 and CCR8. They can also eliminate CCR8-positive cells (e.g., Treg cells) through ADCC, and effectively inhibit tumor growth in various mouse models (e.g., colorectal cancer, lung cancer, breast cancer, and melanoma).

[0008] In one aspect, the present invention provides an anti-CCR8 antibody or an antigen-binding fragment thereof, wherein the anti-CCR8 antibody or the antigen-binding fragment thereof comprises:

[0009] (i) the three complementarity determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in any one of SEQ ID NOs: 21-40, and / or the three complementarity determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in any one of SEQ ID NOs: 42-54;

[0010] (ii) The CDR combination as described in (i), wherein compared with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3, the sequence comprises at least one amino acid addition, substitution or deletion or any combination thereof (for example, 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof), preferably amino acid substitutions, preferably conservative substitutions, and maintains affinity for CCR8.

[0011] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, comprising a heavy chain variable region and / or a light chain variable region, wherein

[0012] The heavy chain variable region comprises:

[0013] (1) HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 or 10 or 11 or 41 and SEQ ID NO: 3, respectively; or HCDR1, HCDR2 and HCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 or 10 or 11 or 41 and SEQ ID NO: 3, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof); or

[0014] (2) HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or HCDR1, HCDR2 and HCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof); or

[0015] (3) HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; or HCDR1, HCDR2 and HCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof);

[0016] and / or

[0017] The light chain variable region comprises:

[0018] (1) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; or LCDR1, LCDR2 and LCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof); or

[0019] (2) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; or LCDR1, LCDR2 and LCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof); or

[0020] (3) LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively; or LCDR1, LCDR2 and LCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof).

[0021] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in any combination of the following tables:

[0022] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, comprising a heavy chain variable region VH and / or a light chain variable region VL, wherein:

[0023] (a) Heavy chain variable region VH

[0024] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 21-40; or

[0025] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 21-40; or

[0026] (iii) comprising an amino acid sequence having at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to the amino acid sequence shown in any one of SEQ ID NOs: 21-40, preferably, the amino acid change occurs in the CDR region, preferably, the amino acid change occurs in the FR region;

[0027] and / or

[0028] (b) Light chain variable region VL

[0029] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 42-54;

[0030] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 42-54; or

[0031] (iii) comprising an amino acid sequence having at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to the amino acid sequence shown in any one of SEQ ID NOs: 42-54, preferably, the amino acid change does not occur in the CDR region, preferably, the amino acid change occurs in the FR region.

[0032] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, comprising a heavy chain variable region VH and a light chain variable region VL as shown in any combination of the following tables:

[0033] In some embodiments, the present invention provides an anti-CCR8 antibody or antigen-binding fragment thereof comprising a heavy chain and / or a light chain, wherein

[0034] (a) Heavy chain

[0035] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 55-74;

[0036] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 55-74; or

[0037] (iii) comprising an amino acid sequence having at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to an amino acid sequence selected from any one of SEQ ID NOs: 55-74, preferably, the amino acid change does not occur in the CDR region of the heavy chain, more preferably, the amino acid change does not occur in the heavy chain variable region, and most preferably, the amino acid change occurs in the heavy chain constant region;

[0038] and / or

[0039] (b) Light chain

[0040] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 75-82;

[0041] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 75-82; or

[0042] (iii) comprising an amino acid sequence having at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to an amino acid sequence selected from any one of SEQ ID NOs: 75-82, preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the amino acid change does not occur in the light chain variable region, most preferably, the amino acid change occurs in the light chain constant region.

[0043] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain constant region and / or a light chain constant region, preferably, the light chain constant region is a lambda chain or a kappa chain constant region; the heavy chain constant region is selected from murine mIgG2a, human IgG1, human IgG2, human IgG3, or IgG4. In some preferred embodiments, the heavy chain constant region is human IgG1 or human IgG4 with an S228P mutation.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, and the sequence of the heavy chain constant region has one or more amino acid substitutions compared to the sequence of the natural human heavy chain constant region, preferably, the one or more amino acid substitutions enhance the ADCC effect of the antibody. In some preferred embodiments, the one or more amino acid substitutions occur at positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 3 01, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439. In some preferred embodiments, the one or more amino acid substitutions occur in one or more of the heavy chain constant region sequence positions L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339, and P396 according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions are selected from one or more of G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions are selected from one or more of N297A substitution, N297Q substitution, L235A substitution together with L237A substitution, L234A substitution together with L235A substitution, E233P substitution, L234V substitution, L235A substitution, C236 deletion, P238A substitution, D265A substitution, A327Q substitution and P329A substitution according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions occur in one or more of positions 235, 239, 243, 292, 300, 330, 332, 396 of the heavy chain constant region sequence according to the EU numbering system.In some preferred embodiments, the one or more amino acid substitutions are at least one selected from S239D, L235V, F243L, R292P, Y300L, A330L, I332E and P396L according to the EU numbering system.

[0045] In some preferred embodiments, the heavy chain constant region has one or more sets of mutations occurring simultaneously at a combination of positions selected from the group consisting of: (1) L235 / F243 / R292 / Y300 / P396, (2) F243 / R292 / Y300 / V305 / P396, (3) D270 / K326 / A330 / K334, (4) S 239 / A330 / I332, (5) S298 / E333 / K334, (6) L234 / L235 / G236 / S239 / H268 / D270 / S298, (7) M252 / S254 / T256, (8) L234 / L235 / D265, (9) G236 / S239 / I332 and (10) S239 / I332.

[0046] In some preferred embodiments, the heavy chain constant region has one or more mutations selected from the following combinations of mutations according to the EU numbering system: (1) L235V / F243L / R292P / Y300L / P396L, (2) F243L / R292P / Y300L / V305I / P396L, (3) D270E / K326D / A330M / K334E, (4) S239D / A330L / I332E, (5) S298A / E333A / K334A, (6)L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, (7)M252Y / S254T / T256 E, (8)L234A / L235A / D265A, (9)L234F / L235E / D265A, (10)G236A / S239D / I332E and (11)S239D / I332E.

[0047] In some embodiments, the CCR8-binding antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain (HC) and a light chain (LC) as shown in any combination of the following tables:

[0048] In some embodiments, the constant regions of the antibodies of the invention are afucosylated or have reduced fucosylation.

[0049] In some embodiments, the antibodies of the invention are monoclonal antibodies.

[0050] In some embodiments, the antibodies of the invention are murine antibodies, chimeric antibodies, or humanized or human antibodies.

[0051] In some embodiments, the antigen-binding fragments of the present invention include the following antibody fragments: Fab, Fab', Fab'-SH, Fv, Fd, single-chain antibody (e.g., scFv) or (Fab')2, single-domain antibody, diabody (dAb) or linear antibody.

[0052] In another aspect, the present invention provides an isolated anti-CCR8 antibody or antigen-binding fragment thereof having one or more of the following properties:

[0053] (1) binding to the same or completely or partially overlapping epitope of the human CCR8 protein as any anti-CCR8 antibody or antigen-binding fragment thereof of the present invention;

[0054] (2) competing with any anti-CCR8 antibody or antigen-binding fragment thereof of the present invention for binding to an epitope of the human CCR8 protein;

[0055] (3) exhibiting the same or similar binding affinity and / or specificity to CCR8 as the antibodies of the present invention;

[0056] (4) Possess one or more biological characteristics of the antibodies of the present invention.

[0057] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have one or more of the following properties:

[0058] (1) Binds to human / cynomolgus monkey CCR8 with high affinity, e.g., exhibiting an EC no greater than 1000 ng / mL 50 Value, EC no higher than 950ng / mL 50 Value, EC no higher than 900ng / mL 50 Value, EC no higher than 850ng / mL 50 Value, EC no higher than 800ng / mL 50 Value, EC no higher than 750ng / mL 50 Value, EC no higher than 700ng / mL 50 Value, EC no higher than 650ng / mL 50 Value, EC no higher than 600ng / mL 50 Value, EC no higher than 550ng / mL 50 Value, EC no higher than 500ng / mL 50 Value, EC no higher than 450ng / mL 50 Value, EC no higher than 400ng / mL 50Value, EC no higher than 350ng / mL 50 Value, EC no higher than 300ng / mL 50 Value, EC no higher than 250ng / mL 50 Value, EC no higher than 200ng / mL 50 Value, EC no higher than 180ng / mL 50 Value, EC no higher than 160ng / mL 50 Value, EC no higher than 150ng / mL 50 Value, EC no higher than 140ng / mL 50 Value, EC no higher than 130ng / mL 50 Value, EC no higher than 120ng / mL 50 Value, EC no higher than 110ng / mL 50 Value, EC no higher than 100ng / mL 50 Value, EC no higher than 95ng / mL 50 Value, EC no higher than 90ng / mL 50 Value, EC no higher than 85ng / mL 50 Value, EC no higher than 80ng / mL 50 Value, EC no higher than 75ng / mL 50 Value, EC no higher than 70ng / mL 50 Value, EC no higher than 65ng / mL 50 Value, EC no higher than 60ng / mL 50 Value, EC no higher than 55ng / mL 50 Value, EC no higher than 50ng / mL 50 Value, EC no higher than 45ng / mL 50 Value, EC no higher than 40ng / mL 50 Value, EC no higher than 35ng / mL 50 Value, EC no higher than 30ng / mL 50 Value, EC no higher than 25ng / mL 50 Value, EC no higher than 20ng / mL 50 Value, EC no higher than 18ng / mL 50 Value, EC no higher than 16ng / mL 50 Value, EC no higher than 15ng / mL 50 Value, EC no higher than 14ng / mL 50 Value, EC no higher than 13ng / mL 50 Value, EC no higher than 12ng / mL 50 Value, EC no higher than 11ng / mL 50Value, EC no higher than 10ng / mL 50 Value, EC no higher than 8ng / mL 50 Value, EC no higher than 6ng / mL 50 Value, EC no higher than 4ng / mL 50 Value, EC no higher than 2ng / mL 50 Value, EC no higher than 1ng / mL 50 Value, or lower EC 50 value.

[0059] (2) Blocking the binding of human / cynomolgus monkey CCR8 to its ligand (e.g., CCL1), for example, exhibiting an IC of no more than 1000 ng / mL 50 Value, IC value not higher than 950ng / mL 50 Value, IC no higher than 900ng / mL 50 Value, IC no higher than 850ng / mL 50 Value, IC no higher than 800ng / mL 50 Value, IC no higher than 750ng / mL 50 Value, IC no higher than 700ng / mL 50 Value, IC no higher than 650ng / mL 50 Value, IC no higher than 600ng / mL 50 Value, IC no higher than 550ng / mL 50 Value, IC no higher than 500ng / mL 50 Value, IC no higher than 450ng / mL 50 Value, IC no higher than 400ng / mL 50 value, IC value not higher than 350ng / mL 50 value, IC value not higher than 300ng / mL 50 value, IC value not higher than 250ng / mL 50 Value, IC no higher than 200ng / mL 50 Value, IC no higher than 180ng / mL 50 value, IC value not higher than 160ng / mL 50 Value, IC value not higher than 150ng / mL 50 Value, IC no higher than 140ng / mL 50 Value, IC no higher than 130ng / mL 50 value, IC value not higher than 120ng / mL 50 value, IC value not higher than 110ng / mL 50 Value, IC value not higher than 100ng / mL 50 Value, IC value not higher than 95ng / mL 50Value, IC no higher than 90ng / mL 50 Value, IC no higher than 85ng / mL 50 Value, IC no higher than 80ng / mL 50 Value, IC no higher than 75ng / mL 50 Value, IC no higher than 70ng / mL 50 Value, IC no higher than 65ng / mL 50 Value, IC no higher than 60ng / mL 50 Value, IC no higher than 55ng / mL 50 Value, IC value not higher than 50ng / mL 50 Value, IC no higher than 45ng / mL 50 Value, IC no higher than 40ng / mL 50 Value, IC no higher than 35ng / mL 50 Value, IC no higher than 30ng / mL 50 Value, IC value not higher than 25ng / mL 50 Value, IC no higher than 20ng / mL 50 Value, IC no higher than 18ng / mL 50 Value, IC no higher than 16ng / mL 50 Value, IC no higher than 15ng / mL 50 Value, IC no higher than 14ng / mL 50 Value, IC no higher than 13ng / mL 50 Value, IC no higher than 12ng / mL 50 Value, IC no higher than 11ng / mL 50 Value, IC value not higher than 10ng / mL 50 Value, IC no higher than 8ng / mL 50 Value, IC no higher than 6ng / mL 50 Value, IC no higher than 4ng / mL 50 Value, IC no higher than 2ng / mL 50 Value, IC value not higher than 1ng / mL 50 Value, or lower IC 50 value.

[0060] (3) Blocking CCL1-induced β-Arrestin recruitment, for example, exhibiting an IC of no more than 10,000 ng / mL 50 Value, IC no higher than 9500ng / mL 50 Value, IC no higher than 9000ng / mL 50 Value, IC no higher than 8500ng / mL 50 Value, IC no higher than 8000ng / mL50 Value, IC no higher than 7500ng / mL 50 Value, IC no higher than 7000ng / mL 50 Value, IC no higher than 6500ng / mL 50 Value, IC no higher than 6000ng / mL 50 Value, IC no higher than 5500ng / mL 50 Value, IC no higher than 5000ng / mL 50 Value, IC no higher than 4500ng / mL 50 Value, IC no higher than 4000ng / mL 50 Value, IC no higher than 3500ng / mL 50 Value, IC no higher than 3000ng / mL 50 Value, IC no higher than 2500ng / mL 50 Value, IC no higher than 2000ng / mL 50 Value, IC no higher than 1800ng / mL 50 Value, IC no higher than 1600ng / mL 50 Value, IC no higher than 1500ng / mL 50 Value, IC no higher than 1400ng / mL 50 Value, IC no higher than 1300ng / mL 50 Value, IC no higher than 1200ng / mL 50 Value, IC no higher than 1100ng / mL 50 Value, IC no higher than 1000ng / mL 50 Value, IC value not higher than 950ng / mL 50 Value, IC no higher than 900ng / mL 50 Value, IC no higher than 850ng / mL 50 Value, IC no higher than 800ng / mL 50 Value, IC no higher than 750ng / mL 50 Value, IC no higher than 700ng / mL 50 Value, IC no higher than 650ng / mL 50 Value, IC no higher than 600ng / mL 50 Value, IC no higher than 550ng / mL 50 Value, IC no higher than 500ng / mL 50 Value, IC no higher than 450ng / mL 50 Value, IC no higher than 400ng / mL 50 value, IC value not higher than 350ng / mL 50value, IC value not higher than 300ng / mL 50 value, IC value not higher than 250ng / mL 50 Value, IC no higher than 200ng / mL 50 Value, IC no higher than 180ng / mL 50 value, IC value not higher than 160ng / mL 50 Value, IC value not higher than 150ng / mL 50 Value, IC no higher than 140ng / mL 50 Value, IC no higher than 130ng / mL 50 value, IC value not higher than 120ng / mL 50 value, IC value not higher than 110ng / mL 50 Value, IC value not higher than 100ng / mL 50 Value, IC no higher than 80ng / mL 50 Value, IC no higher than 60ng / mL 50 Value, IC no higher than 40ng / mL 50 Value, IC no higher than 20ng / mL 50 Value, IC value not higher than 10ng / mL 50 Value, or lower IC 50 value.

[0061] The present invention also provides a multispecific antibody comprising the light chain variable region and / or heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.

[0062] The present invention also provides a single-chain antibody comprising the light chain variable region and the heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.

[0063] The present invention also provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof described herein conjugated to a therapeutic agent or a diagnostic agent.

[0064] In yet another aspect, the present invention provides a polynucleotide molecule encoding any of the anti-CCR8 antibodies described herein or any fragment thereof.

[0065] In another aspect, the present invention provides an expression vector comprising the polynucleotide molecule of the present invention. Preferably, the vector is a eukaryotic expression vector.

[0066] In another aspect, the present invention provides a host cell comprising the polynucleotide molecule of the present invention or the expression vector of the present invention. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0067] In another aspect, the present invention provides a method for preparing the anti-CCR8 antibody or antigen-binding fragment thereof described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0068] In yet another aspect, the present invention provides a pharmaceutical composition comprising the anti-CCR8 antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate described herein, and optionally at least one pharmaceutically acceptable carrier or excipient.

[0069] In yet another aspect, the present invention provides a drug combination comprising an antibody or antigen-binding fragment thereof, a polynucleotide, a vector, a host cell, an immunoconjugate or a pharmaceutical composition as described herein, and one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the additional therapeutic agent is other antibodies. In some embodiments, the additional therapeutic agent is other monoclonal antibodies. In some preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting an immune checkpoint. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-L1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting CTLA4.

[0070] In another aspect, the present invention provides a method for eliminating CCR8-positive Treg cells in vitro or in vivo, the method comprising contacting an anti-CCR8 antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate or pharmaceutical composition described herein with a cell population comprising CCR8-positive Treg cells or administering it to a subject.

[0071] In another aspect, the present invention provides use of the antibodies or antigen-binding fragments thereof, polynucleotides, vectors, host cells, immunoconjugates, pharmaceutical compositions or drug combinations described herein in the preparation of medicaments for treating and / or preventing tumors, autoimmune diseases, or infectious diseases, preferably the tumor is melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer.

[0072] In some embodiments, the present invention provides the use of an antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, or pharmaceutical composition described herein in combination with other therapeutic agents in the preparation of a medicament for treating and / or preventing a tumor, autoimmune disease, or infectious disease, preferably a tumor selected from melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer, or thymic cancer. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the additional therapeutic agent is another antibody. In some embodiments, the additional therapeutic agent is another monoclonal antibody. In some preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting an immune checkpoint. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-L1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting CTLA4.

[0073] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, pharmaceutical composition or drug combination as described herein for use in treating and / or preventing a tumor, an autoimmune disease, or an infectious disease, preferably the tumor is melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer.

[0074] In another aspect, the present invention provides a method for treating and / or preventing tumors, autoimmune diseases, or infectious diseases, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, pharmaceutical composition or drug combination described herein, wherein the tumor is preferably melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer.

[0075] In yet another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, pharmaceutical composition or pharmaceutical combination described herein, preferably further comprising a drug delivery device.

[0076] In yet another aspect, the present invention provides a method for detecting the presence of CCR8 in a sample using the antibody, antigen-binding fragment, or immunoconjugate described herein, or a detection composition containing the antibody, antigen-binding fragment, or immunoconjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 shows the binding activity of the anti-CCR8 antibody of the present invention to human CCR8. Figures AD are the binding curves of the antibody in the purified and quantified monoclonal hybridoma cell culture supernatant to 293F-hCCR8.

[0078] Figure 2 shows the binding activity of the anti-CCR8 antibody of the present invention to cynomolgus monkey CCR8. AB is the binding curve of the purified and quantified monoclonal hybridoma cell culture supernatant antibody to 293T-cynoCCR8.

[0079] Figure 3 shows that multiple antibody clones of the present invention can block the binding of CCL1 to CCR8. AC are inhibition curves of antibodies in purified and quantified monoclonal hybridoma cell culture supernatants blocking 10 nM AlexaFlour-647-labeled human CCL1 and 293F-hCCR8.

[0080] Figure 4 shows the binding activity of the anti-CCR8 chimeric antibodies of the present invention to human CCR8. AB and C are the binding curves of hIgG1 and mIgG2a chimeric antibodies to 293F-hCCR8, respectively.

[0081] Figure 5 shows the binding activity of the anti-CCR8 chimeric antibodies of the present invention to cynomolgus monkey CCR8. Figures A and B are the binding curves of hIgG1 and mIgG2a chimeric antibodies to 293T-cynoCCR8, respectively.

[0082] Figure 6 shows the activity of the anti-CCR8 chimeric antibodies of the present invention in blocking the binding of CCL1. AB and C are the inhibition curves of hIgG1 and mIgG2a chimeric antibodies blocking the binding of 10 nM AlexaFlour-647-labeled human CCL1 to 293F-hCCR8, respectively.

[0083] Figure 7 shows that the CCR8 chimeric antibody of the present invention blocks CCL1-induced β-Arrestin recruitment. A and B are inhibition curves of hIgG1 and mIgG2a chimeric antibodies blocking human CCL1-induced β-Arrestin recruitment, respectively.

[0084] Figure 8 shows the binding activity of the humanized anti-CCR8 antibodies of the present invention to human CCR8. AB, CD, and EG are the binding curves of humanized antibodies cloned 27B9-1G3, 559E1B10, and 563E10E12 to 293F-hCCR8, respectively.

[0085] FIG9 shows the binding activity of the humanized anti-CCR8 antibodies of the present invention to cynomolgus monkey CCR8, as reflected by the binding curves of the humanized antibodies 559E1B10 and 563E10E12 to 293T-cynoCCR8.

[0086] Figure 10 shows the blocking activity of the humanized anti-CCR8 antibodies of the present invention in binding to CCL1. AB, CD, and EG are the inhibition curves of 27B9-1G3, 559E1B10, and 563E10E12 humanized antibodies blocking 10 nM AlexaFlour-647-labeled human CCL1 and 293F-hCCR8, respectively.

[0087] Figure 11 shows an experiment in which the anti-CCR8 humanized antibodies of the present invention block CCL1-induced β-Arrestin recruitment. AB and CE are the inhibition curves of 559E1B10 and 563E10E12 humanized antibodies blocking CCL1-induced β-Arrestin recruitment, respectively.

[0088] FIG12 shows that the chimeric antibody and humanized antibody of 559E1B10 can activate Jurkat-human FcγRⅢa(158V)-NFAT in a dose-dependent manner.

[0089] FIG13 shows that the chimeric antibody and humanized antibody of 563E10E12 can activate Jurkat-human FcγRⅢa(158V)-NFAT in a dose-dependent manner.

[0090] FIG14 shows the binding activity of the anti-CCR8 antibody of the present invention to HuT78.

[0091] Figure 15 shows the specific binding of the anti-CCR8 antibodies of the present invention to hCCR8 and the binding activity of the anti-CCR8 antibodies to CHOK1-hCCR8 (A) and CHO-K1 (B) cells.

[0092] Figure 16 shows the CCR8-dependent activation of Jurkat-human FcγRIIIa(158V)-NFAT by the anti-CCR8 antibody of the present invention. Panels A and B correspond to target cells CHOK1-hCCR8 and CHOK1-Blank, respectively.

[0093] Figure 17 shows the binding of the anti-CCR8 antibodies of the present invention to 293F cells transfected with hCCR4, including binding of anti-CCR8 antibodies to 293F cells transfected with hCCR4-GFP (A, GFP-positive cells) and blank cells (B, all living cells).

[0094] Figure 18 shows activation of Jurkat-human FcγRIIIa(158V)-NFAT by incubation of 559E1B10 and 563E10E12 humanized antibodies with Fc mutations or reduced fucosylation with 293F-human CCR8. AC shows activation of Jurkat-human FcγRIIIa(158V)-NFAT by incubation of 559E1B10_hzH1L1 (A), 563E10E12_hzH1L1_hIgG1 (B), and 563E10E12_hzH1L0_hIgG1 (C), respectively, with 293F-human CCR8.

[0095] FIG19 shows the activation of Jurkat-human FcγRIIIa(158V)-NFAT by incubation of 563E10E12_hzH1L0 with Fc mutation or reduced fucosylation with HuT78.

[0096] Figure 20 shows the ADCC effect induced by healthy human PBMC and anti-CCR8 antibodies. IL-2-activated healthy human PBMCs were incubated with anti-CCR8 antibodies and CHOK1-hCCR8 for approximately 5 hours. The proportion of PI-positive CHOK1-hCCR8 cells was measured, and its relationship with different antibodies and their concentrations is shown in the curve.

[0097] Figure 21 shows that the anti-CCR8 antibody of the present invention eliminates Treg in peripheral PBMC. After IL-2 activated PBMC and anti-CCR8 antibody were incubated for 96 hours, Foxp3 positive cells accounted for CD4 + The proportion of T cells (A) and CD8 + T cells (CD3 + CD4 - ) accounts for CD3 + The proportion of T cells (B).

[0098] Figure 22 shows that the anti-CCR8 antibodies of the present invention inhibit MC38 tumor growth. Humanized CCR8 B-hCCR8 mice were inoculated with MC38 colon cancer cells. 10 mg / kg of the antibody, a negative control (hIgG1), or an equal volume of vehicle (PBS) were subcutaneously injected twice weekly. Tumor volume changes are shown in the curve.

[0099] FIG23 shows the changes in body weight of mice in the MC38 tumor model treated with the anti-CCR8 antibody of the present invention.

[0100] Figure 24 shows that an anti-CCR8 antibody with Fc mutation and reduced fucosylation inhibits MC38 tumor growth. Humanized CCR8 B-hCCR8 mice were inoculated with MC38 colon cancer cells to form tumors. 10 mg / kg of anti-CCR8 antibody or an equal volume of vehicle (PBS) were subcutaneously injected twice weekly. Tumor volume changes are shown in the curve.

[0101] FIG25 shows the changes in body weight of mice in the MC38 tumor model treated with Fc-mutated and fucosylated anti-CCR8 antibodies.

[0102] Figure 26 shows the inhibition of breast cancer EMT-6 tumor growth in mice by the anti-CCR8 antibody of the present invention alone and in combination with an anti-mPD-1 antibody. Each group received 5-10 mg / kg of the antibody (iv), anti-mPD-1 antibody (ip), or an equal volume of vehicle (PBS) control (iv), twice weekly. Tumor volume changes are shown in the graph.

[0103] FIG27 shows the changes in body weight of mice during the treatment of EMT-6 tumor model with the CCR8 antibody of the present invention. DETAILED DESCRIPTION

[0104] definition

[0105] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0106] In order to make it easier to understand the present invention, certain scientific and technological terms are specifically defined below. Unless otherwise clearly defined elsewhere in this article, the scientific and technological terms or expressions used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Regarding the definitions and terms in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel), at least in part. The abbreviations of amino acid residues follow the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes its corresponding plural form, unless otherwise clearly provided in the text.

[0107] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0108] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0109] As used herein, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when explicitly stated to the contrary, such as "only one" or "exactly one" or when used in a claim, "consisting of..." will refer to only one of the listed numbers or one of the elements of the list.

[0110] As used herein, the words "a" and "an" should be understood to mean "at least one" unless the context clearly indicates otherwise.

[0111] The terms "CCR8," "CC motif chemokine receptor type 8," and "chemokine (CC motif) receptor 8" herein refer to any naturally occurring CCR8 produced by expression of CCR8 in a cell. Unless otherwise indicated, the term includes CCR8 from any vertebrate source, such as mammals (e.g., primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats)). For an exemplary human CCR8 protein, see UniProt record P51685, for an exemplary cynomolgus monkey CCR8 protein, see UniProt record G7NYJ2, and for an exemplary mouse CCR8 protein, see UniProt record P56484.

[0112] The term "CCR8" includes variants, subtypes, species homologs of human CCR8, CCR8 of other species, and analogs having at least one common epitope of CCR8, unless otherwise indicated. The term includes unprocessed full-length CCR8 and any form of CCR8 produced as a result of processing in cells. The term encompasses "full-length" unprocessed CCR8 and any form of CCR8 or any fragment thereof, such as splice variants or allelic variants, produced by intracellular processing. In one embodiment, CCR8 refers to the full length or fragment thereof from humans or cynomolgus monkeys (such as mature fragments thereof lacking a signal peptide).

[0113] As used herein, "CCL1" and "CC motif chemokine ligand 1" refer to any naturally occurring CCL1 produced by expression of the CCL1 gene in a cell. As its name suggests, CCL1 belongs to the CC chemokine family and is secreted by activated monocytes / macrophages, T lymphocytes, and endothelial cells. Unless otherwise indicated, the term includes CCL1 from any vertebrate source, such as mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats).

[0114] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which result in the selective damage, destruction, or elimination from the body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0115] The terms "Treg" and "regulatory T cells" refer to a class of T cells that exhibit immunosuppressive effects in immune responses, generally inhibiting or downregulating the induction and proliferation of effector T cells. The impact of Tregs on cancer is complex, but due to the observation that Treg cells tend to be upregulated in individuals with cancer, their apparent recruitment to tumor sites, and the fact that multiple studies suggest that the presence of high numbers of Tregs in the tumor microenvironment is associated with a poor prognosis, it is generally believed that Tregs suppress anti-tumor immunity. Various immunotherapies are being investigated targeting Tregs for the treatment of cancer. In some embodiments of the present invention, tumors contain tumor-infiltrating Treg cells. In some embodiments, tumors contain cells that express CCR8. In some embodiments, the CCR8-expressing cells are Treg cells. In some embodiments, T effector cells do not express or substantially do not express CCR8. In some embodiments, the antibodies of the present invention fully / partially inhibit / eliminate Treg cells. In some embodiments, the antibodies of the present invention fully / partially inhibit / eliminate Treg cells through an ADCC mechanism and / or block the binding of CCL1 to CCR8, thereby inhibiting CCL1-induced signaling pathways. In some embodiments, the antibodies of the present invention treat cancer by completely / partially inhibiting / eliminating Treg cells. In some embodiments, complete / partial inhibition / elimination of Treg cells using the antibodies of the present invention is more effective and / or more efficient than other methods of inhibiting / eliminating Treg cells. In some embodiments, the antibodies of the present invention treat cancer by completely / partially blocking the binding of CCL1 to CCR8. In some embodiments, complete / partial blocking of the binding of CCL1 to CCR8 using the antibodies of the present invention is more effective and / or more efficient than other methods of blocking the binding of CCL1 to CCR8. In some more specific embodiments, the effects include: enhanced anti-tumor immunity, enhanced immune response to tumor antigens, reduced tumor growth, reduced tumor size, increased secretion of anti-tumor cytokines, increased number / function of tumor-infiltrating T effector cells, enhanced long-term anti-tumor immune memory, or any combination of the foregoing. In some embodiments, complete / partial inhibition / elimination of Treg cells using the antibodies of the present invention results in fewer adverse reactions than other methods of inhibiting / eliminating Treg cells. In some more specific embodiments, the adverse reaction is an immune disorder, such as an autoimmune disorder, such as an autoimmune disease.

[0116] The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical cause-effect relationship, typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, for example, the binding of CCL1 (ligand) to CCR8 (receptor), which results in the transmission of a signal from one part of a cell to another part of the cell. Typically, the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that lead to signal transduction. The penultimate process typically involves nuclear events that result in changes in gene expression.

[0117] The terms "activity" or "biological activity", or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize CCR8 activity in vivo or in vitro, IC 50 , the in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies well known in the art include, for example, cross-reactivity (i.e., usually with non-human homologs of the target peptide, or with cross-reactivity of other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured or assessed using techniques well known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from different sources (including humans, primates, or any other source).

[0118] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

[0119] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic applications of the binding compounds described herein.

[0120] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0121] The term "full-length antibody" refers to an immunoglobulin molecule that, when naturally present, comprises at least four peptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conserved regions called framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

[0122] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of antibodies, generally including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments well known in the art; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. In some preferred embodiments of the present invention, the antigen-binding fragment of the present invention is selected from Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity to the antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "function-conservative variants" of the antibody). The term "binding compound" refers to both antibodies and their binding fragments.

[0123] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0124] The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or a light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0125] The term "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen-specificity. However, a bivalent antibody can be bispecific.

[0126] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites.

[0127] The term "murine antibody" or "hybridoma antibody" in the present invention refers to a monoclonal antibody against CCR8 prepared according to the knowledge and skills in the field. During preparation, the test subject is injected with the CCR8 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. Hybridoma technology is achieved by fusing two cells while maintaining the main characteristics of both. These two cells are mouse spleen cells immunized with the antigen and mouse myeloma cells. The main feature of mouse spleen cells (B lymphocytes) immunized with a specific antigen is their antibody secretion function, but they cannot be cultured continuously in vitro. Mouse myeloma cells, on the other hand, can divide and proliferate indefinitely under culture conditions, that is, they have so-called immortality. Under the action of a selective culture medium, only hybrid cells fused with B cells and myeloma cells have the ability to be cultured continuously, forming cell clones that have both the antibody secretion function and the maintenance of cell immortality. In some embodiments, the present invention obtains hybridoma cells that can express positive antibodies by immunizing mice with CCR8 protein, then obtaining spleen cells from the mice and fusing them with myeloma cells.

[0128] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are derived from antibodies ("parent antibodies") of rodents, etc., while the constant domain sequences are derived from human antibodies, such that the resulting chimeric antibodies are less likely to induce adverse immune responses in human subjects compared to parent rodent antibodies. In some embodiments of the invention, the rodent is a mouse or rat. In some preferred embodiments of the invention, the affinity of the chimeric antibody for the antigen is not less than or almost not less than that of the parent mouse antibody.

[0129] The term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Optionally, the humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0130] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0131] The light chain of an antibody can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain. The heavy chain of an antibody can be divided into mainly 5 different types: IgA, IgD, IgE, IgG and IgM according to the amino acid sequence of its heavy chain constant region, and several of these types can be further divided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. An "isotype" antibody refers to the antibody species (e.g., IgM, IgE, IgG such as IgG1, IgG2 or IgG4) provided by the heavy chain constant region gene. Isotypes also include modified forms of one of these species, wherein modifications have been produced to change Fc function, for example to enhance or reduce effector function or the binding to Fc receptors.

[0132] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0133] The term "effector function" herein refers to biological activities mediated by the Fc region of an antibody, which vary with the antibody isotype. Antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; opsonization; downregulation of cell surface receptors; and B cell activation, among others.

[0134] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes are typically composed of various chemically active surface molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0135] The term "cross-reaction" as described herein refers to the combination of antigen fragments of the same target molecule of human, monkey, and / or mouse (mouse or rat). Therefore, "cross-reaction" should be understood as the interspecific reaction between antigen-binding molecules (e.g., antibodies) and similar molecules (e.g., CCR8) expressed in different species. The cross-reaction specificity of monoclonal antibodies recognizing human CCR8, monkey, and / or mouse CCR8 (mouse or rat) can be determined by FACS analysis.

[0136] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D ) represents the equilibrium dissociation constant, which is the dissociation rate constant and the association rate constant (k dis and k on Affinity can be measured by common methods known in the art. In some embodiments of the present invention, surface plasmon resonance (SPR) technology is used to measure affinity, such as the affinity between an antibody of the present invention and an antigen.

[0137] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D ) represents the equilibrium dissociation constant, which is the dissociation rate constant and the association rate constant (k dis and k on Affinity can be measured by common methods known in the art. In some embodiments of the present invention, surface plasmon resonance (SPR) technology is used to measure affinity, such as the affinity between an antibody of the present invention and an antigen.

[0138] The term "not binding" to a protein or cell means not binding to the protein or cell, or not binding to the protein or cell with high affinity, i.e., EC binding to the protein or cell 50 1.0×10 -7 M or higher, more preferably 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or more, more preferably 1.0×10 -3 M or higher.

[0139] The term "high affinity" for IgG antibodies refers to the EC 50 1.0×10 -7 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8M or less, more preferably 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or less, more preferably 5.0×10 -10 M or less, more preferably 1.0×10 -10 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for an IgM subtype refers to EC 50 is 10 -7 M or less, preferably 10 -8 M or less, more preferably 10 -9 M or lower.

[0140] The term "blocking" for IgG antibodies to a receptor refers to the ability of the antibody to compete with the intended ligand of the receptor, thereby inhibiting the binding and interaction between the receptor and the ligand. This inhibition can occur through a variety of mechanisms, including, for example, due to overlapping binding sites on the receptor and / or changes in receptor conformation induced by the antibody, which alter the affinity of the ligand. Antibodies and antibody fragments that are called "functional" are characterized by having such properties. In some embodiments, the ability to "block" refers to the ability of the EC to bind to the ligand. 50 The value is 5.0×10 -5 M or less, 1.0×10 -5 M or lower, 5.0×10 -6 M or less, 1.0×10 -6 M or less, preferably 5.0×10 -7 M or less, more preferably 1.0×10 -7 M or less, more preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower.

[0141] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively lyse target cells that have antibodies bound to surface antigens on their cell membranes.

[0142] The term "complement-dependent cytotoxicity" or "CDC" refers to the effector function of IgG and IgM antibodies that, when bound to surface antigens, trigger the classic complement pathway, including formation of the membrane attack complex and target cell lysis.

[0143] The terms "nucleic acid," "polynucleotide," "nucleic acid molecule," and "polynucleotide molecule" are used interchangeably herein (unless the context indicates otherwise) and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless expressly limited, the terms include nucleic acids containing analogs of known natural nucleotides that have similar binding properties to the reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0144] "Construct" refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule) that can be derived from any source, capable of integrating with a genome or autonomously replicating, and comprising one or more polynucleotide molecules that have been linked (i.e., operably linked) in a functionally operable manner. In some preferred embodiments of the present invention, the recombinant construct comprises a polynucleotide of the present invention operably linked to a transcription initiation regulatory sequence that drives and / or directs transcription of the polynucleotide of the present invention in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to drive and / or direct expression of the polynucleotide of the present invention.

[0145] "Vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors."

[0146] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure vector maintenance and, if desired, to provide for amplification within the host. In preferred embodiments of the present invention, the expression vectors of the present invention comprise a construct of the present invention and / or a polynucleotide of the present invention.

[0147] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0148] "Activation," "stimulation," and "treatment" as applied to the manipulation of a cell or receptor may have the same meaning, e.g., a cell or receptor is activated, stimulated, or treated with a ligand, unless the context dictates otherwise or explicitly. "Ligand" includes natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response," e.g., a response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, rate of gene expression, or differentiation state), where the change is related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.

[0149] In some embodiments of the present invention, the term "tumor" is intended to emphasize malignant tumors, and the terms "cancer" and "tumor" are used interchangeably to refer to any abnormal, uncontrolled cell or tissue growth or proliferation in an animal. As used herein, the terms "cancer" and "tumor" include solid tumors and hematological tumors, and also include precancerous lesions. Specific non-limiting examples of tumors include breast cancer, squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, lung cancer (such as non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, colon cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, thyroid cancer, testicular cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, thymoma and various head and neck cancers. In some embodiments, hematologic neoplasms include mixed B-cell and T-cell leukemia, B-cell lymphoma, granulocytic leukemia (acute and chronic), lymphocytic leukemia (acute and chronic), childhood / juvenile lymphocytic leukemia, myelomonocytic leukemia, diffuse large B-cell lymphoma (DLBC), Hodgkin lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma (MCL), multiple myeloma, myelodysplastic syndrome, etc. In some embodiments, the tumor is a benign tumor.

[0150] As used herein, the term "treatment" or "treating" of any disease or condition refers in one embodiment to slowing down, interrupting, retarding, alleviating, stopping, reducing or reversing the symptoms of a disease, a complication, or the onset of a biochemical sign, alleviating symptoms or preventing or inhibiting the further development of a disease, condition or condition (that is, slowing down or preventing or reducing at least one of the progression of a disease or its clinical symptoms). In another embodiment, "treatment" or "treating" refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient. In another embodiment, "treatment" or "treating" refers to regulating a disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of a physical parameter), or in both aspects. Unless expressly described herein, methods for assessing the treatment and / or prevention of a disease are generally known in the art.

[0151] "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey or a cynomolgus monkey.

[0152] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in either order.

[0153] "Therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of a CCR8 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions, or the progression of such diseases or conditions. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in amelioration of symptoms, e.g., an amount to treat, cure, prevent, or ameliorate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0154] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation or composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0155] anti-CCR8 antibodies

[0156] In one aspect, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CCR8. The terms "anti-CCR8 antibody," "anti-CCR8," "CCR8 antibody," or "antibody that binds to CCR8" refer to antibodies that are capable of binding to a CCR8 protein or fragment thereof with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting CCR8.

[0157] In some embodiments, the antibodies of the invention bind to human or cynomolgus macaque CCR8 proteins. In some embodiments, the antibodies of the invention bind to CHOK1-human CCR8 cells or 293F-cyno CCR8 cells. In some embodiments, the antibodies of the invention inhibit / block the binding of CCR8 to its ligand CCL1. In some embodiments, the antibodies of the invention inhibit / block induced β-Arrestin recruitment.

[0158] Any suitable method for producing antibodies can be used to produce the antibodies of the present invention. Any suitable form of CCR8 can be used as an immunogen (antigen) to produce antibodies. By way of example and not limitation, any CCR8 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing monoclonal anti-CCR8 antibodies of murine origin can be produced by methods well known in the art.

[0159] Antibodies derived from rodents (such as mice) may cause unwanted antibody immunogenicity when used as therapeutic drugs in vivo. Repeated use causes the human body to produce an immune response against the therapeutic antibody. Such immune responses at least lead to loss of therapeutic efficacy, and in severe cases, lead to potentially lethal allergic reactions. One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies, in which mouse variable regions are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, the retention of intact rodent variable regions in chimeric antibodies may still cause harmful immunogenicity in patients. Transplanting the complementary determining region (CDR) loops of rodent variable domains onto human frameworks (i.e., humanization) has been used to further minimize rodent sequences (Jones et al. (1986) Nature 321: 522; Verhoeyen et al. (1988) Science 239: 1534). In some embodiments, the antibodies of the present invention are chimeric antibodies. In some preferred embodiments, the antibodies of the invention are humanized antibodies.

[0160] In some embodiments, chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal hybridoma antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0161] In some embodiments, the chimeric CCR8 antibodies of the present invention can be prepared by operatively linking hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy and light chains. In some embodiments, the constant region comprised by the chimeric antibodies of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0162] In some embodiments, chimeric CCR8 antibodies of the present invention can be obtained by transfecting expression cells with "mixed and matched" chimeric light chain and chimeric heavy chain expression plasmids, and the CCR8 binding of such "mixed and matched" antibodies can be tested using the above-mentioned binding assays and other conventional binding assays (e.g., ELISA).

[0163] As defined herein, "complementarity determining regions" or "CDR regions" or "CDRs" are regions of an antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues.

[0164] The CDRs are primarily responsible for binding to the antigen epitope. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0165] For example, according to different CDR definition schemes, the residues of each CDR are described below.

[0166] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0167] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position of the Kabat numbering system according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). In some embodiments, the HCDRs and LCDRs in the antibodies of the present invention are determined according to the Kabat scheme, respectively.

[0168] Unless otherwise indicated, the boundaries of the CDRs of the antibodies of the invention can be determined by one skilled in the art according to any scheme in the art (eg, different assignment systems or combinations).

[0169] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0170] Antibodies with different specificities (that is, different binding sites for different antigens) have different CDRs. However, although CDR is different between antibodies, there are only a limited number of amino acid positions in the CDR that directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, by the structure of the antibody and protein folding, the residues of the CDR sequence remainder can be determined. Therefore, the present invention also contemplates the variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0171] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0172] The present invention mixes and pairs various chimeric heavy chain and light chain expression plasmids and transfects them into expression cells to produce anti-CCR8 chimeric antibodies.

[0173] The humanized antibodies of the present invention can be prepared by inserting murine CDR regions into human germline framework regions using methods known in the art, such as those described in U.S. Patent Nos. 5,225,539 to Winter et al. and 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0174] In some embodiments, amino acid changes include amino acid deletions, additions, or substitutions. In some embodiments, the anti-CCR8 antibodies or antigen-binding fragments thereof of the present invention include those having amino acid sequences that have been mutated by amino acid deletions, additions, or substitutions, but still have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the above-described antibodies (particularly in the CDR regions depicted in the above sequences). In some embodiments, the antibodies of the present invention have at least one, for example, 1, 2, 3, 4, or 5, amino acid mutations in the CDR regions that have been mutated by amino acid deletions, additions, or substitutions when compared to the CDR regions depicted in the specific sequences. In some embodiments, the antibodies of the present invention have at least one, for example, 1, 2, 3, 4, or 5, amino acid mutations in the framework regions that have been mutated by amino acid deletions, additions, or substitutions when compared to the framework regions in the specific sequences.

[0175] The term "percent (%) amino acid sequence identity," or simply "identity," is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software / algorithms. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the compared sequences.

[0176] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain constant region and / or a light chain constant region, preferably, the light chain constant region is a lambda chain or a kappa chain constant region; the heavy chain constant region is selected from murine mIgG2a, human IgG1, human IgG2, human IgG3, or IgG4, or a modified form thereof. In some preferred embodiments, the heavy chain constant region is human IgG1 or human IgG4 with an S228P mutation. In some preferred embodiments, the modified form of the constant region comprises amino acid sequence modification and / or glycosylation modification.

[0177] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., addition, deletion, or substitution, preferably, substitution) at one or more amino acid positions.

[0178] In some embodiments, the antibody comprises at least one modification that enhances cell killing. In some embodiments, the enhanced cell killing is enhanced antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). In some embodiments, the modification is defucosylation / reduced fucosylation. In some embodiments, the modification is at a position selected from the group consisting of 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 330, 331, 332, 333, 334, 335 315,320,322,324,326,327,328,329,330,331,333,334,335,337,338,340,360,373,376,378,382,388,389,398,414,416,419,430,434,435,437,438 and 439. In this article, if no context otherwise specifies, when referring to constant region amino acid position numbering, reference is made to the EU numbering system. In this article, the mutation in the amino acid sequence can be named as follows: the single letter code of the parent amino acid, followed by the position number, followed by the single letter code of the amino acid after the mutation. For example, the mutation of leucine (L) at position 234 to alanine (A) is represented as "L234A". Sometimes, in the sequence, a slash ( / ) is used to juxtapose multiple alternative options. For example, the deletion of cysteine ​​(C) at position 236 can be represented as "C236 deletion". In some embodiments, the modification is one or more heavy chain constant region mutations at one or more positions selected from L234, L235, G236, S239, F243, D265, H268, D270, R292, S298, Y300, V305, K326, A330, I332, E333, K334 and P396 according to the EU numbering system. In some embodiments, the one or more heavy chain constant region mutations are selected from an N297A substitution, an N297Q substitution, an L235A substitution together with an L237A substitution, an L234A substitution together with an L235A substitution, an E233P substitution, an L234V substitution, an L235A substitution, a C236 deletion, a P238A substitution, a D265A substitution, an A327Q substitution, and a P329A substitution according to the EU numbering system.In some embodiments, the modification is one or more heavy chain constant region mutations selected from the group consisting of G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L according to the EU numbering system. In some embodiments, the one or more heavy chain constant region mutations are one or more heavy chain constant region mutations selected from the group consisting of L235V, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330L, A330M, I332E, E333A, K334A, K334E, and P396L according to the EU numbering system. In some embodiments, the one or more heavy chain constant region mutations are one or more heavy chain constant region mutations selected from M252Y, S254T, and T256E according to the EU numbering system.

[0179] In some embodiments, the heavy chain constant region has one or more sets of mutations selected from the following combinations of positions where mutations occur simultaneously: (1) L235 / F243 / R292 / Y300 / P396, (2) F243 / R292 / Y300 / V305 / P396, (3) D270 / K326 / A330 / K334, (4) S239 / A330 / I332, (5) S298 / E333 / K334, (6) L234 / L235 / G236 / S239 / H268 / D270 / S298, (7) M252 / S254 / T256, (8) L234 / L235 / D265, (9) G236 / S239 / I332, and (10) S239 / I332.

[0180] In some embodiments, the heavy chain constant region has one or more mutations selected from the following combinations: (1) L235V / F243L / R292P / Y300L / P396L, (2) F243L / R292P / Y300L / V305I / P396L, (3) D270E / K326D / A330M / K334E, (4) S239D / A330L / I332E, (5) S298A / E 333A / K334A, (6)L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, (7)M252Y / S254T / T256E, ( 8)L234A / L235A / D265A, (9)L234F / L235E / D265A, (10)G236A / S239D / I332E and (11)S239D / I332E.

[0181] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region (CH1 domain) to alter the number (e.g., increase or decrease) of cysteine ​​residues in the hinge region.

[0182] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties that are known and readily available in the art.

[0183] Antibody expression

[0184] In another aspect, the present invention provides a polynucleotide molecule encoding an anti-CCR8 antibody described herein or any fragment thereof. The polynucleotide molecule may comprise a polynucleotide molecule encoding an amino acid sequence of the light chain variable region and / or heavy chain variable region of the antibody, or at least a portion thereof, or a polynucleotide molecule encoding an amino acid sequence of the light chain and / or heavy chain of the antibody, or at least a portion thereof.

[0185] For example, the polynucleotide molecules of the present invention comprise a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-95, or a nucleic acid encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 1-95.

[0186] In some embodiments, the polynucleotide molecules encoding the antibodies of the present invention include polynucleotide molecules that have been mutated by nucleotide deletion, addition or substitution, but still have at least about 60, 70, 80, 90, 95 or 100% identity with the CDR corresponding coding regions depicted in the sequences described above.

[0187] In yet another aspect, the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably, the vector is a eukaryotic expression vector. In some embodiments, the polynucleotide molecule as described herein is contained in one or more expression vectors.

[0188] In another aspect, the present invention provides a host cell comprising the polynucleotide molecule as described herein or the expression vector as described herein. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0189] In another aspect, the present invention provides a method for preparing an anti-CCR8 antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell as described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0190] The present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention or any fragment thereof, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These especially include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by measuring which cell line has high expression levels.

[0191] In one embodiment, the present invention provides a method for preparing an anti-CCR8 antibody, wherein the method comprises, upon introducing an expression vector into a mammalian host cell, producing the antibody by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown.

[0192] Standard protein purification methods can be used to recover antibodies from the culture medium. Antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0193] It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from one another. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies. Likewise, in certain embodiments, non-fucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts and are unlikely to be immunogenic because their carbohydrate structures are normal components of natural human serum IgG.

[0194] Pharmaceutical compositions and pharmaceutical preparations

[0195] In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-CCR8 antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, or an immunoconjugate as described herein, and a pharmaceutically acceptable carrier or excipient. It should be understood that the anti-CCR8 antibody or pharmaceutical composition provided herein can be incorporated into a formulation with suitable carriers, excipients, and other agents for co-administration, thereby providing improved transfer, delivery, tolerance, and the like.

[0196] The term "pharmaceutical composition" refers to a preparation that permits the active ingredient contained therein to exist in biologically effective form, and that contains no additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.

[0197] Pharmaceutical formulations comprising the anti-CCR8 antibodies described herein can be prepared by mixing the anti-CCR8 antibodies of the invention having the desired degree of purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of aqueous solutions or lyophilized formulations.

[0198] The pharmaceutical compositions or formulations of the present invention may also contain one or more other active ingredients as required for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. In some embodiments, the pharmaceutical compositions of the present invention further comprise a composition of polynucleotide molecules encoding anti-CCR8 antibodies.

[0199] The pharmaceutical composition of the present invention may also include one or more additional therapeutic agents, which are required for the specific indication being treated, encompassing any substance that is effective in preventing or treating tumors (e.g., cancer), preferably with those therapeutic agents that do not adversely affect each other's activity. For example, chemotherapeutic agents, hormones, and the like. The therapeutic agents are suitably combined in an amount effective for the intended application. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the additional therapeutic agent is other antibodies. In some embodiments, the additional therapeutic agent is other monoclonal antibodies. In some preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting an immune checkpoint. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-L1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting CTLA4.

[0200] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, an immunoconjugate described herein, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0201] In yet another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, the immunoconjugate described herein, the pharmaceutical composition described herein, or the pharmaceutical combination described herein.

[0202] Combination products or kits

[0203] In some embodiments, the present invention also provides a combination product comprising an anti-CCR8 antibody or fragment thereof of the present invention and one or more additional therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, anti-infective agents, small molecule drugs, or immunomodulators, etc.).

[0204] In some embodiments, the combination product is used to prevent or treat diseases associated with CCR8 / immune checkpoints and / or diseases mediated by CCR8 and / or immune checkpoints. In some embodiments, the additional therapeutic agent is an existing standard of care. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the additional therapeutic agent is other antibodies. In some embodiments, the additional therapeutic agent is other monoclonal antibodies. In some preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting an immune checkpoint. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting PD-L1. In some more preferred embodiments, the additional therapeutic agent is a monoclonal antibody targeting CTLA4.

[0205] In some embodiments, two or more components of the combination product may be co-administered to a subject sequentially, separately or simultaneously.

[0206] In some embodiments, the present invention also provides a kit comprising an anti-CCR8 antibody or fragment thereof, pharmaceutical composition or combination product of the present invention, and optionally a package insert directing administration.

[0207] In some embodiments, the present invention also provides a pharmaceutical product comprising the anti-CCR8 antibody or fragment thereof, pharmaceutical composition or combination product of the present invention, optionally further comprising a package insert for instructions for administration.

[0208] Medical uses and treatments

[0209] Any of the anti-CCR8 antibodies or corresponding immunoconjugates provided herein can be used in therapeutic methods. It should also be understood that when discussing "antibodies," compositions comprising the antibodies are also included. The anti-CCR8 antibodies of the present invention can be used in therapeutic or prophylactic methods described in any embodiment of the present invention in a therapeutically effective amount or a prophylactically effective amount.

[0210] In yet another aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof, a polynucleotide, an expression vector, a host cell, an immunoconjugate, or a pharmaceutical composition as described herein in the preparation of a medicament. The present invention also provides the use of a drug combination as described herein in the preparation of a medicament. Thus, the present invention essentially provides the use of an effective amount of an antibody or antigen-binding fragment thereof, a polynucleotide, an expression vector, a host cell, an immunoconjugate, or a pharmaceutical composition as described herein in combination with other therapeutic agents in the preparation of a medicament. Wherein, the medicament is used to prevent and / or treat a tumor, an autoimmune disease, or an infectious disease in a subject, and the tumor is preferably, for example, melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer, or thymic cancer and its metastatic cancer.

[0211] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof, polynucleotide, expression vector, host cell, immunoconjugate, pharmaceutical composition or drug combination as described herein for use in treating and / or preventing a tumor, autoimmune disease, or infectious disease, wherein the tumor is preferably, for example, melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer and metastatic cancer thereof.

[0212] In another aspect, the present invention provides a method for treating and / or preventing tumors, autoimmune diseases, or infectious diseases, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody or antigen-binding fragment thereof, polynucleotide, expression vector, host cell, immunoconjugate, pharmaceutical composition or drug combination described herein, wherein the tumor is preferably, for example, melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer and metastatic cancer thereof.

[0213] In some embodiments, the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or local administration to the lesion, etc.

[0214] The term "treatment" refers to a clinical intervention intended to change the natural course of a disease in an individual being treated. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any adverse feelings or direct or indirect pathological consequences of the disease, reducing the rate of progression of the disease, improving or alleviating the disease state, and alleviating or improving the prognosis. For the antibodies of the present invention, the ability to reduce the severity of the disease in one or more aspects is to exert a therapeutic effect. In certain embodiments, this is manifested as one or more of the following: an increase in the average lifespan (survival) of the patient; a delay in disease progression; a decrease in the need for medical care.

[0215] The present invention also provides for co-administering a therapeutically effective amount of one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to a subject. The antibodies of the present invention can be used alone or in combination with other therapeutic agents in a therapy. In some embodiments, the antibodies of the present invention are co-administered with at least one additional therapeutic agent.

[0216] Methods for diagnosis and detection

[0217] In another aspect, the present invention provides a method for detecting the presence of CCR8 in a sample using the antibodies or antigen-binding fragments thereof described herein. The term "detection" as used herein includes quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissues. In certain embodiments, CCR8 is human CCR8 or cynomolgus monkey CCR8. The method includes the steps of contacting the sample with an antibody or antigen-binding fragment thereof described herein or a detection composition containing the antibody or antigen-binding fragment thereof, and detecting the presence of a conjugate or binding signal generated by the binding of the antibody or antigen-binding fragment to CCR8. When used for detection purposes, the antibodies or antigen-binding fragments thereof described herein may be labeled to indicate whether the conjugate has been formed. In certain embodiments, the method may be an in vitro or in vivo method.

[0218] In some embodiments, CCR8 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval. In one embodiment, an anti-CCR8 antibody or antigen-binding fragment thereof is provided for use in a diagnostic or detection method.

[0219] The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0220] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0221] Example

[0222] The present invention is illustrated by the following examples, but is not intended to be limiting thereof. The present invention has been described in detail herein, and specific embodiments thereof are disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention.

[0223] Example 1: Animal Immunization and Preparation of Mouse Hybridoma Antibodies Producing Anti-Human CCR8

[0224] Mice are immunized with the full-length human CCR8 gene (DNA or mRNA) and an engineered cell line that overexpresses human CCR8. Immunization or surge immunization also uses the full-length monkey CCR8 gene (DNA or mRNA) and cells that overexpress monkey CCR8. SP2 / 0 cells are fused and positive clones are screened by flow cytometry. The specific method is as follows:

[0225] Animal Immunization: The full-length gene encoding human CCR8 (UniProtKB / Swiss-Prot: P51685) was constructed into the PCDNA3.1 or PCDNA3.4 plasmid (produced by Shanghai Ruizhi Chemical Research Co., Ltd.) as an immunogen. Balb / c female mice were immunized with DNA at a dose of 4 μg / mouse. DNA immunization was repeated 6-7 times at 2-week intervals. Ten days after the last immunization, 293F cells expressing human CCR8 (produced by Shanghai Ruizhi Chemical Research Co., Ltd., 293F-hCCR8) were injected intraperitoneally for pulse immunization. Three days later, the mouse spleens were harvested for cell fusion.

[0226] Balb / c female mice were intraperitoneally immunized with 293F cells expressing human CCR8 (293F-hCCR8) as immunogens (2-5x10 6 Cell immunization was repeated three times at intervals of 2 weeks. Ten days after the last immunization, 293F-hCCR8 cells (2-5x10 6 cells / mouse) for pulse immunization, and 3 days later, the spleens of the mice were taken for cell fusion.

[0227] Balb / c female mice were immunized with full-length mRNA encoding human CCR8 (UniProtKB / Swiss-Prot: P51685) as an immunogen (prepared by Shanghai Hongcheng Pharmaceutical Co., Ltd., 50 μg / mouse). mRNA immunization was repeated three or four times at 3-week intervals. Ten days after the final immunization, 293F-hCCR8 cells were injected intraperitoneally for pulse immunization. Three days later, the spleens of the mice were harvested for cell fusion.

[0228] Cell fusion: Mouse spleen cells and SP2 / 0 cells (ATCC No. CRL-1581) were electrofused at a ratio of 2:1 (BTX electrofusion instrument: ECM2001 + ), cultured in 96-well culture plates with HAT medium (GIBCO, catalog number: H0262), and hybridoma cell supernatant antibody screening was performed after 10 days.

[0229] Screening of human CCR8 and cynomolgus monkey CCR8 specific positive clones: cells expressing human CCR8 (CHOK1-hCCR8, constructed by Shanghai Ruizhi Chemical Research Co., Ltd.) and cynomolgus monkey CCR8 (CHOK1-cynoCCR8, constructed by Shanghai Ruizhi Chemical Research Co., Ltd.) were plated at 5x10 7Cells were plated at a density of 100 μL / well in a 96-well U-shaped assay plate. 100 μL of hybridoma cell culture supernatant was added and incubated at 4°C for 1 hour. Cells were washed twice by centrifugation at 300 g in FACS buffer (PBS containing 1% FBS). Alexa488-labeled anti-mouse IgG antibody was added and incubated at 4°C for 1 hour. Cells were washed three times by centrifugation at 300 g in FACS buffer. Mean fluorescence intensity (MFI) was measured using a flow cytometer (BECKMAN COULTER cytoFLEX). Positive clones were screened by comparison with a negative control (CHOK1 cells that do not express CCR8). The clones in Table 1 showed higher mean fluorescence intensity (MFI) for binding to human CCR8 (CHOK1-hCCR8) and cynomolgus macaque CCR8 (CHOK1-cynoCCR8) than the blank control cells (CHOK1), indicating that these clones specifically bind to human and cynomolgus macaque CCR8.

[0230] Table 1 Binding of positive clone hybridoma supernatants to human CCR8 (CHOK1-hCCR8), cynomolgus monkey CCR8 (CHOK1-cynoCCR8) and blank cell CHOK1 (mean fluorescence intensity, MFI)

[0231] Example 2: Preparation and identification of mouse monoclonal antibodies against human CCR8

[0232] Hybridoma cells with binding activity to human CCR8 and cynomolgus macaque CCR8 listed in Table 1 were cultured in serum-free medium. After 10 days, the culture supernatant was collected and purified using a Protein A column (Borgron (Shanghai) Biotechnology Co., Ltd., Catalog No. AA0272) to obtain purified mouse monoclonal antibodies. Flow cytometry was used to determine the binding activity of the anti-human CCR8 antibodies and their blocking activity against CCL1.

[0233] 2.1 Binding activity of anti-human CCR8 antibodies to human CCR8 and cynomolgus monkey CCR8

[0234] Saturation binding assay of anti-CCR8 antibodies to cells expressing human CCR8 or cynomolgus monkey CCR8:

[0235] The cell concentration of 293F cell line expressing human CCR8 (293F-hCCR8, constructed by Shanghai Ruizhi Chemical Research Co., Ltd.) or 293T cell line expressing cynomolgus macaque CCR8 (293T-cyno CCR8, constructed by Kangyuan Broad Technology Co., Ltd.) was adjusted to 1×10 6100 μL / well of the diluted solution was placed in a 96-well U-bottom plate and centrifuged. Anti-human CCR8 antibody, control antibody (anti-human CCR8 antibody 433H, clone 433H, BD Pharmingen, catalog number 624092; 10A11 is the sequence from patent WO2020138489 (light chain variable region sequence number: 59, heavy chain variable region sequence number: 41), fused with the human and mouse constant region sequences listed in Table 5 to generate 10A11_hIgG1 and 10A11_mIgG2a antibodies, respectively), and IgG1 isotype (Baiying Biotechnology Co., Ltd., catalog number: B117901) were diluted to the starting working concentration in FACS buffer (PBS containing 1% FBS). A serial dilution was then performed in FACS buffer. Cells were resuspended in 100 μL / well of the serially diluted antibody solution, pipetted to mix, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. 100 μL of secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 647, Invitrogen, Cat. No. A21445) diluent was used to resuspend the cell pellet, pipetted to mix, and incubated at 4°C for approximately 45 minutes. After incubation, the cells were centrifuged, washed three times, and resuspended in 100 μL / well of FACS buffer. The mean fluorescence intensity (MFI) was measured using a flow cytometer (BECKMAN COULTER cytoFLEX). The experimental data were analyzed using Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis, and the corresponding MFI value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve and calculate the EC. 50 .

[0236] As shown in Figures 1 and 2 and Table 2 , antibody clones such as 27B9-1G3, 559E1B10, 563E10E12, 569D11B5, and 589D7C7 had strong binding activity to 293F-hCCR8, and EC 50 The EC values ​​of 433H and 10A11_mIgG2a for binding to 293F-hCCR8 were 107.0-360.6 ng / mL. 50 The antibody clones 27B9-1G3, 559E1B10, 563E10E12, 569D11B5 and 589D7C7 all had strong binding activity to cynomolgus monkey CCR8, EC 50The binding activity of control antibodies 433H and 10A11_mIgG2a to cynomolgus monkey CCR8 was very weak, EC 50 They were 15312.5 and 5770.5 ng / mL respectively.

[0237] Table 2 Binding activity of anti-CCR8 antibodies to human CCR8 and cynomolgus monkey CCR8

[0238] N / A: Not applicable, no valid EC 50

[0239] 2.2 Anti-CCR8 antibody blocking CCL1 binding experiment

[0240] CCL1 is the main ligand of CCR8, and CCR8 is the only known receptor of CCL1. High expression of CCL1 in tumor tissue is negatively correlated with tumor prognosis. CCL1 can induce the migration of Treg through CCR8, and blocking the binding of CCL1 to CCR8 can inhibit the migration of Treg and tumor growth (see Klarquist J et al. Ccl22 Diverts T Regulatory Cells and Controls the Growth of Melanoma. Cancer Res. 2016 Nov 1; 76(21): 6230-6240 and Xu Y et al. Sox2 Communicates with Tregs Through CCL1to Promote the Stemness Property of Breast Cancer Cells. Stem Cells. 2017 Dec; 35(12): 2351-2365.). Anti-CCR8 antibodies with the function of blocking the binding of CCL1 to CCR8 may have stronger anti-tumor activity. A competition binding assay was performed to detect the activity of anti-CCR8 antibodies in blocking the binding of AlexaFlour-647-labeled human CCL1 to 293F-hCCR8. The specific method is as follows:

[0241] The cell concentration of 293F cell line expressing human CCR8 (293F-hCCR8, constructed by Shanghai Ruizhi Chemical Research Co., Ltd.) was adjusted to 1×10 6100 μL / well of the diluted solution was placed in a 96-well U-bottom plate at 100 μL / well. After centrifugation, the supernatant was discarded. Anti-CCR8 antibody, control antibody (anti-human CCR8 antibody, clone 433H, BD Pharmingen, catalog number 624092), and hIgG1 isotype (Bio-Ying Biotechnology Co., Ltd., catalog number B117901) were diluted to 2x the starting working concentration in FACS buffer (PBS containing 1% FBS). Serial dilutions were then performed using FACS buffer. Cells were resuspended in 50 μL / well of the serially diluted antibody solution and mixed by pipetting. Subsequently, 50 μL / well of a 20 nM human CCL1-AlexaFlour-647 (Almac, CAF-7) solution was added, mixed by pipetting, and incubated at 4°C for 1 hour. After incubation, cells were centrifuged, washed three times, and resuspended in 100 μL / well of FACS buffer. The mean fluorescence intensity (MFI) was read by flow cytometry (BECKMAN COULTER cytoFLEX). The experimental data were analyzed using Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis and the corresponding MFI value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve and calculate the IC 50 .

[0242] As shown in Figure 3 and Table 3 , antibody clones 27B9-1G3, 559E1B10, 563E10E12, 569D11B5, and 589D7C7 can all block the binding of CCL1 to CCR8, with the IC 50 The IC value of 433H blocking activity was 75.0-131.7 ng / mL. 50 It is 94.3ng / mL.

[0243] Table 3 Anti-CCR8 antibodies blocking CCL1 binding activity

[0244] N / A: No blocking activity or poor blocking activity, no effective IC 50 value.

[0245] Example 3: Anti-CCR8 Antibody Sequencing and Chimeric Antibody Functional Identification

[0246] 3.1 Anti-CCR8 Antibody Sequencing and Chimeric Antibody Construction, Expression, and Purification

[0247] The hybridoma clones that performed well in the above activity identification were sequenced, and the sequences are shown in Table 4.

[0248] The sequenced light and heavy chain variable regions (see Table 4) were constructed onto human constant regions (IgG1 / κ, see Table 5) or murine constant regions (mIgG2a) for in vitro functional characterization or in vivo efficacy studies. Sequencing confirmed that the constructed hIgG1 and mIgG2a chimeric antibodies were consistent with the sequences in Tables 4 and 5.

[0249] The protein was expressed in Expi293F cells (Thermofisher Catalog No. A1452) and purified using a Protein A column. The specific method is as follows:

[0250] Expi293 cells expressing antibodies: One day before transfection, dilute the Expi293 cells to a density of 1.5 × 10 6 cells / mL, and cultured in a shaker at 120 rpm at 37°C and 8% CO2. The next day, the viable cell density and survival rate were determined. The cell transfection density should be 3×10 6 cells / mL, with a cell viability greater than 95%. Prepare the PEI / plasmid complex: Mix PEI (1 mg / mL, Polysciences, Catalog No. 24765-1) by inverting. Dilute the plasmid in OPM-293 CD05 Medium (OPM, Catalog No. 81075-001) to a concentration of 1 μg / mL per transfection volume. The volume of medium used for the plasmid dilution should be 1 / 20 of the transfection volume. Mix gently, and the heavy chain to light chain ratio should be 1:1.5. Dilute the PEI reagent in OPM-293 CD05 Medium to twice the amount of plasmid used. The volume of medium used for the dilution should be 1 / 20 of the transfection volume. Mix gently by inverting, and incubate at room temperature for 5 minutes. Add the diluted PEI reagent to the diluted plasmid and mix gently by inverting. Incubate the PEI / plasmid complex at room temperature for 15 minutes, then slowly add the solution dropwise to the transfer shaker flask, gently swirling the shaker during addition. After transfection, incubate the flask in a shaker at 37°C, 8% CO2, and 120 rpm. On the second day after transfection (24 hours after transfection), add 10% OPM-293 ProFeed (OPM, Catalog No. F081918) to the flask. Gently swirl the flask during addition. Return the flask to the shaker and continue incubation for 5-7 days. Harvest the supernatant.

[0251] Protein A column for antibody purification: Prepare a gravity chromatography column. Open the column cap, place the gasket at the bottom of the column, and tighten. Prepare Protein A (Cytiva, Cat. No. 17549801). Accurately calculate the required filler suspension volume based on the target filler volume and filler suspension ratio: Required filler suspension volume = target filler volume / filler suspension ratio. Vortex the filler thoroughly to ensure complete suspension. Add the filler suspension to the bottom of the gravity chromatography column. Add at least 10 CV of equilibration buffer (PBS) to the column. After equilibration, check the pH at the outlet. If the target pH has not been reached, continue adding equilibration buffer until it reaches the target pH. Slowly add a desired volume of sample to the column. Add at least 10 CV of wash buffer to the column. Slowly add 5 CV of elution buffer (10-50 mM NaAc, pH 3.0-pH 3.5) to the column. Incubate for 3-5 minutes and collect the eluate. Repeat the elution step as needed. Neutralization: Adjust the pH to the target pH with neutralization buffer (1M Tris). Determine the protein concentration using a Nanodrop. Replace the buffer containing the antibody with PBS by ultrafiltration.

[0252] Table 4 Anti-CCR8 antibody sequences

[0253] Table 5 Chimeric and humanized antibody constant region sequences

[0254] 3.2 Binding activity of anti-CCR8 chimeric antibodies to human CCR8 and cynomolgus monkey CCR8

[0255] The saturation binding assay described in Example 2-2.1 was used to detect the binding activity of anti-CCR8 chimeric antibodies to human and cynomolgus monkey CCR8. The results are shown in Figures 4 and 5 and Table 6. The EC values ​​for the binding activity of 27B9-1G3, 559E1B10, 563E10E12, 569D11B5, and 589D7C7 hIgG1 or mIgG2a chimeric antibodies to 293F-hCCR8 cells were 0. 50 The EC value of 433H binding activity to 293F-hCCR8 cells was 27.3-273.4 ng / mL. 50 The hIgG1 chimeric antibody 27B9-1G3 and the mIgG2a chimeric antibodies 563E10E12, 569D11B, 589D7C7 and 559E1B10 had strong binding activity to cynomolgus monkey CCR8, EC 50 The binding activity of 433H and 10A11_hIgG1 to cynomolgus monkey CCR8 was weaker, EC50 14580.5 and 6309.0 ng / ml.

[0256] Table 6 Binding activity of anti-CCR8 chimeric antibodies to human CCR8 and cynomolgus monkey CCR8

[0257] NT: Not tested; N / A: No binding activity or poor binding activity, no effective EC 50 value.

[0258] 3.3 Anti-CCR8 chimeric antibody competition for CCL1 binding

[0259] The competition binding assay method described in Example 2-2.2 was used to test the competition binding of anti-CCR8 chimeric antibodies to CCL1. The results are shown in Figure 6 and Table 7. Both hIgG1 and mIgG2a chimeric antibodies can dose-dependently block the binding of CCL1 to 293F-hCCR8. The IC 50 The IC values ​​for blocking by 433H and 10A11_hIgG1 were 29.5-273.4 ng / mL. 50 70.5 and 54.6 ng / mL.

[0260] Table 7 Anti-CCR8 chimeric antibody blocking activity of CCL1 binding

[0261] N / A: No binding activity, no effective EC was obtained 50 value

[0262] 3.4 Anti-CCR8 chimeric antibody blocks CCL1-induced β-Arrestin recruitment

[0263] CCL1 induces chemotaxis and receptor endocytosis through CCR8, the latter of which depends on the expression and recruitment of β-arrestin. β-arrestin analysis is used to detect the activation function of CCL1 on CCR8 (James M Fox et al. Structure / function relationships of CCR8 agonists and antagonists. Amino-terminal extension of CCL1 by a single amino acid generates a partial agonist. J Biol Chem. 2006 Dec 1; 281(48): 36652-61. doi: 10.1074 / jbc.M605584200. and Libao Liu et al. Biological characterization of ligands targeting the human CC chemokine receptor 8 (CCR8) reveals the biased signaling properties of small molecule agonists. Biochem Pharmacol. 2021 Jun;188:114565.doi:10.1016 / j.bcp.2021.114565.). Tango-H_CCR8-CHO-K1 cells (Jiman Biotech (Shanghai) Co., Ltd., GM-C09028) were used to detect the effect of anti-CCR8 antibodies on CCL1-induced β-arrestin recruitment. Anti-CCR8 antibodies were used to detect the effect of anti-CCR8 antibodies on CCL1-induced β-arrestin recruitment according to the experimental protocol provided by Jiman Biotech (Shanghai) Co., Ltd. The specific method is as follows:

[0264] Tango-H_CCR8-CHO-K1 cells were inoculated into cell culture flasks, and Doxycycline (Selleck, S4163-100 mg) was added to the culture medium at a final concentration of 10 μg / mL. The culture was continued for about 48 hours to induce human CCR8 expression using Doxycycline. The induced Tango-H_CCR8-CHO-K1 cells were digested with trypsin containing 0.25% EDTA (Gibco, 25200072), centrifuged at 200 × g for 5 minutes, and the supernatant was removed. The cells were resuspended in fresh F12K complete medium (Gibco, 21127022), the cell viability was detected and counted, and the cell concentration was adjusted to 5 × 10 5Cells / mL were plated in a white opaque plate at 100 μL / well and cultured overnight in a 37°C, 5% CO2 incubator to allow the cells to adhere. Anti-CCR8 antibody, positive control antibody, and hIgG1 isotype (Bio-Ying Biotechnology Co., Ltd., B117901) were diluted to 2 times the starting working mass concentration using F12K complete medium, and then serially diluted using F12K complete medium. After gently aspirating the medium from the Tango-H_CCR8-CHO-K1 cell culture plate, serially diluted antibodies were added, 50 μL per well. Subsequently, 50 μL of human CCL1 (R&D, 272-I) solution diluted in F12K complete medium to a final concentration of 20 nM was added to each well. After incubation at 37°C, 5% CO2 for 6 hours, the cells were equilibrated at room temperature for at least 15 minutes. 100 μL of luciferase substrate solution (Vazyme, DD1203) was added to each well. After mixing, the cells were incubated in the dark at room temperature for 5 minutes. Relative light units (RLU) were read on the cell culture plate using a microplate reader. Graphpad Prism 8.0 software was used to analyze the data. A four-parameter regression model was used to fit the antibody dose-response curve, with the logarithm of the antibody concentration on the x-axis and the corresponding RLU value on the y-axis.

[0265] As shown in Figure 7 and Table 8 , hIgG1 or mIgG2a chimeric antibodies 27B9-1G3, 563E10E12, 569D11B5, 589D7C7, 559C12G12, and 559E1B10 were able to block CCL1-induced β-Arrestin recruitment, IC 50 The IC values ​​of 433H, 10A11_hIgG1, and 10A11_mIgG2a were 260.2-1757.0 ng / mL. 50 They were 1291.5, 3268.0 and 3250.0 ng / mL.

[0266] Table 8 Anti-CCR8 chimeric antibodies block CCL1-induced β-Arrestin recruitment

[0267] N / A: No blocking activity or weak blocking activity, no effective IC 50 value

[0268] Example 4: Humanization of anti-CCR8 antibodies

[0269] Using Kabat numbering to identify CDRs, the human germline gene with the highest homology to the mouse sequence was selected as the acceptor framework, and the mouse CDRs were transplanted into the human framework. Based on the importance of amino acids, backmutations were performed, remutating key amino acids in the transplanted framework region to their corresponding mouse counterparts. Several variants were designed for each heavy and light chain.

[0270] 4.1 Humanization of anti-CCR8 antibody 27B9-1G3

[0271] The mouse variable region sequences and the selected human germline gene amino acid sequences are shown in Table 9 below; the humanized heavy and light chain sequences designed therefrom are shown in Table 10.

[0272] The humanized heavy and light chains were combined to obtain the following humanized antibodies:

[0273] 4.2 Humanization of anti-CCR8 antibody 559E1B10

[0274] The mouse variable region sequences and the selected human germline gene amino acid sequences are shown in Table 11 below; the humanized heavy and light chain sequences designed therefrom are shown in Table 12.

[0275] The humanized heavy and light chains were combined to obtain the following humanized antibodies:

[0276] 4.3 Humanization of anti-CCR8 antibodies 563E10E12, 569D11B5, and 589D7C7

[0277] The sequences of the three antibody clones, 563E10E12, 569D11B5, and 589D7C7, are similar and considered a family. Therefore, humanization of these three antibodies was considered to be performed together. The murine variable region sequences and the amino acid sequences of the selected human germline genes are shown in Table 13 below; the humanized heavy and light chain sequences designed from these sequences are shown in Table 14.

[0278] The humanized heavy and light chains were combined to obtain the following humanized antibodies:

[0279] Example 5: Functional identification of anti-CCR8 humanized antibodies

[0280] The light and heavy chain variable regions of the humanized antibodies 27B9-1G3, 559E1B10, and 563E10E12 were constructed onto human constant regions (hIgG1 / κ, sequence shown in Table 5), gene synthesis was performed, and sequencing confirmed consistency with the designed sequence. The proteins were expressed in Expi293 cells using the method described in Example 3 and purified using a Protein A column. Their functions were characterized by binding to human and cynomolgus monkey CCR8, blocking CCL1 binding, and blocking CCL1-induced β-Arrestin recruitment.

[0281] 5.1 Anti-CCR8 humanized antibody saturation binding experiment

[0282] The binding activity of the anti-CCR8 humanized antibodies to human CCR8 and cynomolgus monkey CCR8 was tested using the method described in Example 2-2.1. As shown in Figures 8 and 9 and Table 15, the binding activity of the 27B9-1G3 humanized antibody to 293F-hCCR8 was substantially comparable to that of its parent chimeric antibody. The binding activity of most 559E1B10 humanized antibodies to 293F-hCCR8 was substantially comparable to that of its parent chimeric antibody. The binding activity of most 563E10E12 humanized antibodies to 293F-hCCR8 was substantially comparable to that of its parent chimeric antibody. The humanized antibodies 559E1B10_hzH1L1, 563E10E12_hzH1L0 and 563E10E12_hzH1L1 tested had strong binding activity to 293T-cynoCCR8, while 433H and 10A11_hIgG1 had almost no binding activity to cynomolgus monkey CCR8.

[0283] Table 15 EC values ​​of humanized anti-CCR8 antibodies binding to human CCR8 (293F-hCCR8) and cynomolgus monkey CCR8 (293T-cynoCCR8) 50 value

[0284] NT: Not tested; N / A: No binding activity or poor binding activity, no effective EC 50 value.

[0285] 5.2 Anti-CCR8 humanized antibody competition for CCL1 binding

[0286] The competition binding assay described in Example 2-2.2 was used to test the humanized anti-CCR8 antibodies for CCL1 binding. The results, as shown in Figure 10 and Table 16, indicate that the blocking activity of some 27B9-1G3 humanized antibodies, such as 27B9-1G3_hzH3L1 and 27B9-1G3_hzH3L2, was substantially comparable to that of their parent chimeric antibodies. The blocking activity of most 559E1B10 humanized antibodies, such as 559E1B10_hzH2L1, 559E1B10_hzH3L1, 559E1B10_hzH1L2, 559E1B10_hzH3L2, 559E1B10_hzH0L1, and 559E1B10_hzH0L2, was comparable to that of their parent chimeric antibodies. The blocking activity of all humanized antibodies against 563E10E12 was comparable to that of its parent chimeric antibody. The blocking activity of the humanized antibodies against 559E1B10, 559E1B10_hzH3L1, 559E1B10_hzH1L2, 559E1B10_hzH0L1, and 559E1B10_hzH0L2, and all humanized antibodies against 563E10E12 was superior to or similar to that of 433H.

[0287] Table 16 IC of anti-CCR8 humanized antibodies blocking CCL1 binding on human CCR8 (293F-hCCR8) 50 value

[0288] N / A: The binding activity is poor and no effective EC is obtained. 50 value.

[0289] 5.3 Anti-CCR8 humanized antibody blocking CCL1-induced β-Arrestin recruitment

[0290] The method described in Example 3-3.4 was used to test the blocking activity of anti-CCR8 humanized antibodies in CCL1-induced β-Arrestin recruitment. As shown in Figure 11 and Table 17, the blocking activity of some 27B9-1G3 humanized antibodies, such as 27B9-1G3_hzH3L1 and 27B9-1G3_hzH3L2, was substantially equivalent to that of their parent chimeric antibodies. The blocking activity of most 559E1B10 humanized antibodies, such as 559E1B10_hzH2L1, 559E1B10_hzH3L1, 559E1B10_hzH1L2, 559E1B10_hzH3L2, 559E1B10_hzH0L1, and 559E1B10_hzH0L2, was comparable to that of their parent chimeric antibodies. The blocking activity of all humanized antibodies against 563E10E12 was comparable to that of its parent chimeric antibody. The blocking activity of the humanized antibodies against 559E1B10, 559E1B10_hzH3L1, 559E1B10_hzH1L2, 559E1B10_hzH0L1, and 559E1B10_hzH0L2, and all humanized antibodies against 563E10E12 was superior to or similar to that of 433H.

[0291] Table 17 IC of anti-CCR8 humanized antibodies blocking CCL1-induced β-Arrestin recruitment 50 value

[0292] N / A: Not applicable, no blocking activity.

[0293] 5.4 Determination of humanized anti-CCR8 antibody-dependent FcγRⅢa activation using Jurkat-human FcγRⅢa(158V)-NFAT

[0294] In studies on mouse models, it was found that anti-CCR8 antibodies mainly eliminate Tregs in tumor tissues through ADCC to inhibit tumor growth (Helena Van Damme et al. Therapeutic depletion of CCR8+tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy. J Immunother Cancer. 2021Feb; 9(2):e001749. doi:10.1136 / jitc-2020-001749.). Jurkat-human FcγRⅢa(158V)-NFAT was incubated with CCR8-expressing cells to determine the activation of FcγRⅢa by anti-CCR8 antibodies. The Fab end of the CCR8 antibody binds to the target on the target cell, and its Fc end binds to the FcγRIIIa receptor on the effector cell, thereby activating the NFAT signaling pathway in the effector cell. The luciferase produced by the activation of the NFAT pathway is quantified to reflect the ADCC activity of the antibody. The specific method is as follows:

[0295] 293F-human CCR8 (293F-hCCR8, Shanghai Ruizhi Chemical Research Co., Ltd.) cells were digested with trypsin (Gibco, 25200072) containing 0.25% EDTA, centrifuged at 300 g for 5 minutes, and the cell concentration was adjusted to 3×10 5 Cells / mL. 100 μL per well was inoculated into a white opaque plate and cultured overnight in a 37°C, 5% CO2 incubator to allow the cells to adhere to the wall and be used as target cells. Anti-CCR8 antibody, positive control antibody, and hIgG1isotype (Baiying Biotechnology Co., Ltd., B117901) were diluted with 1640 medium to 2 times the starting working mass concentration, and then diluted 5 times with culture medium. The collected Jurkat-human FcγRⅢa (158V)-NFAT (Jiman Biotechnology (Shanghai) Co., Ltd., GM-C05619) cells were centrifuged at 300g for 5 minutes, the supernatant was discarded, and 1640 medium was added to adjust the cell concentration to 3×10 6cells / mL. The 293F-hCCR8 cell culture plate was removed, the supernatant was gently aspirated, and 50 μL of Jurkat-human FcγRⅢa(158V)-NFAT cells and 50 μL of serially diluted anti-CCR8 antibody were added to each well. After induction at 37°C, 5% CO₂ for 6 hours, the cells were equilibrated at room temperature for at least 15 minutes. 100 μL of luciferase substrate solution (Vazyme, DD1203) was added to each well, mixed, and incubated at room temperature in the dark for 5 minutes. Relative light units (RLU) were read on the cell culture plate using a microplate reader. Graphpad Prism 8.0 software was used to analyze the experimental data, with the logarithm of the anti-CCR8 antibody concentration as the x-axis and the corresponding RLU value as the y-axis. A four-parameter regression model was used to fit the dose-effect curve of the anti-CCR8 antibody.

[0296] As shown in Figures 12 and 13, and Tables 18 and 19, chimeric and humanized antibodies 559E1B10 and 563E10E12 activated Jurkat-human FcγRIIIa(158V)-NFAT in a dose-dependent manner. The activities of humanized antibodies 559E1B10_hzH0L1, 559E1B10_hzH1L1, 559E1B10_hzH2L1, 559E1B10_hzH3L1, 559E1B10_hzH0L2, 559E1B10_hzH1L2, 559E1B10_hzH2L2, and 559E1B10_hzH3L2 were comparable to those of their parent chimeric antibody, 559E1B10_hIgG1. All humanized clones of 563E10E12 tested, such as 563E10E12_hzH0L0, 563E10E12_hzH1L0, and 563E10E12_hzH0L1, showed comparable activity to their parent chimeric antibodies.

[0297] Table 18 EC activation of 559E1B10 humanized antibody against Jurkat-human FcγRⅢa(158V)-NFAT 50 value

[0298] Table 19 EC activation of 563E10E12 humanized antibody against Jurkat-human FcγRⅢa(158V)-NFAT 50

[0299] Example 6. Binding activity of anti-CCR8 antibodies to HuT78 cells expressing endogenous CCR8

[0300] Human T cell lymphoma cell line HuT78 and activated human Treg both express CCR8 (James M Fox et al. and Yiftah Barsheshet et al. CCR8+FOXp3+T reg cells as master drivers of immune regulation. Proc Natl Acad Sci US A. 2017 Jun 6; 114(23): 6086-6091. doi: 10.1073 / pnas.1621280114.). FACS was used to detect the binding activity of anti-CCR8 antibody to endogenously expressed CCR8. HuT78 (Cobioer, CBP60267) cells were adjusted to a cell density of 1×10 6 Cells / mL, 100 μL per well was placed in a 96-well U-bottom plate, and the supernatant was removed by centrifugation. Anti-CCR8 antibodies, control antibodies 433H, 10A11_hIgG1, B16_hIgG1:7-B16.001 (sequence in patent US20210277129A1, light chain variable region sequence number: 81, heavy chain variable region sequence number: 80) and hIgG1isotype (Baiying Biotechnology Co., Ltd., B117901) were serially diluted with PBS solution containing 2% FBS (FACS buffer), with a starting concentration of 10 μg / mL and 4-fold serial dilution. Resuspend the cells in 100 μL / well of the serially diluted antibody, pipet and mix well, incubate at 4°C for 1 hour, and wash three times with FACS buffer after incubation. Resuspend the cells with 100 μL / well of the secondary antibody AF647-Goat anti-Human IgG (H+L) (Invitrogen, A21445) or AF647-Donkey anti-Mouse IgG (H+L) (Invitrogen, Cat. No.: A31571; for detecting mouse antibody 433H) diluted 1:1000 in FACS buffer, pipette to mix, and incubate at 4°C for approximately 45 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer. Resuspend the cells with 100 μL / well FACS buffer, and read the mean fluorescence intensity (MFI) using a flow cytometer. Graphpad Prism 8.0 software was used to analyze the experimental data. The logarithm of the anti-CCR8 antibody concentration was used as the x-axis, and the corresponding MFI value was used as the y-axis. A four-parameter regression model was used to fit the dose-effect curve of the anti-CCR8 antibody, and the EC was calculated. 50 .

[0301] As shown in FIG14 and Table 20, humanized antibodies 563E10E12_hzH1L0 and 563E10E12_hzH1L1 bound to HuT78 cells with endogenous CCR8 expression in a dose-dependent manner. 50 It is comparable to 433H, B16_hIgG1, and 10A11_hIgG1, and its maximum binding MFI is higher than those of B16_hIgG1 and 10A11_hIgG1. However, 559E1B10_hzH1L1 has essentially no binding activity to HuT78.

[0302] Table 20 EC of binding activity of anti-CCR8 antibodies to HuT78 50

[0303] N / A: No activity or no effective EC was obtained 50 value.

[0304] Example 7: Anti-CCR8 Antibodies Specifically Bind to Human CCR8

[0305] 7.1 Anti-CCR8 Antibodies Specifically Bind to CHO-K1 Cells Expressing Human CCR8

[0306] CHO-K1 cells were used in the mouse immunization and screening process of CCR8 antibodies. The binding of anti-CCR8 antibodies to cell surface CCR8 was detected using a CHO-K1 cell line overexpressing human CCR8 (constructed by Ruizhi Chemical, CHOK1-hCCR8) and a wild-type CHO-K1 cell line (Jiman Bio, GM-15570) and the experimental method in Example 2-2.1.

[0307] As shown in FIG15 and Table 21, 559E1B10_hzH1L1, 563E10E12_hzH1L0, and 563E10E12_hzH1L1 all bound to CHOK1-hCCR8 in a dose-dependent manner, but did not bind to CHOK1-null cells.

[0308] Table 21 ECs specifically binding to hCCR8 on the cell surface by anti-CCR8 antibodies 50

[0309] N / A: No activity or no effective EC was obtained 50 value.

[0310] 7.2 Anti-CCR8 Antibody Mediates CCR8-Dependent Activation of Jurkat-human FcγRⅢa(158V)-NFAT

[0311] The human CCR8-overexpressing CHO-K1 cell line (constructed by Ruizhi Chemical, CHOK1-hCCR8) and the CHO-K1 blank cell line (Jiman Bio, GM-15570) were used to determine that the activation of Jurkat-human FcγRⅢa(158V)-NFAT by anti-CCR8 antibodies was dependent on CCR8. CHOK1-hCCR8 cells and CHOK1 blank cells were adjusted to a cell density of 3×10 5 Cells / mL were used, and the method described in Example 5-5.4 was used to detect the activation of Jurkat-human FcγRⅢa(158V)-NFAT by anti-CCR8 antibodies. 4A19 is the sequence described in patent WO2021194942A1, with the light chain variable region sequence number: 18 and the heavy chain variable region sequence number: 6. As shown in Figure 16 and Table 22, in the presence of CHOK1-hCCR8 cells, anti-CCR8 antibodies activated Jurkat-human FcγRⅢa(158V)-NFAT in a dose-dependent manner; however, in the presence of CHO-K1 blank cells, anti-CCR8 antibodies failed to activate Jurkat-human FcγRⅢa(158V)-NFAT, demonstrating that anti-CCR8 antibodies can specifically recognize CCR8 and mediate CCR8-dependent ADCC.

[0312] Table 22 EC activation of Jurkat-human FcγRⅢa(158V)-NFAT by anti-CCR8 antibodies CCR8-dependent 50

[0313] N / A: No activity or no effective EC was obtained 50 value.

[0314] 7.3 Anti-CCR8 Antibodies Do Not Bind to Human CCR4

[0315] The gene with the highest homology to CCR8 in humans is CCR4 (Antonio Recchiuti et al. Pro-Resolving Lipid Mediators (SPMs) and Their Actions in Regulating miRNA in Novel Resolution Circuits in Inflammation. Front Immunol. 2012 Oct 22; 3:298. doi:10.3389 / fimmu.2012.00298.eCollection 2012.). However, CCR4 and CCR8 share only 46% homology (NCBI blast results). The binding activity of anti-CCR8 antibodies to human CCR4 was tested using the following method. TM 293F cells were transiently transfected with hCCR4-GFP (Sino, HG13064-ACG) using the 293 transfection kit (Gibco, A14524). The cell density after transfection was adjusted to 1×10 6 cells / ·mL, 100 μL per well was placed in a 96-well U-bottom plate, and the cells were obtained by centrifugation. Anti-CCR8 antibody was diluted to the starting working mass concentration with PBS solution containing 2% FBS (FACS buffer), the starting concentration was 30 μg / mL, and then diluted 4 times with FACS buffer solution. Resuspend the cells in 100 μL per well, mix well by pipetting, incubate at 4°C for 1 hour, and then wash three times with FACS buffer. Resuspend the cells in 100 μL per well of the secondary antibody AF647-Goat anti-Human IgG (H+L) (Invitrogen, A21445) diluted 1:1000 in FACS buffer, mix well by pipetting, and incubate at 4°C for 40 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer. According to LIVE / DEAD TMAfter labeling dead cells according to the instructions of Fixable Near IR (780) Viability Kit (Invitrogen, L34992), cells were resuspended in 100 μL FACS buffer per well and the mean fluorescence intensity (MFI) was read using a flow cytometer. The expression of CCR4 was determined using an anti-CCR4 antibody (Biolegend, 359412) in cells transfected with hCCR4-GFP. The binding of GFP-positive cells (CCR4-expressing cells) transfected with hCCR4-GFP and live cells transfected with a blank plasmid to anti-CCR8 antibodies was analyzed separately. As shown in Figure 17, the chimeric and humanized antibodies of 563E10E12 did not bind to cells transfected with hCCR4 (GFP-positive cells) or 293F cells, while the chimeric and humanized antibodies of 559E1B10 had weak binding to hCCR4 at high concentrations.

[0316] 7.4 Binding of anti-CCR8 antibodies to immune cells in PBMCs

[0317] CCR8 is not expressed on other immune cells in the peripheral blood of healthy humans, except for a small amount on Tregs (George Plitas et al. Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer. Immunity. 2016 Nov 15; 45(5): 1122-1134. doi: 10.1016 / j.immuni.2016.10.032.). PBMCs from healthy donors were used to test whether anti-CCR8 antibodies bind nonspecifically to peripheral immune cells. Frozen PBMCs from healthy donors (Allcells, catalog numbers Z0060 and Y1446) were thawed and the cell concentration was adjusted to 2×10 6 cells / mL, 100 μL was placed in each well of a 96-well U-bottom plate, and the supernatant was removed by centrifugation. TMDead cells were labeled according to the instructions of Fixable Near IR (780) Viability Kit (Invitrogen, L34992). 50 μL FcX reagent (Biolegend, 422302) diluted 1:25 in FACS buffer was added to each well to block the nonspecific staining of Fc receptors by the antibody and incubated at room temperature for 15 minutes. PE anti-human CD3 Antibody (Biolegend-317308, 1:50 dilution), FITC-anti-human CD8 (Biolegend-301006, 1:50 dilution), Pacific Blue TM anti-human CD19 (Biolegend-363036, 1:50 dilution) and Brilliant Violet 605 TM Add 40 μL of a mixture of anti-human CD14 (Biolegend-367126, 1:50 dilution) to the cells per well and mix thoroughly. Dilute the anti-CCR8 antibody and hIgG1 isotype (Bio-Ying Biotechnology Co., Ltd., B117901) labeled with the AF647 labeling kit (Invitrogen, A20186) to a starting concentration of 100 μg / mL in FACS buffer. Dilute the mixture 5-fold in FACS buffer and add 10 μL per well to the cells. Incubate at 4°C for 1 hour. After incubation, wash three times with FACS buffer and resuspend the cells in 100 μL of FACS buffer per well. Measure the mean fluorescence intensity (MFI) using a flow cytometer.

[0318] As shown in Table 23, the anti-CCR8 antibodies 559E1B10_hzH1L1_hIgG1, 563E10E12_hzH1L0_hIgG1 and 563E10E12_hzH1L1_hIgG1 showed no significant difference in the expression of CCR8 antibodies. + ,CD4 + (CD3 + CD8 - )T cells, CD14 + Monocytes, CD19 + B cells and other CD3 - CD14 - CD19 - There was no significant binding of immune cells.

[0319] Table 23 Binding intensity of anti-CCR8 antibodies to immune cells in PBMC (mean fluorescence intensity, MFI)

[0320] Example 8: Fc mutation and reduced fucosylation enhance ADCC effect mediated by anti-CCR8 antibodies

[0321] 8.1 S239D / I332E, S239D / A330L / I332E, L235V / F243L / R292P / Y300L / P396L mutations and reduced fucosylation enhance activation of Jurkat-human FcγRⅢa(158V)-NFAT by anti-CCR8 antibodies and 293F-human CCR8

[0322] Antibody constant region (Fc) mutations S239D / I332E, S239D / A330L / I332E, or L235V / F243L / R292P / Y300L / P396L can increase affinity for FcγRⅢa and enhance antibody-mediated ADCC (Greg A Lazar et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci US A. 2006 Mar 14; 103(11):4005-10. doi:10.1073 / pnas.0508123103. and Jeffrey L Nordstrom et al. Anti-tumor activity and toxicokinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with enhanced Fcγreceptor binding properties. Breast Cancer). Res. 2011; 13(6): R123. doi: 10.1186 / bcr3069.). These groups of mutations have been used clinically to enhance the ADCC effect of antibodies (Rena Liu et al. Fc-Engineering for Modulated Effector Functions-Improving Antibodies for Cancer Treatment. Antibodies (Basel). 2020 Nov 17; 9(4): 64. doi: 10.3390 / antib9040064.).

[0323] The Fc of humanized anti-CCR8 antibodies was mutated to S239D / I332E (DE), S239D / A330L / I332E (DLE), or L235V / F243L / R292P / Y300L / P396L (VLPLL) to enhance the ADCC effect induced by them. Defucosylation or reduced fucosylation can increase the ADCC effect of antibodies. Many antibodies with defucosylation or reduced fucosylation have been approved for marketing or are in the late stages of clinical research (Natasha A Pereira et al. The "less-is-more" in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity. MAbs. 2018 Jul; 10(5): 693-711. doi: 10.1080 / 19420862.2018.1466767.). To inhibit fucosylation, ExpiCHO cells were passaged and expanded using ExpiCHO Expression Medium (Gibco, Catalog No.: A2910001) containing 100 μM 2F-peracetyl-fucose (MILLIPORE, Catalog No.: 344827-10MGCN) before transient transfection. On the day of transient transfection, cells were diluted to 6 × 10 6 Cells were transiently transfected using the Expi CHO Transfection Kit (Gibco, Catalog No. A29129) at a concentration of 1 μg plasmid / mL of cell suspension. Transiently transfected cells were fed (Gibco, Catalog No. A29129) 18-22 hours after transient transfection and continued to be cultured at 37°C, 8% CO2, and 100 rpm in a shaker. Seven days after transient transfection, the cell culture medium was harvested, centrifuged, and filtered through 0.45 μm to obtain the supernatant, which was then purified using Protein A media to obtain the antibody.

[0324] The method described in Example 5-5.4 was used to detect the activation of Jurkat-human FcγRⅢa(158V)-NFAT by incubation of anti-CCR8 antibodies with 293F-hCCR8. As shown in Figure 18 and Table 24, compared with 559E1B10_hzH1L1_hIgG1, the Fc mutants 559E1B10_hzH1L1_DE and 559E1B10_hzH1L1_VLPLL enhanced the activation of Jurkat-human FcγRⅢa(158V)-NFAT. 50Compared with 563E10E12_hzH1L0_hIgG1, the Fc mutants 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE, and 563E10E12_hzH1L0_VLPLL and the reduced fucosylation 563E10E12_hzH1L0_AF enhanced the activation of Jurkat-human FcγRⅢa(158V)-NFAT. 50 Compared with 563E10E12_hzH1L1_hIgG1, the Fc mutants 563E10E12_hzH1L1_DE and 563E10E12_hzH1L1_VLPLL enhanced the activation of Jurkat-human FcγRⅢa(158V)-NFAT, EC 50 The activity of the Fc-mutated 559E1B10 and 563E10E12 humanized antibodies or the reduced fucosylation 563E10E12_hzH1L0 was comparable to that of the reduced fucosylation B16 (B16_AF).

[0325] Table 24 Activation of Jurkat-human FcγRⅢa(158V)-NFAT by incubation of anti-CCR8 antibodies with Fc mutation or reduced fucosylation with 293F-humanCCR8 (EC 50 ,μg / mL)

[0326] N / A: No activity or no effective EC was obtained 50 value.

[0327] 8.2 Fc mutation and reduced fucosylation enhance activation of Jurkat-human FcγRⅢa(158V)-NFAT by anti-CCR8 antibody and HuT78

[0328] The anti-CCR8 antibodies with different Fc mutations or reduced fucosylation were incubated with Hut78 and Jurkat-human FcγRⅢa(158V)-NFAT cells to detect the effects of S239D / I332E, S239D / A330L / I332E, L235V / F243L / R292P / Y300L / P396L mutations or reduced fucosylation on the activation of Jurkat-human FcγRⅢa(158V)-NFAT by anti-CCR8 antibodies and HuT78. The cell density of HuT78 cells was adjusted to 3×10 5cells / mL were used as target cells, and the activation of Jurkat-human FcγRⅢa(158V)-NFAT by anti-CCR8 antibody was detected using the method in Example 5-5.4.

[0329] As shown in Figure 19 and Table 25, incubation of Fc-mutated antibodies 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE, 563E10E12_hzH1L0_VLPLL and reduced fucosylation antibody 563E10E12_hzH1L0_AF with HuT78 increased the activation of Jurkat-human FcγRⅢa(158V)-NFAT by 563E10E12_hzH1L0, EC 50 The signal was reduced by 2-6 fold and the maximum signal increased by 4-5 fold. 563E10E12_hzH1L0 with Fc mutation or reduced fucosylation activated Jurkat-human FcγRⅢa(158V)-NFAT at the same level as B16 with reduced fucosylation.

[0330] Table 25 Effect of Fc mutation or reduced fucosylation 563E10E12_hzH1L0 on the EC activation of Jurkat-human FcγRⅢa(158V)-NFAT by incubation with Hut78 50

[0331] N / A: No activity or no effective EC was obtained 50 value.

[0332] Example 9: ADCC effect induced by healthy human PBMC and anti-CCR8 antibody

[0333] PBMCs were used as effector cells and CHOK1-hCCR8 as target cells to detect the ADCC effect of anti-CCR8 antibodies. PBMCs (Allcells) from healthy donors were resuscitated and the cell concentration was adjusted to 2×10 cells / mL using 1640 complete medium containing 200 IU / mL IL-2 (Peprotech, Cat. No. 200-02). 6 Cells / mL were cultured in a 37°C, 5% CO2 incubator overnight. The next day, non-adherent PBMCs were aspirated, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 1640 medium was added to adjust the cell concentration to approximately 3×10 6Anti-CCR8 antibody and hIgG1 isotype (Bio-Ying Biotechnology Co., Ltd., catalog number: B117901) were diluted to 2 times the working mass concentration in 1640 medium, starting at 0.8 μg / mL and diluted 5-fold. The collected CHOK1-hCCR8 cells were centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the cell concentration was adjusted to 2 × 10 cells / mL with DPBS. 6 cells / mL, add CellTrace according to the instructions TM CHOK1-hCCR8 was labeled with Violet (Invitrogen, Cat. No. C34557) and the CellTrace TM Adjust the cell density of Violet-labeled CHO-hCCR8 cells to approximately 1.5 × 10 cells using 1640 medium. 5 cells / mL. 50 μL CellTrace TM Violet-labeled CHOK1-hCCR8, 50 μL PBMC, and 100 μL diluted anti-CCR8 antibody were mixed and incubated in a 37°C, 5% CO2 incubator for approximately 5 hours. 2 μL PI (Invitrogen, Cat. No. 006990-50) dye was added to each well, mixed, and incubated in the dark for 5 minutes at room temperature to label dead cells. The percentage of PI-positive cells was measured by flow cytometry using CellTrace. TM The percentage of Violet positive cells was calculated using lysis% = CellTrace TM The antibody-induced ADCC effect was calculated by measuring the proportion of Violet-labeled PI-positive cells. Graphpad Prism 8.0 software was used to analyze the experimental data. A four-parameter regression model was used to fit the dose-effect curve of the anti-CCR8 antibody, with the logarithm of the anti-CCR8 antibody concentration as the x-axis and the corresponding lysis percentage as the y-axis.

[0334] As shown in Figure 20 and Table 26, compared to 563E10E12_hzH1L0_hIgG1, the humanized antibody 563E10E12_hzH1L0_AF with reduced fucosylation and the Fc-mutated antibodies 563E10E12_hzH1L0_DE and 563E10E12_hzH1L0_DLE increased their ADCC activity induced by PBMCs. The ADCC activity induced by the humanized antibody 563E10E12_hzH1L0_hIgG was comparable to that induced by B16_hIgG1. The ADCC activity induced by the humanized antibody 563E10E12_hzH1L0 with reduced fucosylation and an Fc-mutated antibody 563E10E12_hzH1L0 with PBMCs was similar to that induced by B16 with reduced fucosylation.

[0335] Table 26 EC of ADCC effect induced by human PBMC and anti-CCR8 antibody 50

[0336] N / A: Not applicable

[0337] Example 10: Anti-CCR8 antibodies specifically eliminate CCR8-positive Tregs in peripheral PBMCs

[0338] Healthy human PBMCs were used to detect the specific depletion of CCR8-expressing Treg cells in PBMCs by anti-CCR8 antibodies. Since the proportion of Treg cells expressing CCR8 in healthy human PBMCs is low, IL-2 was used in this experiment to promote the proliferation and activation of Tregs. PBMCs (Allcells) from healthy donors were resuscitated and the cell concentration was adjusted to 2×10 cells / mL using 1640 complete medium containing 200 IU / mL IL-2 (Peprotech, Cat. No. 200-02). 6 Cells / mL, 100 μL per well was added to a 96-well U-bottom plate and incubated at 37°C, 5% CO2 for 48 hours. Anti-CCR8 antibody and hIgG1isotype (Bio-Ying Biotechnology Co., Ltd., catalog number: B117901) were diluted in 1640 complete medium with a starting concentration of 20 μg / mL and 4-fold serial dilution. 100 μL per well of the diluted antibody was added to the above 96-well plate, pipetted to mix, and incubated at 37°C, 5% CO2 for 96 hours. Cells were harvested by centrifugation and incubated according to the LIVE / DEAD method. TMDead cells were labeled according to the instructions of the Fixable Near IR (780) Viability Kit (Invitrogen, Catalog No.: L34992). 50 μL of a 1:25 diluted FcX reagent (Biolegend, Catalog No.: 422302) was added to each well and incubated at room temperature for 15 minutes. 50 μL of a 1:50 diluted AF700 Anti-Human CD3 (Biolegend, Catalog No.: 317340) and Percp cy5.5 Anti-Human CD4 (Biolegend, Catalog No.: 300530) mixed staining solution was added to each well and incubated at 4°C for 1 hour. The cells were washed three times with FACS buffer. According to the instructions of the Foxp3 / Transcription Factor Staining Kit (Invitrogen, Catalog No.: 00-5523-00), cells were fixed and labeled with PE anti-human Foxp3 (Biolegend, Catalog No.: 320208), and washed three times with FACS buffer. Resuspend the cells in 100 μL FACS buffer per well, read the relevant information using flow cytometry, and analyze the experimental data using Graphpad Prism 8.0 software. + CD4 + ) cells, and the proportion of CCR8-positive cells in Treg was 31.5%.

[0339] As shown in FIG21 , 563E10E12_hzH1L0_AF with reduced fucosylation and 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE, 563E10E12_hzH1L0_VLPLL and B16_AF with reduced fucosylation dose-dependently partially eliminated Tregs in PBMCs, but did not affect CD8 T cells that did not express CCR8. + T(CD3 + CD4 - ) cells. 563E10E12_hzH1L0_hIgG1 and B16_hIgG1 only slightly eliminated Tregs at higher concentrations. This suggests that after human peripheral blood mononuclear cells (PBMCs) are activated with IL-2, Tregs express a certain level of CCR8 and can be eliminated by anti-CCR8 antibodies, while CD8 T cells that do not express CCR8 are not eliminated.

[0340] Example 11: Anti-CCR8 Antibody Inhibition Experiment in Mouse Tumor Model

[0341] 11.1 Anti-CCR8 Antibody Inhibits MC38 Colon Cancer Growth in a CCR8 Humanized Mouse Model

[0342] Human CCR8 humanized mice (C57BL / 6-Ccr8 tm1(CCR8) / Bcgen, B-hCCR8, Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd., Catalog No.: 110096) mice were used to establish the MC38 (Beijing Biocytogen Pharmaceutical Technology Co., Ltd.) colon cancer animal model and to test the in vivo efficacy of anti-CCR8 antibodies. MC38 cells (5×10 6 100 μL of 500 cells / mL) was inoculated subcutaneously on the right side of B-hCCR8 mice. When the average tumor volume reached about 100 mm 3 At 6-8 days, mice were divided into groups and subcutaneously injected twice weekly with 10 mg / kg of anti-CCR8 antibody, a negative control (hIgG1) (Bio-Ying Biotechnology Co., Ltd., Catalog No. B117901), or an equal volume of vehicle (PBS). Following administration, the mice were housed normally, and their survival was observed. Body weight and tumor volume were recorded.

[0343] Compared to the vehicle control and negative control (hIgG1), the anti-CCR8 antibodies 559E1B10_hzH1L1_mIgG2a, 563E10E12_hzH1L0_mIgG2a, 563E10E12_hzH1L1_mIgG2a, and 10A11_mIgG2a all inhibited the growth of MC38 colon cancer cells (Figure 22). On day 20 after administration, the tumor inhibition rates (TGI) of the anti-CCR8 antibodies 559E1B10_hzH1L1_mIgG2a, 563E10E12_hzH1L0_mIgG2a, 563E10E12_hzH1L1_mIgG2a, and 10A11_mIgG2a were 40.1%, 54.0%, 22.5%, and 53%, respectively. During the experiment, all animals were active and ate well during the administration period, and their body weight increased to a certain extent, indicating that the animals tolerated the anti-CCR8 antibody well ( FIG23 ).

[0344] 11.2 Anti-CCR8 Antibodies with Fc Mutation and Reduced Fucosylation Inhibit the Growth of MC38 Colon Cancer in a CCR8 Humanized Mouse Model

[0345] The MC38 colon cancer model was established in CCR8 humanized mice B-hCCR8 using the method described in Example 11-11.1. When the average tumor size was about 100 mm 3Mice were divided into groups of 5 and subcutaneously injected with 10 mg / kg of anti-CCR8 antibody or an equal volume of vehicle (PBS) twice weekly. After administration, the mice were housed normally, and their survival was observed. Their body weight and tumor volume were recorded.

[0346] As shown in Figure 24, compared to the vehicle, the anti-CCR8 antibody 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_DE and 563E10E12_hzH1L0_DLE, and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF all inhibited the growth of MC38 colon cancer cells. On day 25 after administration, the tumor inhibition rates (TGI) of 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_DE and 563E10E12_hzH1L0_DLE, and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF were 60.1%, 57.8%, 60.2%, and 52.9%, respectively. All mice in the treatment groups showed no behavioral abnormalities or weight loss, indicating that the tumor-bearing mice had good tolerance to the anti-CCR8 antibody at this dose (Figure 25).

[0347] 11.3 In human CD34 + Anti-CCR8 antibodies inhibit the growth of human non-small cell lung cancer cells in HSC-humanized mice and human PBMC-humanized mice

[0348] 11.3.1 Anti-CCR8 Antibodies in Human CD34 + HSCs inhibit the growth of non-small cell lung cancer cell line A549 in humanized mice

[0349] Human hematopoietic cells hCD34 + HSCs were transplanted into irradiated NCG mice for immune reconstitution to obtain human CD34 + HSC humanized mice (Jicui Yaokang). Human non-small cell lung cancer A549 cells were inoculated with human CD34 + HSC humanized mice were inoculated subcutaneously on the right side of the body with a volume of 0.2 mL / mouse containing 30% Matrigel. When the average tumor volume was approximately 100 mm 3At the same time, mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg / kg), pembrolizumab (1-20 mg / kg), a combination of CCR8 antibody (1-20 mg / kg) and pembrolizumab (1-20 mg / kg), and negative control (PBS), twice a week for 4 weeks. After administration, the mice were housed normally and the tumor volume was recorded. At the end of the experiment, the tumor samples were harvested, minced with dissecting laboratory scissors, and enzymatically dissociated with the human tumor dissociation kit (Miltenyi Biotech) combined with the gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions. The cell suspension was filtered through a 70 μm MACS smart filter to obtain a single cell suspension. The immune cell subsets (Treg and CD8 + T cells) were analyzed.

[0350] 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.

[0351] 11.3.2 CCR8 Antibody Inhibits the Growth of the Human Non-Small Cell Lung Cancer Cell Line NCI-H1299 in a Humanized Human PBMC Mouse Model

[0352] Human non-small cell lung cancer NCI-H1299 cells were inoculated subcutaneously on the right side of human PBMC humanized mice (Jicui Yaokang) with an inoculation volume of 0.2 mL / mouse containing 30% Matrigel. When the average tumor volume was approximately 100 mm 3 At the same time, the mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg / kg), pembrolizumab (1-20 mg / kg), a combination of CCR8 antibody (1-20 mg / kg) and pembrolizumab (1-20 mg / kg), and negative control (PBS) twice a week for 4 weeks. After administration, the mice were housed normally and the tumor volume was recorded. At the end of the experiment, the tumor samples were harvested, minced with dissecting laboratory scissors, and the tumor tissue was enzymatically dissociated using a human tumor dissociation kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions. The cell suspension was filtered through a 70 μm MACS smart filter to obtain a single cell suspension. The immune cell subsets (Treg and CD8 + T cells) were analyzed.

[0353] 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.

[0354] 11.3.3 CCR8 Antibody Inhibits Growth of Human Non-Small Cell Lung Cancer NCI-H292 in a Humanized PBMC Mouse Model

[0355] Human non-small cell lung cancer NCI-H292 cells were inoculated subcutaneously on the right side of human PBMC humanized mice (Jicui Yaokang) with an inoculation volume of 0.2 mL / mouse containing 30% Matrigel. When the average tumor volume was approximately 100 mm 3 At the same time, mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg / kg), pembrolizumab (1-20 mg / kg), a combination of CCR8 antibody (1-20 mg / kg) and pembrolizumab (1-20 mg / kg), and negative control (PBS) twice a week for 3 weeks. After administration, the mice were housed normally and the tumor volume was recorded. At the end of the experiment, tumor samples were harvested, minced with dissecting laboratory scissors, and enzymatically dissociated with the human tumor dissociation kit (Miltenyi Biotech) combined with the gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions. The cell suspension was filtered through a 70 μm MACS smart filter to obtain a single cell suspension. Immune cell subsets and functional biomarkers were analyzed by flow cytometry. Immune cell subsets (Treg and CD8 + T cells) were analyzed.

[0356] 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth compared with the negative control group.

[0357] 11.4 Combination of CCR8 Antibody and Pembrolizumab Inhibits the Growth of Human Colon Cancer HT29 in a Human PBMC Humanized Mouse Model

[0358] Human rectal cancer HT29 cells were inoculated subcutaneously on the right side of human PBMC-humanized mice (Shanghai Model Organisms) at a volume of 0.1 mL / mouse containing 30% Matrigel. When the average tumor volume was approximately 100 mm 3 At the same time, mice were randomly divided into groups and given anti-CCR8 antibody (10 mg / kg), pembrolizumab (3.25 mg / kg), a combination of CCR8 antibody (10 mg / kg) and pembrolizumab (3.25 mg / kg), and negative control (PBS) twice a week for 4 weeks. After administration, the mice were housed normally and the tumor volume was recorded. At the end of the experiment, the tumor samples were harvested, minced with dissecting laboratory scissors, and the tumor tissue was enzymatically dissociated using a human tumor dissociation kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions. The cell suspension was filtered through a 70 μm MACS smart filter to obtain a single cell suspension. Immune cell subsets (Treg and CD8 + T cells) were analyzed.

[0359] 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.

[0360] 11.5 Efficacy of anti-CCR8 antibodies in the human CCR8 humanized mouse breast cancer EMT-6 tumor model

[0361] Murine breast cancer EMT-6 cells were inoculated subcutaneously on the right side of humanized CCR8 mice (Shanghai Model Organisms) at a volume of 0.1 mL / mouse (in PBS). When the average tumor volume was approximately 50-100 mm 3 At the same time, mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg / kg) and negative control (PBS) twice a week for 3 weeks. After administration, the mice were raised normally and the tumor volume was recorded. At the end of the experiment, the tumor samples were harvested, minced with dissecting laboratory scissors, and enzymatically dissociated with the human tumor dissociation kit (Miltenyi Biotech) combined with the gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions. The cell suspension was filtered through a 70 μm MACS smart filter to obtain a single cell suspension. The immune cell subsets (Treg and CD8 + T cells) were analyzed.

[0362] 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.

[0363] Then, EMT-6 breast cancer cells were inoculated subcutaneously on the right side of humanized CCR8 mice (Shanghai Model Organisms) at a volume of 0.1 mL / mouse (in PBS). When the average tumor volume was approximately 50-100 mm 3 At the same time, mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg / kg), anti-mouse PD-1 (mPD-1) antibody (Leinco Technologies, P372) and negative control (PBS) twice a week. After administration, the mice were housed normally and the tumor volume was recorded. At the end of the experiment, the tumor samples were harvested, minced with dissecting laboratory scissors, and enzymatically dissociated with the human tumor dissociation kit (Miltenyi Biotech) combined with the gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions. The cell suspension was filtered through a 70 μm MACS smart filter to obtain a single cell suspension. The immune cell subsets (Treg and CD8 + T cells) were analyzed.

[0364] The results are shown in Figure 26. Compared with the PBS control group, 563E10E12_hzH1L0_DLE alone at doses of 5 mg / kg and 10 mg / kg showed significant anti-tumor activity in B-hCCR8 humanized mice transplanted with EMT-6 cells, significantly inhibiting the increase in tumor volume (p<0.05) (Figure 26). Furthermore, at the same 5 mg / kg dose, the tumor inhibition rate (TGI = 94.2%) of 563E10E12_hzH1L0_DLE combined with anti-mPD-1 antibody (5 mg / kg) was superior to that of 563E10E12_hzH1L0_DLE alone (TGI = 53.8%) and anti-mPD-1 antibody alone (TGI = 76.4%).

[0365] During the experiment, no obvious clinical symptoms were found in any group of mice. Similar to the PBS control group, the weight of the animals in the drug-treated groups gradually increased, indicating that the animals tolerated the test substance well (Figure 27). At the end of the experiment, compared with the PBS control group, the Treg clearance rates in the tumors of the 5mg / kg 563E10E12_hzH1L0_DLE group, the anti-mPD-1 antibody group, the combined drug group, and the 10mg / kg 563E10E12_hzH1L0_DLE group were 26.3%, 24.9%, 23.4%, and 27.3%, respectively; and the CD8+T cells / Treg ratio increased by 32.5%, 22.6%, 46.8%, and 11.5%, respectively.

[0366] 11.6 Efficacy of anti-CCR8 antibodies in the human CCR8 humanized mouse melanoma B16F10 tumor model

[0367] Murine melanoma B16F10 cells were inoculated subcutaneously on the right side of humanized CCR8 mice (Biocytogen) at a volume of 0.1 mL / mouse (in PBS). When the average tumor volume was approximately 50-100 mm 3 At the same time, mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg / kg), anti-mouse PD1 monoclonal antibody (1-20 mg / kg), a combination of anti-CCR8 antibody (1-20 mg / kg) and anti-mouse PD1 monoclonal antibody (1-20 mg / kg) and negative control (PBS) twice a week for 3 weeks. After administration, the mice were housed normally and the tumor volume was recorded. At the end of the experiment, the tumor samples were harvested, minced with dissecting laboratory scissors, and the tumor tissue was enzymatically dissociated using a human tumor dissociation kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions. The cell suspension was filtered through a 70 μm MACS smart filter to obtain a single cell suspension. Immune cell subsets (Treg and CD8 + T cells) were analyzed.

[0368] 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.

[0369] 11.7 Efficacy of Anti-CCR8 Antibodies in the Human CCR8 Humanized Mouse Colorectal Cancer CT26 Tumor Model

[0370] Mouse colorectal cancer CT26 cells were inoculated subcutaneously on the right side of humanized CCR8 mice (Shanghai Model Organisms) at a volume of 0.1 mL / mouse (in PBS). When the average tumor volume was approximately 50-100 mm 3 At the same time, mice were randomly divided into groups and given anti-CCR8 antibody (1-20 mg / kg), anti-mouse PD1 monoclonal antibody (1-20 mg / kg), a combination of anti-CCR8 antibody (1-20 mg / kg) and anti-mouse PD1 monoclonal antibody (1-20 mg / kg) and negative control (PBS) twice a week for 3 weeks. After administration, the mice were housed normally and the tumor volume was recorded. At the end of the experiment, the tumor samples were harvested, minced with dissecting laboratory scissors, and the tumor tissue was enzymatically dissociated using a human tumor dissociation kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions. The cell suspension was filtered through a 70 μm MACS smart filter to obtain a single cell suspension. Immune cell subsets (Treg and CD8 + T cells) were analyzed.

[0371] 563E10E12_hzH1L0_hIgG1, its Fc mutant antibodies 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE and the fucosylation-reducing antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth relative to the negative control group.

[0372] Various modifications and variations of the methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the present invention as claimed should not be unduly limited to such specific embodiments. In fact, various modifications of the described modes for implementing the present invention will be apparent to those skilled in the art of molecular biology, immunology, or related fields and are intended to be within the scope of the appended claims.

[0373] Sequence Listing

Claims

1. An antibody or antigen-binding fragment thereof that binds to CCR8, having one or more of the following properties: (1) With an EC no higher than 500 ng / mL 50 Specific binding to human CCR8 and / or cynomolgus monkey CCR8; (2) blocking the binding of human and / or cynomolgus monkey CCR8 to the ligand CCL1; (3) blocking CCL1-induced β-Arrestin recruitment; (4) Clearing CCR8 + Treg cells; (5) Inhibit tumor growth, preferably without affecting body weight.

2. An antibody or antigen-binding fragment thereof that binds to CCR8, comprising (i) the three complementarity determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in any one of SEQ ID NOs: 21-40, and / or the three complementarity determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in any one of SEQ ID NOs: 42-54; (ii) The CDR combination as described in (i), wherein compared with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3, the sequence comprises at least one amino acid addition, substitution or deletion or any combination thereof (for example, 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof), preferably amino acid substitutions, preferably conservative substitutions, and maintains affinity for CCR8.

3. An antibody or antigen-binding fragment thereof that binds to CCR8, comprising a heavy chain variable region and / or a light chain variable region, wherein The heavy chain variable region comprises: (1) HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 or 10 or 11 or 41 and SEQ ID NO: 3, respectively; or HCDR1, HCDR2 and HCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 or 10 or 11 or 41 and SEQ ID NO: 3, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof); or (2) HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or HCDR1, HCDR2 and HCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof); or (3) HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; or HCDR1, HCDR2 and HCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof); and / or The light chain variable region comprises: (1) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; or LCDR1, LCDR2 and LCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof); or (2) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; or LCDR1, LCDR2 and LCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof); or (3) LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively; or LCDR1, LCDR2 and LCDR3 that are identical to the amino acid sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, or have at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof).

4. An antibody or antigen-binding fragment thereof that binds to CCR8, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in any combination of the following table:

5. An antibody or antigen-binding fragment thereof that binds to CCR8, comprising a heavy chain variable region VH and / or a light chain variable region VL, wherein: (a) Heavy chain variable region VH (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 21-40; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 21-40; or (iii) an amino acid sequence comprising at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to the amino acid sequence shown in any one of SEQ ID NOs: 21-40, preferably, the amino acid change does not occur in the CDR region, preferably, the amino acid change occurs in the FR region; and / or (b) Light chain variable region VL (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 42-54; (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 42-54; or (iii) comprising an amino acid sequence having at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to the amino acid sequence shown in any one of SEQ ID NOs: 42-54, preferably, the amino acid change does not occur in the CDR region, preferably, the amino acid change occurs in the FR region.

6. An antibody or antigen-binding fragment thereof that binds to CCR8, comprising a heavy chain variable region VH and a light chain variable region VL as shown in any combination of the following tables:

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising a heavy chain constant region and / or a light chain constant region, preferably, the light chain constant region is a lambda chain or a kappa chain constant region; the heavy chain constant region is selected from murine mIgG2a, human IgG1, human IgG2, human IgG3 or IgG4 type; more preferably, the heavy chain constant region is human IgG1 type or human IgG4 type with an S228P mutation.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising a heavy chain and / or a light chain, wherein (a) Heavy chain (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 55-74; (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 55-74; or (iii) comprising an amino acid sequence having at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to an amino acid sequence selected from any one of SEQ ID NOs: 55-74, preferably, the amino acid change does not occur in the CDR region of the heavy chain, more preferably, the amino acid change does not occur in the heavy chain variable region, and most preferably, the amino acid change occurs in the heavy chain constant region; and / or (b) Light chain (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 75-82; (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 75-82; or (iii) comprises an amino acid sequence having at least one amino acid addition, substitution or deletion or any combination thereof (e.g., 1, 2 or 3 amino acid additions, substitutions or deletions or any combination thereof) compared to an amino acid sequence selected from any one of SEQ ID NOs: 75-82, preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the amino acid change does not occur in the light chain variable region, most preferably, the amino acid change occurs in the light chain constant region.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, and the sequence of the heavy chain constant region has one or more amino acid substitutions compared to the sequence of a native human heavy chain constant region, preferably, the one or more amino acid substitutions enhance the ADCC effect of the antibody, more preferably, the one or more amino acid substitutions occur at positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325 one or more of 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the one or more amino acid substitutions occur in one or more of the heavy chain constant region sequence positions L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339, and P396 according to the EU numbering system.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein the one or more amino acid substitutions are selected from one or more of G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T and P396L substitutions according to the EU numbering system.

13. The antibody or antigen-binding fragment thereof of claim 10, wherein the one or more amino acid substitutions are selected from one or more of an N297A substitution, an N297Q substitution, an L235A substitution together with an L237A substitution, an L234A substitution together with an L235A substitution, an E233P substitution, an L234V substitution, an L235A substitution, a C236 deletion, a P238A substitution, a D265A substitution, an A327Q substitution, and a P329A substitution according to the EU numbering system.

14. The antibody or antigen-binding fragment thereof of claim 10, wherein the one or more amino acid substitutions occur in one or more of positions 235, 239, 243, 292, 300, 330, 332, 396 of the heavy chain constant region sequence according to the EU numbering system.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the one or more amino acid substitutions are at least one selected from S239D, L235V, F243L, R292P, Y300L, A330L, I332E and P396L according to the EU numbering system.

16. The antibody or antigen-binding fragment thereof of claim 10, wherein the heavy chain constant region has one or more sets of mutations occurring simultaneously at a combination of positions selected from the group consisting of: (1) L235 / F243 / R292 / Y300 / P396, (2) F243 / R292 / Y300 / V305 / P396, (3) D270 / K326 / A330 / K3 34, (4) S239 / A330 / I332, (5) S298 / E333 / K334, (6) L234 / L235 / G236 / S239 / H268 / D270 / S298, (7) M252 / S254 / T256, (8) L234 / L235 / D265, (9) G236 / S239 / I332, and (10) S239 / I332.

17. The antibody or antigen-binding fragment thereof of claim 10, wherein the heavy chain constant region has one or more mutations selected from the following combinations of mutations according to the EU numbering system: (1) L235V / F243L / R292P / Y300L / P396L, (2) F243L / R292P / Y300L / V305I / P396L, (3) D270E / K326D / A330M / K334E, (4) S239D / A330L / I336 32E, (5)S298A / E333A / K334A, (6)L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, (7)M252Y / S254T / T256E, (8)L234A / L235A / D265A, (9)L234F / L235E / D265A, (10)G236A / S239D / I332E and (11)S239D / I332E.

18. An antibody or antigen-binding fragment thereof that binds to CCR8, comprising a heavy chain (HC) and a light chain (LC) as shown in any combination of the following tables:

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the constant region of the antibody or antigen-binding fragment thereof is afucosylated or has reduced fucosylation.

20. The CCR8-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 19, wherein the antibody is a monoclonal antibody.

21. The antibody or antigen-binding fragment thereof of any one of claims 1 to 19, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fd, Fab'-SH, Fv, single-chain antibody (e.g., scFv) or (Fab')2, single domain antibody, diabody (dAb) or linear antibody.

22. An isolated polynucleotide encoding any one or more chains of the CCR8-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 21.

23. A vector comprising the polynucleotide of claim 22, preferably said vector is an expression vector.

24. A host cell comprising the nucleic acid of claim 22 or the vector of claim 23, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., the host cell is a CHO cell, such as a CHO-K1 cell or an expiCHO cell, or the host cell is a 293 cell, such as a HEK293 cell) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

25. A method for preparing an antibody or antigen-binding fragment thereof that binds to CCR8, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 21 under conditions suitable for expression of the antibody, and optionally further comprising recovering the antibody or antigen-binding fragment thereof from the host cell.

26. An immunoconjugate comprising the CCR8-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 21 and other substances, such as therapeutic agents or labels.

27. A pharmaceutical composition comprising the CCR8-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 21 or the immunoconjugate according to claim 20, and optionally one or more other therapeutic agents, and optionally pharmaceutically acceptable excipients.

28. A pharmaceutical combination comprising the CCR8-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 21 or the immunoconjugate according to claim 20, and one or more other therapeutic agents, preferably, the other therapeutic agent is a monoclonal antibody, more preferably, the other therapeutic agent is a monoclonal antibody targeting an immune checkpoint, and most preferably, the other therapeutic agent is a monoclonal antibody targeting PD-1.

29. A method of enhancing an immune response (e.g., an anti-tumor immune response) in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 21, or the polynucleotide of claim 22, or the vector of claim 23, or the host cell of claim 24, or the immunoconjugate of claim 26, or the pharmaceutical composition of claim 27, or the combination product of claim 28.

30. A method for preventing and / or treating a tumor, an autoimmune disease or an infectious disease in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, or the polynucleotide according to claim 22, or the vector according to claim 23, or the host cell according to claim 24, or the immunoconjugate according to claim 26, or the pharmaceutical composition according to claim 27, or the combination product according to claim 28, preferably, the disease is a tumor, such as melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer and metastatic cancer thereof.

31. The method of claim 30, further comprising co-administering one or more therapies, such as treatment modalities and / or other therapeutic agents, to the subject, preferably, treatment modalities include surgery and / or radiation therapy.

32. Use of an effective amount of the anti-CCR8 antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, or the polynucleotide according to claim 22, or the vector according to claim 23, or the host cell according to claim 24, or the immunoconjugate according to claim 26, or the pharmaceutical composition according to claim 27, or the pharmaceutical combination according to claim 28 in the preparation of a medicament for preventing and / or treating tumors, autoimmune diseases or infectious diseases in a subject.

33. Use of an effective amount of the anti-CCR8 antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, or the polynucleotide according to claim 22, or the vector according to claim 23, or the host cell according to claim 24, or the immunoconjugate according to claim 26, or the pharmaceutical composition according to claim 27, in combination with other therapeutic agents in the preparation of a medicament for preventing and / or treating tumors, autoimmune diseases, or infectious diseases in a subject. Preferably, the other therapeutic agent is a monoclonal antibody, more preferably, the other therapeutic agent is a monoclonal antibody targeting an immune checkpoint, and most preferably, the other therapeutic agent is a monoclonal antibody targeting PD-1.

34. The use of claim 32 or 33, wherein the tumor is a solid tumor.

35. The use of claim 34, wherein the tumor is melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, lung cancer, ovarian cancer, kidney cancer, bladder cancer, liver cancer or thymic cancer and metastatic cancer thereof.

36. The use of any one of claims 32-35, further comprising co-administering one or more other therapies to the subject.

37. The use of claim 36, wherein the therapy comprises a treatment modality and / or other therapeutic agents.

38. A method for detecting CCR8 in a sample, comprising the step of using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 21 or the immunoconjugate according to claim 26.

39. A detection kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 21 or the immunoconjugate according to claim 26.

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