Anti-CCR8 antibodies and uses thereof

Abstract

The present invention provides an antibody that targets CCR8 or an antigen-binding fragment thereof, and uses thereof. The antibody that targets CCR8 or an antigen-binding fragment thereof specifically binds to CCR8. Furthermore, the present invention provides uses of the antibody to inhibit the binding of CCL1 to CCR8, inhibit the signaling pathway induced thereby, reduce CCR8-positive Treg cells through the ADCC effect, and enhance anti-tumor immunity.

Description

[Technical Field]

[0001] The present invention relates to the field of monoclonal antibodies and / or modified antibodies. In particular, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CCR8, and compositions containing the same. Furthermore, the present invention also provides 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 thereof using the antibodies or antigen-binding fragments thereof of the present invention. [Background technology]

[0002] The immune system in the body is a core defense against tumor development and progression, and the inability of the immune system to recognize and eliminate malignant cells plays a key role in the pathogenesis of cancer. In the tumor microenvironment, high expression of multiple immune checkpoints reduces immune activation and suppresses antitumor immunity. Monoclonal antibodies against the immune suppressive checkpoints CTLA-4 and PD-1 / PD-L1 exhibit remarkable antitumor effects by enhancing immune activation in the tumor microenvironment. Currently, many monoclonal antibodies against CTLA-4 and PD-1 / PD-L1, including the anti-CTLA-4 antibody ipilimumab, the anti-PD-1 antibodies nivolumab and pembrolizumab, and the anti-PD-L1 antibody atezolizumab, have been clinically approved for the treatment of various solid tumors. Compared to conventional treatments (such as chemotherapy, radiation therapy, and surgery), cancer immunotherapy has significantly improved patient survival and quality of life (K Esfahaniwait, 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] Immune checkpoint inhibitors are clinically approved for the treatment of various solid tumors, but response rates vary significantly across tumor types, with overall response rates low. The overall response rate (ORR) of immune checkpoint inhibitors in non-small cell lung cancer (NSCLC), urothelial cancer, and head and neck cancer is only around 20%, and in solid tumors such as gastric cancer, it remains below 10% (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). While some patients experience durable responses to immune checkpoint inhibitors, many patients who initially respond subsequently develop acquired resistance. For example, 50% of patients with NSCLC and gastric cancer develop acquired resistance (Schoenfeld AJ and Hellmann MD). Unfortunately, some patients with solid tumors not only fail to benefit from immune checkpoint inhibitors but also experience hyperprogression. Tumor hyperprogression occurs at different rates in patients with various solid tumors after immune checkpoint therapy. For example, 14% of NSCLC patients experience 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). The development of new immune checkpoint inhibitors or immunomodulators to complement or overcome the shortcomings and limitations of currently used immune checkpoint inhibitors in clinical practice remains a key challenge.

[0004] The overall low response rate to immune checkpoint inhibitors and the development of acquired resistance and hyperprogression are associated with multiple mechanisms in tumor cell signaling pathways and the tumor microenvironment (Baxter MA et al., Resistance to immune checkpoint inhibitors in advanced gastroesophageal cancers. Br J Cancer. 2021 Oct; 125(8): 1068-1079. doi: 10.1038 / s41416-021-01425-7). Regulatory T cells (Tregs) play a key role in immune regulation and are closely associated with tumor initiation and progression. Compared with the periphery, Treg cells are more abundant and more functional in the tumor microenvironment. Tregs suppress antitumor immunity through mechanisms such as secretion of inhibitory cytokines such as IL-10, IL-2 depletion, and direct contact suppression, and are closely associated with intrinsic and acquired resistance to immune checkpoint inhibitors (Baxter MA et al.). PD-1 blockade induces the recovery of dysfunctional PD-1+ CD8+ T cells but may also enhance immune suppression by PD-1+ Treg cells. If the number of PD-1-expressing Treg cells exceeds the number of PD-1+ CD8+ T cells in the tumor microenvironment, PD-1 blockade may lead to tumor hyperprogression (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). Studies in preclinical animal models and clinical settings have shown that elimination of Treg cells in the tumor microenvironment promotes antitumor immunity and enhances the therapeutic efficacy of PD-1 / PD-L1 blockade. Clinically, the combination of the anti-CTLA-4 antibody ipilimumab and the anti-PD-1 antibody nivolumab has been shown to have a survival benefit in patients with melanoma and renal cell carcinoma.

[0005] Treg-depleting antibodies currently under clinical development, including antibodies against targets such as CTLA-4, CD25, the costimulator OX40, and GITR, have shown 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 Treg cells and effector T cells, meaning that simultaneous depletion of peripheral Treg cells and effector T cells reduces clinical efficacy and increases toxicity, limiting their clinical application. In recent years, it has been revealed that the chemokine receptor CCR8 is highly expressed on Treg cells in human tumors (such as breast cancer, non-small cell lung cancer, colorectal cancer, melanoma, hepatocellular carcinoma, and pancreatic ductal adenocarcinoma), but is expressed at low levels or absent on Treg cells 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 expression of CCR8 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 more strongly suppress 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, CCR8 antibodies have been shown to specifically eliminate Treg cells in the tumor microenvironment, suppress tumor growth, and exhibit synergistic effects with antibodies against PD-1 (US Patent Application No. US20190071508 and Van Damme H. et al., Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits anti-tumor 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 to human CCR8 is expected to enhance the therapeutic effects of immune checkpoints in clinical settings and have important application value. Summary of the Invention

[0006] The present invention provides an anti-human CCR8-specific antibody obtained by immunizing mice with cells overexpressing human CCR8 and a nucleic acid encoding CCR8. The anti-CCR8 antibody has high affinity for human CCR8 and monkey CCR8, inhibits the binding of CCL1 to CCR8 and the signal transduction pathway induced thereby, and effectively suppresses tumor growth in various in vivo mouse models (e.g., models of colorectal cancer, lung cancer, breast cancer, melanoma, etc.) by eliminating CCR8-positive cells (e.g., Treg cells) through the ADCC effect.

[0007] According to one aspect, the present invention provides an anti-CCR8 antibody or antigen-binding fragment, comprising: (i) three complementarity-determining regions HCDR1, HCDR2, and HCDR3 of a heavy chain variable region set forth in any of SEQ ID NOs: 21 to 40, and / or three complementarity-determining regions LCDR1, LCDR2, and LCDR3 of a light chain variable region set forth in any of SEQ ID NOs: 42 to 54; (ii) A sequence in which the CDR combination according to (i) further comprises 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), preferably amino acid substitutions, and preferably conservative amino acid substitutions, compared with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3, and the affinity to CCR8 is maintained. The present invention provides an anti-CCR8 antibody or antigen-binding fragment comprising:

[0008] According to one aspect, the present invention provides a CCR8-binding antibody or antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising: (1) HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, 10, 11, or 41, and SEQ ID NO: 3, respectively; or HCDR1, HCDR2, and HCDR3 that are identical to, or have at least one amino acid addition, substitution, or deletion, or any combination thereof (e.g., one, two, or three amino acid additions, substitutions, or deletions, or any combination thereof) when compared to, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, 10, 11, or 41, and SEQ ID NO: 3, respectively; or (2) HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or HCDR1, HCDR2, and HCDR3 that are identical to, or have at least one amino acid addition, substitution, or deletion, or any combination thereof (e.g., one, two, or three amino acid additions, substitutions, or deletions, or any combination thereof) when compared to the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or (3) HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; or HCDR1, HCDR2, and HCDR3 that are identical to, or have at least one amino acid addition, substitution, or deletion, or any combination thereof (e.g., 1, 2, or 3 amino acid addition, substitution, or deletion, or any combination thereof) when compared to the amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; Including, and / or the light chain variable region comprising: (1) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; or LCDR1, LCDR2, and LCDR3 that are identical to, or have at least one amino acid addition, substitution, or deletion, or any combination thereof (e.g., one, two, or three amino acid additions, substitutions, or deletions, or any combination thereof) compared to, the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; or (2) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; or LCDR1, LCDR2, and LCDR3 that are identical to, or have at least one amino acid addition, substitution, or deletion, or any combination thereof (e.g., one, two, or three amino acid additions, substitutions, or deletions, or any combination thereof) when compared to the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; or (3) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth 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 set forth in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, or that have at least one amino acid addition, substitution, or deletion, or any combination thereof (e.g., 1, 2, or 3 amino acid addition, substitution, or deletion, or any combination thereof) when compared to the amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively. The present invention provides an anti-CCR8 antibody or antigen-binding fragment comprising:

[0009] According to one aspect, the present invention provides a composition comprising: [Table 1] The present invention provides a CCR8-binding antibody or an antigen-binding fragment thereof comprising any one of the combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 shown in

[0010] According to one aspect, the present invention provides a CCR8-binding antibody or antigen-binding fragment thereof comprising a heavy chain variable region VH and / or a light chain variable region VL, (a) the heavy chain variable region VH is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the amino acid sequences selected from SEQ ID NOs: 21 to 40; or (ii) comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 21 to 40; or (iii) 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 any one of the amino acid sequences selected from SEQ ID NOs: 21 to 40, wherein preferably, the amino acid changes occur within the CDRs, and preferably, the amino acid changes do not occur within the framework regions (FRs); and / or (b) the light chain variable region VL is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the amino acid sequences selected from SEQ ID NOs: 42 to 54; (ii) comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 42 to 54; or (iii) 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 any one of the amino acid sequences selected from SEQ ID NOs: 42 to 54, wherein preferably, the amino acid changes do not occur within the CDRs, and preferably, the amino acid changes do not occur within the FRs. CCR8-binding antibodies or antigen-binding fragments thereof are provided.

[0011] According to one aspect, the present invention provides a method for producing a compound according to the following table: [Table 2] The present invention provides a CCR8-binding antibody or an antigen-binding fragment thereof, which comprises any one of the combinations of heavy chain variable region VH and light chain variable region VL described in the above.

[0012] According to one aspect, the present invention provides an anti-CCR8 antibody or antigen-binding fragment comprising a heavy chain and / or a light chain, (a) the heavy chain is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the amino acid sequences selected from SEQ ID NOs: 55 to 74; (ii) comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 55 to 74; or (iii) 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 any one of the amino acid sequences selected from SEQ ID NOs: 55 to 74, wherein preferably, the amino acid change does not occur within the CDR of the heavy chain variable region, more preferably, the amino acid change does not occur within the heavy chain variable region, and most preferably, the amino acid change occurs within the heavy chain constant region; and / or (b) the light chain is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the amino acid sequences selected from SEQ ID NOs: 75 to 82; (ii) comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 75 to 82; or (iii) 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 any one of the amino acid sequences selected from SEQ ID NOs: 75 to 82, wherein preferably, the amino acid change does not occur within the CDR of the light chain variable region, preferably, the amino acid change does not occur within the light chain variable region, and most preferably, the amino acid change occurs within the light chain constant region. Anti-CCR8 antibodies or antigen-binding fragments are provided.

[0013] According to one aspect, the present invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region and / or a light chain constant region. Preferably, the light chain constant region is a λ or κ chain constant region, and the heavy chain constant region is selected from mouse mIgG2a, human IgG1, human IgG2, human IgG3, or IgG4 subclasses. According to a preferred aspect, the heavy chain constant region is a constant region of the human IgG1 subclass or a human IgG4 subclass having an S228P mutation.

[0014] According to one aspect, there is provided an antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, the sequence of which has one or more amino acid substitutions compared to the sequence of a native human heavy chain constant region, and preferably, the one or more amino acid substitutions are substitutions that enhance the ADCC activity of the antibody. According to a preferred aspect, the one or more amino acid substitutions are at the following positions in the sequence of the heavy chain constant region, as numbered according to the EU numbering system: 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, Occurs in one or more of 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. In a preferred embodiment, the one or more amino acid substitutions occur at one or more of the following positions in the heavy chain constant region sequence, as numbered according to the EU numbering system: L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339, and P396. In a preferred embodiment, the one or more amino acid substitutions are one or more selected from the following substitutions numbered according to the EU numbering system: G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L.In a preferred embodiment, the one or more amino acid substitutions are selected from one or more of the following substitutions numbered according to the EU numbering system: N297A substitution, N297Q substitution, L235A and L237A substitution, L234A and L235A substitution, E233P substitution, L234V substitution, L235A substitution, C236 deletion, P238A substitution, D265A substitution, A327Q substitution, and P329A substitution. In a preferred embodiment, the one or more amino acid substitutions occur in one or more of the following positions numbered according to the EU numbering system in the sequence of the heavy chain constant region: 235, 239, 243, 292, 300, 330, 332, and 396. In one preferred embodiment, the at least one amino acid substitution is at least one selected from the following substitutions numbered according to the EU numbering system: S239D, L235V, F243L, R292P, Y300L, A330L, I332E, and P396L.

[0015] In a preferred embodiment, the heavy chain constant region contains the following sets of simultaneous mutations, numbered according to the EU numbering system: (1) L235 / F243 / R292 / Y300 / P396, (2) F243 / R292 / Y300 / V305 / P396, (3) D270 / K326 / A330 / K334, and (4) S239 / A330 / I333. 2, (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.

[0016] In a preferred embodiment, the heavy chain constant region contains one of the following mutation sets numbered 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 / E333 (5) L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, (6) M252Y / S254T / T256E, (7) L234A / L235A / D265A, (8) L234F / L235E / D265A, (9) G236A / S239D / I332E, and (11) S239D / I332E.

[0017] According to one embodiment, the following table: [Table 3] The present invention provides a CCR8-binding antibody or an antigen-binding fragment thereof, which comprises any one of the combinations of heavy chain (HC) and light chain (LC) described above.

[0018] In certain aspects, the constant regions of the antibodies of the invention are afucosylated or hypofucosylated.

[0019] According to one embodiment, the antibody of the present invention is a monoclonal antibody.

[0020] In certain aspects, the antibodies of the present invention are murine, chimeric, humanized, or human antibodies.

[0021] In some embodiments, antigen-binding fragments of the invention include the following antibody fragments: Fab, Fab', Fd, Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies, diabodies (dAbs), or linear antibodies.

[0022] According to another aspect, the present invention provides an isolated anti-CCR8 antibody or antigen-binding fragment thereof, comprising: (1) binds to an epitope of the human CCR8 protein that is identical to or completely or partially overlaps with any of the anti-CCR8 antibodies or antigen-binding fragments thereof of the present invention; (2) competes with any of the anti-CCR8 antibodies of the present invention or antigen-binding fragments thereof for binding to an epitope of human CCR8 protein; (3) exhibits the same or similar binding affinity and / or specificity for CCR8 as the antibodies of the present invention; (4) having one or more biological characteristics of the antibody of the present invention; The present invention provides an isolated anti-CCR8 antibody or antigen-binding fragment thereof having one or more of the following:

[0023] According to one aspect, there is provided an antibody or antigen-binding fragment thereof of the invention, comprising: (1) Binds to human / cynomolgus monkey CCR8 with high affinity, e.g., EC 50 value less than 1000 ng / mL, EC 50 value less than 950 ng / mL, EC 50 value less than 900 ng / mL, EC 50 value less than 850 ng / mL, EC 50 value less than 800 ng / mL, EC 50 value less than 750 ng / mL, EC 50 value less than 700 ng / mL, EC 50 value less than 650 ng / mL, EC 50 value less than 600 ng / mL, EC 50 value less than 550 ng / mL, EC 50 value less than 500 ng / mL, EC 50 value less than 450 ng / mL, EC 50 value less than 400 ng / mL, EC 50 value less than 350 ng / mL, EC 50 value less than 300 ng / mL, EC 50 value less than 250 ng / mL, EC 50 value less than 200 ng / mL, EC 50 value less than 180 ng / mL, EC 50 value less than 160 ng / mL, EC 50 value less than 150 ng / mL, EC 50 value less than 140 ng / mL, EC 50value less than 130 ng / mL, EC 50 value less than 120 ng / mL, EC 50 value less than 110 ng / mL, EC 50 Value less than 100 ng / mL, EC 50 value less than 95 ng / mL, EC 50 value less than 90 ng / mL, EC 50 value less than 85 ng / mL, EC 50 value less than 80 ng / mL, EC 50 value <75 ng / mL, EC 50 value less than 70 ng / mL, EC 50 value less than 65 ng / mL, EC 50 value less than 60 ng / mL, EC 50 value less than 55 ng / mL, EC 50 value less than 50 ng / mL, EC 50 value less than 45 ng / mL, EC 50 value less than 40 ng / mL, EC 50 value less than 35 ng / mL, EC 50 value less than 30 ng / mL, EC 50 value less than 25 ng / mL, EC 50 value less than 20 ng / mL, EC 50 value less than 18 ng / mL, EC 50 value less than 16 ng / mL, EC 50 value less than 15 ng / mL, EC 50 value less than 14 ng / mL, EC 50 value less than 13 ng / mL, EC 50 value less than 12 ng / mL, EC 50 value less than 11 ng / mL, EC 50 Value less than 10 ng / mL, EC 50 value less than 8 ng / mL, EC 50 value less than 6 ng / mL, EC 50 value less than 4 ng / mL, EC 50 Value less than 2 ng / mL, EC 50 EC values ​​less than 1 ng / mL or even lower 50 Indicate the value, (2) Inhibit the binding of human / cynomolgus monkey CCR8 to its ligand (e.g., CCL1), e.g., IC 50 Value less than 1000ng / mL, IC 50 value less than 950 ng / mL, IC 50value less than 900 ng / mL, IC 50 value less than 850 ng / mL, IC 50 value less than 800 ng / mL, IC 50 value less than 750 ng / mL, IC 50 value less than 700 ng / mL, IC 50 value less than 650 ng / mL, IC 50 value less than 600 ng / mL, IC 50 value less than 550 ng / mL, IC 50 value less than 500 ng / mL, IC 50 value less than 450 ng / mL, IC 50 value less than 400 ng / mL, IC 50 value less than 350 ng / mL, IC 50 value less than 300 ng / mL, IC 50 value less than 250 ng / mL, IC 50 value less than 200 ng / mL, IC 50 value less than 180 ng / mL, IC 50 value less than 160 ng / mL, IC 50 value less than 150 ng / mL, IC 50 value less than 140 ng / mL, IC 50 value less than 130 ng / mL, IC 50 value less than 120 ng / mL, IC 50 value less than 110 ng / mL, IC 50 Values ​​less than 100 ng / mL, IC 50 Values ​​less than 95 ng / mL, IC 50 value less than 90 ng / mL, IC 50 value less than 85 ng / mL, IC 50 value less than 80 ng / mL, IC 50 value less than 75 ng / mL, IC 50 value less than 70 ng / mL, IC 50 value less than 65 ng / mL, IC 50 value less than 60 ng / mL, IC 50 value less than 55 ng / mL, IC 50 value less than 50 ng / mL, IC 50 value less than 45 ng / mL, IC 50 value less than 40 ng / mL, IC 50 value less than 35 ng / mL, IC 50 value less than 30 ng / mL, IC 50 value less than 25 ng / mL, IC50 value less than 20 ng / mL, IC 50 value less than 18 ng / mL, IC 50 value less than 16 ng / mL, IC 50 value less than 15 ng / mL, IC 50 value less than 14 ng / mL, IC 50 value less than 13 ng / mL, IC 50 value less than 12 ng / mL, IC 50 value less than 11 ng / mL, IC 50 value less than 10 ng / mL, IC 50 Value less than 8ng / mL, IC 50 Value less than 6ng / mL, IC 50 Value less than 4ng / mL, IC 50 Value less than 2 ng / mL, IC 50 IC values ​​less than 1 ng / mL or even lower 50 Indicate the value, (3) inhibit CCL1-induced β-arrestin recruitment, e.g., IC 50 Value less than 10,000 ng / mL, IC 50 value less than 9500 ng / mL, IC 50 value less than 9000ng / mL, IC 50 value less than 8500ng / mL, IC 50 value less than 8000ng / mL, IC 50 value less than 7500 ng / mL, IC 50 value less than 7000ng / mL, IC 50 value less than 6500 ng / mL, IC 50 value less than 6000 ng / mL, IC 50 value less than 5500 ng / mL, IC 50 value less than 5000 ng / mL, IC 50 value less than 4500 ng / mL, IC 50 value less than 4000ng / mL, IC 50 value less than 3500 ng / mL, IC 50 value less than 3000 ng / mL, IC 50 value less than 2500 ng / mL, IC 50 value less than 2000 ng / mL, IC 50 value less than 1800 ng / mL, IC 50 value less than 1600 ng / mL, IC 50 value less than 1500 ng / mL, IC50 value less than 1400 ng / mL, IC 50 value less than 1300 ng / mL, IC 50 value less than 1200 ng / mL, IC 50 value less than 1100 ng / mL, IC 50 Value less than 1000ng / mL, IC 50 value less than 950 ng / mL, IC 50 value less than 900 ng / mL, IC 50 value less than 850 ng / mL, IC 50 value less than 800 ng / mL, IC 50 value less than 750 ng / mL, IC 50 value less than 700 ng / mL, IC 50 value less than 650 ng / mL, IC 50 value less than 600 ng / mL, IC 50 value less than 550 ng / mL, IC 50 value less than 500 ng / mL, IC 50 value less than 450 ng / mL, IC 50 value less than 400 ng / mL, IC 50 value less than 350 ng / mL, IC 50 value less than 300 ng / mL, IC 50 value less than 250 ng / mL, IC 50 value less than 200 ng / mL, IC 50 value less than 180 ng / mL, IC 50 value less than 160 ng / mL, IC 50 value less than 150 ng / mL, IC 50 value less than 140 ng / mL, IC 50 value less than 130 ng / mL, IC 50 value less than 120 ng / mL, IC 50 value less than 110 ng / mL, IC 50 Values ​​less than 100 ng / mL, IC 50 value less than 80 ng / mL, IC 50 value less than 60 ng / mL, IC 50 value less than 40 ng / mL, IC 50 value less than 20 ng / mL, IC 50 IC values ​​less than 10 ng / mL or even lower 50 Indicate the value, Also provided are antibodies or antigen-binding fragments thereof having one or more of:

[0024] The present invention also provides multispecific antibodies comprising the light chain variable region and / or the heavy chain variable region of an antibody or antigen-binding fragment thereof described in this disclosure.

[0025] The present invention also provides single chain antibodies comprising the light chain variable region and the heavy chain variable region of an antibody or antigen-binding fragment thereof described in this disclosure.

[0026] The present invention also provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof described in this disclosure and a therapeutic or diagnostic agent conjugated thereto.

[0027] According to yet another aspect, the present invention provides a polynucleotide molecule encoding an anti-CCR8 antibody or any fragment thereof according to the present disclosure.

[0028] According to another aspect, the present invention provides an expression vector comprising a polynucleotide molecule according to the present invention, wherein said vector is preferably a eukaryotic expression vector.

[0029] According to another aspect, the present invention provides a host cell comprising a polynucleotide molecule according to the invention or an expression vector according to the invention. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0030] According to yet another aspect, the present invention provides a method for preparing an anti-CCR8 antibody or antigen-binding fragment thereof described herein, comprising expressing the antibody or antigen-binding fragment thereof described herein in a host cell under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0031] According to yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-CCR8 antibody or antigen-binding fragment thereof, a polynucleotide, a vector, a host cell, an immunoconjugate described herein, and optionally at least one pharmaceutically acceptable carrier or excipient.

[0032] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, or pharmaceutical composition described herein and one or more additional therapeutic agents. According to one embodiment, the additional therapeutic agent is a chemotherapeutic agent. According to one embodiment, the additional therapeutic agent is another antibody. According to one embodiment, the additional therapeutic agent is another monoclonal antibody. According to a preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets an immune checkpoint. According to a more preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets PD-1. According to a more preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets PD-L1. According to a more preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets CTLA-4.

[0033] According to yet another aspect, the present invention provides a method for depleting CCR8-positive Treg cells, the method comprising exposing an anti-CCR8 antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, or pharmaceutical composition described herein to a cell population containing CCR8-positive Treg cells or administering the same to the body of a subject, in vitro or in vivo.

[0034] According to yet another aspect, the present invention provides use of an antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, pharmaceutical composition, or pharmaceutical combination described herein in the preparation of a medicament for treating and / or preventing a tumor, autoimmune or infectious disease, 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.

[0035] According to one aspect, the present invention provides use of an antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, or pharmaceutical composition described herein in combination with another therapeutic agent in the preparation of a medicament for treating and / or preventing a tumor, autoimmune disease, or infectious disease. 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. According to one aspect, the additional therapeutic agent is a chemotherapeutic agent. According to one aspect, the additional therapeutic agent is another antibody. According to one aspect, the additional therapeutic agent is another monoclonal antibody. According to one preferred aspect, the additional therapeutic agent is a monoclonal antibody targeting an immune checkpoint. According to a more preferred aspect, the additional therapeutic agent is a monoclonal antibody targeting PD-1. According to an even more preferred aspect, the additional therapeutic agent is a monoclonal antibody targeting PD-L1. In a more preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets CTLA4.

[0036] According to yet another aspect, the present invention provides use of an antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, pharmaceutical composition, or pharmaceutical combination described herein for the treatment and / or prevention of tumor, autoimmune or infectious disease, 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.

[0037] According to yet another aspect, the present invention provides a method for treating and / or preventing a tumor, autoimmune disease, or infectious disease, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, pharmaceutical composition, or pharmacological combination described herein, 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.

[0038] According to yet another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, immunoconjugate, pharmaceutical composition, or pharmaceutical combination described herein, and preferably further comprising a drug delivery device.

[0039] According to another aspect, the present invention provides a method for detecting the presence of CCR8 in a sample using an antibody, antigen-binding fragment thereof, or immunoconjugate described herein, or a detection composition comprising said antibody, antigen-binding fragment thereof, or immunoconjugate. [Brief explanation of the drawings]

[0040] [Figure 1A-B] Figure 1 shows the binding activity of the anti-CCR8 antibodies of the present invention to human CCR8. A to D are binding curves for 293F-hCCR8 of antibodies purified and quantified from monoclonal hybridoma cell culture supernatants. [Figure 1C-D] Same as above. [Figure 2] Figure 2 shows the binding activity of the anti-CCR8 antibody of the present invention to cynomolgus monkey CCR8. AB are binding curves of the antibody purified and quantified from the monoclonal hybridoma cell culture supernatant to 293T-cynoCCR8. [Figure 3]Figure 3. Several antibody clones of the present invention are shown to have the ability to inhibit the binding of CCL1 to CCR8. (A-C) Inhibition curves showing that antibodies purified and quantified from monoclonal hybridoma cell culture supernatants blocked the binding of 10 nM AlexaFluor-647-labeled human CCL1 to 293F-hCCR8. [Figure 4] Figure 4 shows the binding activity of anti-CCR8 chimeric antibodies of the present invention to human CCR8. Lines A to B and C are binding curves of hIgG1- and mIgG2a-subclass chimeric antibodies to 293F-hCCR8. [Figure 5] Figure 5 shows the binding activity of anti-CCR8 chimeric antibodies of the present invention to cynomolgus monkey CCR8. A and B are binding curves of hIgG1- and mIgG2a-subclass chimeric antibodies to 293T-cynoCCR8, respectively. [Figure 6] Figure 6 shows the activity of anti-CCR8 chimeric antibodies of the present invention to inhibit binding to CCL1. A to B and C are curves showing that hIgG1- and mIgG2a-subclass chimeric antibodies inhibited the binding of 10 nM AlexaFluor-647-labeled human CCL1 to 293F-hCCR8, respectively. [Figure 7] Figure 7 shows that CCL1-induced β-arrestin recruitment was inhibited by the CCR8 chimeric antibody of the present invention. A and B are curves showing that human CCL1-induced β-arrestin recruitment was inhibited by hIgG1- and mIgG2a-subclass chimeric antibodies, respectively. [Figure 8A-D] Figure 8 shows the binding activity of humanized anti-CCR8 antibodies of the present invention to human CCR8. A to B, C to D, and E to G are binding curves of humanized antibodies derived from clones 27B9-1G3, 559E1B10, and 563E10E12 to 293F-hCCR8, respectively. [Figure 8E-G] Same as above. [Figure 9]Figure 9 shows the binding activity of anti-CCR8 humanized antibodies of the present invention to cynomolgus monkey CCR8. Binding curves of 559E1B10- and 563E10E12-derived humanized antibodies to 293T-cynoCCR8. [Figure 10A-C] Figure 10 shows inhibition of CCL1-binding activity by humanized anti-CCR8 antibodies of the present invention. A to B and C are curves showing that binding of 10 nM AlexaFluor-647-labeled human CCL1 to 293F-hCCR8 was inhibited by humanized antibodies derived from 27B9-1G3, 559E1B10, and 563E10E12, respectively. [Figure 10D-G] Same as above. [Figure 11A-C] Figure 11. Experiments demonstrating that CCL1-induced β-arrestin recruitment is inhibited by the anti-CCR8 humanized antibodies of the present invention. A-B and C-E are curves showing that human CCL1-induced β-arrestin recruitment was inhibited by the 559E1B10- and 563E10E12-derived humanized antibodies, respectively. [Figure 11D-E] Same as above. [Figure 12] FIG. 12. Chimeric and humanized antibodies derived from 559E1B10 have the ability to activate Jurkat-human FcγRIIIa(158V)-NFAT in a dose-dependent manner. [Figure 13] FIG. 13. Chimeric and humanized antibodies derived from 563E10E12 have the ability to activate Jurkat-human FcγRIIIa(158V)-NFAT in a dose-dependent manner. [Figure 14] Figure 14 shows the binding activity of the anti-CCR8 antibodies of the present invention to HuT78. [Figure 15] Figure 15 shows the specific binding of the anti-CCR8 antibodies of the present invention to hCCR8. Binding activity of the anti-CCR8 antibodies to CHOK1-hCCR8 (A) and CHO-K1 (B) cells. [Figure 16]Figure 16 shows the CCR8-dependent activation of Jurkat-human FcγRIIIa(158V)-NFAT by the anti-CCR8 antibody of the present invention. The target cells corresponding to panels A and B are CHOK1-hCCR8 and CHOK1-blank, respectively. [Figure 17] Figure 17 shows the binding activity of the anti-CCR8 antibody of the present invention to 293F cells transfected with hCCR4. Binding of the anti-CCR8 antibody to 293F cells transfected with hCCR4-GFP (A, GFP-positive cells) and blank 293F cells (B, total viable cells). [Figure 18] Figure 18 shows activation of Jurkat-human FcγRIIIa(158V)-NFAT by incubation of Fc mutant or defucosylated humanized antibodies derived from clones 559E1B10 and 563E10E12 with 293F-human CCR8. Panels A to C show activation of Jurkat-human FcγRIIIa(158V)-NFAT by incubation of Fc mutant or defucosylated antibodies 559E1B10_hzH1L1 (A), 563E10E12_hzH1L1_hIgG1 (B), and 563E10E12_hzH1L0_hIgG1 (C), respectively, with 293F-human CCR8. [Figure 19] Figure 19. Activation of Jurkat-human FcγRIIIa(158V)-NFAT by incubation of Fc variants or defucosylated antibody 563E10E12_hzH1L0 with HuT78. [Figure 20] Figure 20. ADCC induced by healthy human PBMCs and anti-CCR8 antibodies. Healthy human PBMCs were activated with IL-2 and incubated with anti-CCR8 antibodies and CHOK1-hCCR8 cells for 5 hours. The ratio of PI-positive CHOK1-hCCR8 cells was then determined, and the relationship between this ratio and different antibodies and their concentrations was plotted as a curve. [Figure 21]Figure 21 shows the depletion of Treg cells in peripheral PBMCs by anti-CCR8 antibodies of the present invention. (A) The ratio of Foxp3-positive cells among CD4+ T cells and (B) the ratio of CD8+ T cells (CD3+CD4-) among CD3+ T cells after 96 hours of incubation of PBMCs activated with IL-2 and anti-CCR8 antibodies. [Figure 22] Figure 22. Inhibition of MC38 tumor growth by anti-CCR8 antibodies of the invention. CCR8-humanized B-hCCR8 mice were inoculated with MC38 colon cancer cells. Mice were injected subcutaneously with 10 mg / kg of antibody, or a negative control (hIgG1), or an equal volume of vehicle control (PBS). Administration was performed twice weekly. The change in tumor volume is shown in the curves. [Figure 23] Figure 23. Changes in mouse body weight when the MC38 tumor model was treated with the anti-CCR8 antibodies of the present invention. [Figure 24] Figure 24. Inhibition of MC38 tumor growth by Fc variant and defucosylated anti-CCR8 antibodies. CCR8-humanized B-hCCR8 mice were inoculated with MC38 colon cancer cells to induce tumor development. Mice were injected subcutaneously with 10 mg / kg of anti-CCR8 antibody or an equal volume of vehicle control (PBS). Treatment was administered twice weekly. Changes in tumor volume are shown in the curves. [Figure 25] Figure 25. Shows changes in mouse body weight in the MC38 tumor model treated with Fc variant and defucosylated anti-CCR8 antibodies. [Figure 26] Figure 26 shows the inhibition of tumor growth in mouse breast cancer EMT-6 when the anti-CCR8 antibody of the present invention was administered alone or in combination with an anti-mPD-1 antibody. The dose for each group was 5-10 mg / kg of antibody (intravenous administration), anti-mPD-1 antibody (intraperitoneal administration), or an equal volume of vehicle (PBS) (intravenous administration). Administration was performed twice weekly. The curves show the changes in tumor volume. [Figure 27] Figure 27. Shows changes in mouse body weight when the EMT-6 tumor model was treated with the anti-CCR8 antibodies of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0041] definition 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, all of which are within the skill of the art.

[0042] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless expressly defined elsewhere herein, technical and scientific terms used in this disclosure have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. For definitions and terms in the art, experts can refer, at least in part, to "Current Protocols in Molecular Biology (Ausubel)." Abbreviations for amino acid residues follow the three-letter and / or one-letter codes used in the art to represent one of the 20 common L-amino acids. In this disclosure (including the claims), unless otherwise specified, singular forms include the corresponding plural forms.

[0043] The term "about" when used in conjunction with a numerical value is intended to include ranges from a lower limit of 5% less than the stated numerical value to an upper limit of 5% more than the stated numerical value, for example, but not limited to, ±5%, ±2%, ±1%, ±0.1%, as such variations are appropriate for the practice of the disclosed methods.

[0044] The term "and / or" should be construed to mean any one alternative or any two or more alternatives in combination.

[0045] In this disclosure, the term "or" should be interpreted in the same sense as "and / or" as defined above. For example, when separating alternatives in a list, "or" and "and" should be interpreted in an open manner, i.e., to include at least one, but also more than one, of the list of quantities or elements, and, where appropriate, to include additional unlisted items. Only when the term is clearly stated to the contrary, such as "only one" or "indeed one," or when the claim states "composed of ...," will the term refer to only one specified number or element in the list.

[0046] Unless the context clearly indicates otherwise, as used in this disclosure, "1" and "one" should be understood to mean "at least one."

[0047] As used herein, the terms "CCR8," "CC motif chemokine receptor type 8," "chemokine (CC motif) receptor 8," and the like refer to any naturally occurring CCR8 produced by expressing CCR8 in a cell. Unless otherwise specified, the term includes CCR8 derived from any vertebrate, e.g., mammals (e.g., primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats)). See UniProt entry P51685 for an exemplary human CCR8 protein, UniProt entry G7NYJ2 for an exemplary cynomolgus monkey CCR8 protein, and UniProt entry P56484 for an exemplary mouse CCR8 protein.

[0048] Unless otherwise specified, the term "CCR8" includes variants, subtypes, species homologs, and analogs of human CCR8 or CCR8 from other species, including those that share at least one common epitope with CCR8. The term includes full-length unprocessed CCR8 and all intracellularly processed forms of CCR8. The term encompasses not only "full-length unprocessed CCR8" but also all intracellularly processed forms of CCR8 or fragments thereof (e.g., splice variants or allelic variants). In one embodiment, CCR8 refers to full-length CCR8 from human or cynomolgus monkey or a fragment thereof (e.g., a mature fragment lacking a signal peptide).

[0049] In the present disclosure, "CCL1" and "CC motif chemokine ligand 1" refer to naturally occurring CCL1 produced by expression of the CCL1 gene in cells. As the name suggests, CCL1 belongs to the CC chemokine family and is secreted by activated monocytes / macrophages, T lymphocytes, and endothelial cells. Unless otherwise specified, this term includes CCR8 from any vertebrate, such as mammals, including primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats).

[0050] The term "immune response" refers to the selective damage, destruction, or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues, for example, by the action of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (e.g., antibodies, cytokines, complement, etc.) produced by these cells or the liver.

[0051] The terms "Treg" and "regulatory T cells" refer to a type of T cell that exerts an immunosuppressive effect in immune responses and typically suppresses or downregulates the induction and proliferation of effector T cells. Treg cells have complex effects on cancer. Because Treg cells tend to be upregulated in cancer patients and appear to be recruited to tumor sites, many studies have suggested that the presence of Treg cells in the tumor environment is an indicator of poor prognosis. Treg cells are thought to suppress human anti-tumor immunity. Various immunotherapies targeting Treg cells are being investigated as cancer treatment methods. According to one embodiment of the present invention, tumors contain tumor-infiltrating Treg cells. According to one embodiment, tumors contain cells expressing CCR8. According to one embodiment, the CCR8-expressing cells are Treg cells. According to one embodiment of the present invention, effector T cells do not express CCR8 or barely express CCR8. According to one embodiment, the antibodies of the present invention completely / partially suppress / eliminate Treg cells. According to certain aspects, the antibodies of the present invention completely / partially suppress / eliminate Treg cells via the ADCC mechanism and / or inhibit the binding of CCL1 to CCR8, thereby suppressing the CCL1-induced signaling pathway. According to certain aspects, the antibodies of the present invention are used to treat cancer by completely / partially suppressing / eliminating Treg cells. According to certain aspects, the antibodies of the present invention are used to completely / partially suppress / eliminate Treg cells more effectively and / or efficiently than other methods of completely / partially suppressing / eliminating Treg cells. According to certain aspects, the antibodies of the present invention are used to treat cancer by completely / partially inhibiting the binding of CCL1 to CCR8. According to certain aspects, the antibodies of the present invention are used to completely / partially inhibit the binding of CCL1 to CCR8 more effectively and / or efficiently than other methods of inhibiting the binding of CCL1 to CCR8. In more specific embodiments, the effect includes enhanced anti-tumor immunity, enhanced immune response to tumor antigens, delayed tumor growth, reduced tumor size, increased secretion of anti-tumor cytokines, enhanced number or function of tumor-infiltrating T effector cells, enhanced long-term memory effect of anti-tumor immunity, or a combination thereof.In one embodiment, the antibodies of the present invention are used to completely or partially suppress / deplete Treg cells in a manner that has fewer side effects than other methods of suppressing / depleting Treg cells. In a more specific embodiment, the side effect is an immune disorder, e.g., an autoimmune disorder, e.g., an autoimmune disease.

[0052] The terms "signaling pathway" or "signaling activity" refer to a biochemical causal relationship that is typically initiated by a protein-protein interaction, such as the binding of a growth factor to a receptor, e.g., CCL1 (ligand) to CCR8 (receptor), resulting in the transmission of a signal from one part of a cell to another part of the cell. This transmission usually involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a cascade of reactions that leads to signal transduction. The penultimate process often involves nuclear events that lead to changes in gene expression.

[0053] In the present disclosure, the terms "active" or "biological activity" or "biological properties" or "biological characteristics" refer to the affinity and specificity for an epitope / antigen, the ability to neutralize or antagonize the activity of CCR8 in vitro or in vivo, IC 50These include, but are not limited to, antibody specificity, in vivo antibody stability, antibody immunogenicity, etc. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (usually with non-human homologs of the target peptide or with other proteins or tissues) and the ability to maintain high expression levels of the protein in mammalian cells. The above properties or characteristics may be observed, measured, or assessed using techniques known in the art, such as ELISA, FACS, or BIACORE plasmon resonance analysis, unlimited neutralization assays in vitro or in vivo, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections of different origins (including human, primate, and other origins).

[0054] The term "antibody" refers to any form of antibody having the desired biological activity. Thus, when used in the broadest sense, the term 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.

[0055] The term "isolated antibodies" refers to a purified state of the binding compounds, meaning in this case that such molecules are substantially free of nucleic acids, proteins, lipids, sugars, or other substances such as cellular 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 would significantly interfere with the experimental or therapeutic use of the binding compounds described in this disclosure.

[0056] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies target a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically contain many antibodies directed against (or specific for) several different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and does not require that the antibody be produced by any particular method.

[0057] The term "full antibody" refers to a naturally occurring immunoglobulin molecule comprising at least four polypeptide chains (two heavy (H) and two light (L) chains linked by disulfide bonds). Each heavy chain is composed of a heavy chain variable region (abbreviated as VH in this disclosure) and a heavy chain constant region (abbreviated as CH in this disclosure). 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 as VL in this disclosure) 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 hypervariable regions, called complementarity-determining regions (CDRs), and more highly conserved regions, called framework regions (FRs), intervening between them. Each VH or VL region is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and host tissues and factors, such as the first component (C1q) of the classical complement system.

[0058] The term "antigen-binding fragment" of an antibody (a "parent antibody") includes antibody fragments or derivatives, typically including at least a fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody, and fragments that retain at least a portion of the binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., sc-Fv), nanobodies (nanobodies), and multispecific antibodies formed from antibody fragments, all of which are well known in the art. In a preferred embodiment 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 (e.g., sc-Fv), nanobodies formed from antibody fragments, and multispecific antibodies. When antigen-binding activity is expressed in molar concentration, a binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, a 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. Antigen-binding fragments of antibodies are also expected to contain conservative or non-conservative amino acid substitutions (also called "conservative variants" or "functionally conservative variants") that do not significantly alter their biological activity. "Binding compounds" refers to both antibodies and binding fragments thereof.

[0059] A "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 further contains a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0060] A "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the variable region of either the heavy or light chain. In some cases, two or more VH regions may be covalently linked via a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same antigen or different antigens.

[0061] The term "bivalent antibody" contains two antigen-binding sites. In some cases, the two antigen-binding sites may have the same antigen specificity. However, a bivalent antibody may also be bispecific.

[0062] "Diabodies" are small antibody fragments with two antigen-binding sites, comprising a heavy chain variable region (VH) and a light chain variable region (VL) bound to 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, these domains can pair with the complementary domains on another chain to form two antigen-binding sites.

[0063] In the present invention, "murine-derived antibody" or "hybridoma antibody" refers to an anti-CCR8 monoclonal antibody prepared using knowledge and techniques within the field. To prepare such antibodies, a CCR8 antigen is administered to a subject, and hybridomas expressing antibodies with the desired sequence or function are isolated. Hybridoma technology involves fusing two types of cells while retaining their primary characteristics. These two types of cells are mouse spleen cells immunized with an antigen and mouse myeloma cells. Mouse spleen cells (B lymphocytes) primarily secrete antibodies after immunization with a specific antigen, but are characterized by their inability to be continuously cultured outside the body. In contrast, mouse myeloma cells divide and proliferate indefinitely under culture conditions, possessing so-called immortality. Under selective culture media, only hybrid cells obtained by fusing B cells and myeloma cells form cell clones that retain antibody secretion and maintain cellular immortality, and acquire the ability to be continuously cultured. In some embodiments, the present invention is carried out by a method of immunizing mice with CCR8 protein, collecting spleen cells from the immunized mice, and fusing them with myeloma cells to obtain hybridoma cells that express positive antibodies.

[0064] A "chimeric antibody" refers to an antibody having the variable region of a first antibody and the constant region of a second antibody derived from different species. Typically, the variable region is obtained from an antibody of an animal, such as a rodent (the "parent antibody"), while the constant region sequence is obtained from a human antibody. This makes the resulting chimeric antibody less likely to provoke a harmful immune response in a human subject compared to the parent rodent antibody. According to one aspect of the invention, the rodent is a mouse or a rat. According to a preferred aspect of the invention, the affinity of the chimeric antibody for the antigen is not, or is not significantly, reduced compared to the affinity of the parent mouse antibody.

[0065] The term "humanized antibodies" refers to forms of antibodies that contain sequences derived from human and non-human (e.g., murine or rat) antibodies. Generally, humanized antibodies will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops of the variable domain correspond to non-human immunoglobulin sequences and all or substantially all of the framework (FR) regions correspond to human immunoglobulin sequences. Optionally, the humanized antibody will also comprise at least a portion of a human immunoglobulin constant region (Fc).

[0066] The term "fully human antibodies" refers to antibodies that contain only human immunoglobulin protein sequences. Fully human antibodies may contain mouse glycosylation if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, "mouse antibodies" refers to antibodies that contain only mouse immunoglobulin protein sequences. Meanwhile, fully human antibodies may contain rat glycosylation if produced in a rat, a rat cell, or a hybridoma derived from a rat cell. Similarly, "rat antibodies" refers to antibodies that contain only rat immunoglobulin protein sequences.

[0067] Antibody light chains are classified into two types (called kappa (κ) and lambda (λ)). Antibody heavy chains are classified into five major classes, IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant region. Some of these classes are further classified into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. An "isotype" (antibody isotype) refers to the antibody class (IgM, IgE, IgG (IgG1, IgG2, IgG4, etc.)) encoded by the heavy chain constant region gene. Isotypes also include variants of these classes that have been modified to alter Fc function, for example, to enhance or reduce effector function or binding to Fc receptors.

[0068] The term "Fc region" (Fc-region) as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, comprising at least a portion of the constant region. This term includes native Fc regions and variant Fc regions. According to some embodiments, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, with the exception that the C-terminal lysine (Lys447) of the Fc region may or may not be present (note that the numbering in this paragraph is according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0069] The term "effector functions" refers to biological activities mediated by the Fc region of an antibody and vary depending on 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; down-regulation of cell surface receptors; and B-cell activation.

[0070] The term "epitope" refers to a protein determinant that specifically binds to an antibody. Epitopes usually consist of chemically active surface molecules such as amino acids or sugar side chains and have specific three-dimensional structural and charge characteristics. Conformational epitopes are distinguished from nonconformational epitopes in that the former lose their binding in the presence of denaturing solvents, whereas the latter do not.

[0071] As used herein, "cross-reaction" refers to binding to antigen fragments of the same target molecule derived from humans, monkeys, and / or rodents (mouse or rats). Therefore, "cross-reaction" should be understood as an interspecies reaction between an antigen-binding molecule (e.g., an antibody) and a molecule of the same class (e.g., CCR8) expressed in a different species. The cross-reaction specificity of a monoclonal antibody that recognizes human CCR8, monkey CCR8, and / or rodent (mouse or rat) CCR8 can be determined by FACS analysis.

[0072] The term "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 a partner Y is generally determined by the equilibrium dissociation constant (K D ), which is expressed as the dissociation rate constant (k dis ) and the binding rate constant (k on ) The affinity can be measured by known common methods. According to one embodiment of the present invention, the affinity, for example, the affinity between the antibody of the present invention and an antigen, is measured using surface plasmon resonance (SPR) technology.

[0073] "No binding to" a protein or cell means that it does not bind to the protein or cell, or does not bind with high affinity, i.e., the EC 50 is 1.0×10 -7 M or more, preferably 1.0 × 10 -6 M or more, preferably 1.0 × 10 -5 M or more, preferably 1.0 × 10 -4 M or more, preferably 1.0 × 10 -3 This means that it is M or more.

[0074] The term "high affinity" in reference to IgG antibodies refers to the EC 50 is 1.0×10 -7 M or less, preferably 5.0 × 10 -8 M or less, preferably 1.0 × 10-8 M or less, preferably 5.0 × 10 -9 M or less, preferably 1.0 × 10 -9 M or less, preferably 5.0 × 10 -10 M or less, and more preferably 1.0 × 10 -10 For other antibody subtypes, "high affinity" binding can vary. For example, for IgM subtypes, "high affinity" binding means binding with an EC 50 is 10 -7 M or less, preferably 10 -8 M or less, preferably 10 -9 This means that it is less than or equal to M.

[0075] The term "blocking" in relation to IgG antibodies means that administration of the antibody competes with the receptor's natural ligand, inhibiting the binding and interaction between the receptor and the ligand. This inhibition can occur through various mechanisms, such as overlapping of the binding site on the receptor and / or antibody-induced conformational changes in the receptor that alter affinity for the ligand. So-called "functional" antibodies and antibody fragments are characterized by having such properties. In one embodiment, the "blocking" ability refers to the ability to inhibit EC 50 The value is 5.0 x 10 -5 M or less, 1.0×10 -5 M or less, 5.0×10 -6 M or less, 1.0×10 -6 M or less, preferably 5.0 × 10 -7 M or less, preferably 1.0 × 10 -7 M or less, preferably 5.0 × 10 -8 M or less, and more preferably 1.0 × 10 -8 This means that it is less than or equal to M.

[0076] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cellular immune defense in which effector cells of the immune system actively lyse target cells that have antibodies bound to surface antigens on their cell membranes.

[0077] The term "complement-dependent cytotoxicity" or "CDC" refers to the effector functions of IgG and IgM antibodies that result from antibody binding to surface antigens and induction of the classical complement pathway, including the formation of membrane attack complexes and lysis of target cells.

[0078] In this disclosure, the terms "nucleic acids," "polynucleotide," "nucleic acid molecules," and "polynucleotide molecules" are used interchangeably (unless the context dictates otherwise) and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and single- or double-stranded polymers thereof. Unless expressly limited, the terms include nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to natural nucleotides (see U.S. Pat. No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced with pseudouridine, methods for synthesizing such mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses not only the explicitly stated sequence but also its conservatively varied variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. 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)).

[0079] "Construct" refers to any recombinant polynucleotide molecule, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular, single- or double-stranded DNA or RNA polynucleotide molecule, from any source, linked by functional operations (i.e., operative linkage) to one or more polynucleotide molecules, capable of integration into a genome or of autonomous replication. According to certain preferred aspects of the present invention, the recombinant construct includes 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. Expression of the polynucleotide of the present invention can be driven and / or directed by heterologous and non-heterologous (i.e., endogenous) promoters.

[0080] A "vector" refers to any recombinant polynucleotide construct that can be used for transformation (i.e., introducing heterologous DNA into a host cell). One type of vector, a "plasmid," refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector, a viral vector, can ligate additional DNA segments into the viral genome. Moreover, some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell after introduction into the host cell, and thereby are replicated along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to in the present disclosure as "expression vectors."

[0081] In the present disclosure, the term "expression vector" refers to a nucleic acid molecule that is capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. An expression vector contains one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if necessary, amplification within the host. In a preferred embodiment of the present invention, the expression vector of the present invention comprises a construct of the present invention and / or a polynucleotide of the present invention.

[0082] The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced and its progeny. Host cells include "transformants" and "transformed cells." These include the primary transformed cell and the progeny thereof. Host cells are any type of cell line that can be used to produce the antibody molecules of the invention, and include 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 in transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0083] The terms "activation," "stimulation," and "treatment with," when used with respect to the manipulation of cells or receptors, can have the same meaning, for example, as activating, stimulating, or treating a cell or receptor with a ligand, unless the context clearly dictates otherwise. "Ligand" includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and antibody-derived binding compounds. "Ligand" also includes small molecules, such as cytokine peptidomimetics and antibody peptidomimetics. "Activation" refers to the activation of cells regulated by internal mechanisms, external factors, or environmental factors. "Response / reaction," such as the response of a cell, tissue, organ, or organism, refers to a change in biochemical or physiological behavior (e.g., concentration, density, adhesion, migration, gene expression rate, differentiation state, etc., within a biological compartment). Such changes are related to internal mechanisms, such as activation, stimulation, treatment, or genetic programs.

[0084] According to one aspect 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 the abnormal, uncontrolled growth or proliferation of cells or tissues in an animal. In this disclosure, the terms "cancer" and "tumor" include solid tumors and hematologic tumors, as well as precancerous lesions. 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 (e.g., non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, etc.), peritoneal cancer, hepatocellular carcinoma, gastric cancer, colorectal 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 one embodiment, hematological tumors include mixed B / 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's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma (MCL), multiple myeloma, myelodysplastic syndrome, etc. In one embodiment, the tumor is a benign neoplasm.

[0085] In the present disclosure, the term "treating" or "healing" of any disease or condition refers, according to one aspect, to slowing, interrupting, preventing, ameliorating, arresting, alleviating, or reversing the onset of symptoms, complications, or biochemical manifestations of the disease, alleviating symptoms, or preventing or inhibiting further progression of the disease, condition, or disorder (i.e., slowing, arresting, or alleviating the progression of the disease or at least one of its clinical symptoms). In another aspect, "treating" or "healing" refers to alleviating or improving at least one physical parameter, including physical parameters that are not discernible to the patient. In another aspect, "treating" or "healing" refers to modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Unless otherwise specified in this disclosure, methods for assessing the treatment and / or prevention of disease are generally known in the art.

[0086] A "subject" includes a human or non-human animal. The term "non-human animal" includes all vertebrates, including mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In this disclosure, the terms "cyno" or "cynomolgus" refer to cynomolgus monkeys or those derived from cynomolgus monkeys.

[0087] Administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0088] The terms "therapeutically effective amount," "therapeutic effective dose," and "effective amount" refer to an amount of a CCR8 antibody or antigen-binding fragment thereof of the present invention administered to a cell, tissue, or subject, alone or in combination with other therapeutic agents, that is effective in preventing or ameliorating symptoms of one or more diseases or conditions, or inhibiting the progression of a disease or condition. A therapeutically effective dose also refers to the amount of the antibody or antigen-binding fragment thereof sufficient to result in symptomatic improvement, e.g., treating, curing, preventing, or ameliorating an associated medical condition, or increasing the rate of treatment, cure, prevention, or amelioration. When a single active ingredient is administered to an individual, the therapeutically effective dose refers to that ingredient alone. In the case of combined administration, the therapeutically effective dose refers to the combined amount of active ingredients that results in a therapeutic effect, regardless of combined, sequential, or simultaneous administration. An effective amount of a therapeutic agent is an amount that results in an increase in a diagnostic criterion or parameter by at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% or more.

[0089] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation or composition, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, preservatives, etc.

[0090] Anti-CCR8 antibody In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CCR8. The terms "anti-CCR8 antibody," "anti-CCR8," "CCR8 antibody," and "an antibody that binds to CCR8" refer to an antibody that can bind to CCR8 protein or a fragment thereof with sufficient affinity to be used as a diagnostic and / or therapeutic agent that targets CCR8.

[0091] In certain embodiments, the antibodies of the present invention bind to human or cynomolgus monkey CCR8 protein. In certain embodiments, the antibodies of the present invention bind to CHOK1-human CCR8 cells or 293F-cynomolgus monkey CCR8 cells. In certain embodiments, the antibodies of the present invention inhibit / block the binding of CCR8 to its ligand CCL1. In certain embodiments, the antibodies of the present invention inhibit / block the induced recruitment of β-arrestin.

[0092] Any suitable method for producing an antibody of the present invention can be used. Any suitable form of CCR8 can be used as the immunogen (antigen) for producing the antibody. For example, a CCR8 mutant or a fragment thereof can be used as the immunogen. In one embodiment, hybridoma cells producing mouse-derived monoclonal anti-CCR8 antibodies can be generated by methods known in the art.

[0093] Antibodies derived from rodents (e.g., mice) can cause undesirable antibody immunogenicity when used as therapeutic agents in vivo. Repeated use of these antibodies can trigger an immune response against the therapeutic antibody in humans. Such an immune response can result in, at the very least, a loss of therapeutic efficacy and, in severe cases, potentially fatal allergic reactions. One approach to reducing the immunogenicity of rodent antibodies is to create chimeric antibodies by fusing mouse variable regions with human constant regions (Liu et al., (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, maintaining the rodent variable regions intact in chimeric antibodies can potentially cause adverse immunogenicity in patients. The technique of grafting complement-determining region (CDR) loops of rodent variable regions 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 one embodiment, the antibody of the present invention is a chimeric antibody. In one preferred embodiment, the antibody of the present invention is a humanized antibody.

[0094] In one embodiment, chimeric or humanized antibodies of the invention can be prepared based on the sequences of the murine monoclonal hybridoma antibodies prepared as described above. Using standard molecular biology techniques, DNA encoding heavy and light immunoglobulin chains can be obtained from the murine hybridoma of interest and modified to contain non-murine (e.g., human) immunoglobulin sequences.

[0095] According to one embodiment, the chimeric CCR8 antibody described in the present invention can be prepared by operatively linking a heavy chain variant region and a light chain variant region of a hybridoma-derived immunoglobulin to a human IgG constant region using methods known in the art (see, for example, U.S. Pat. No. 4,816,567 (Cabilly et al.)). According to one embodiment, the constant region contained in the chimeric antibody of the present invention can be selected from any IgG subclass, such as IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0096] In one embodiment, chimeric CCR8 antibodies can be obtained by introducing "mixed and matched" expression plasmids of chimeric light chains and chimeric heavy chains into expression cells. The binding of the resulting "mixed and matched" antibodies to CCR8 can be tested using the binding assays described above and other conventional binding assays (e.g., ELISA).

[0097] As defined in this disclosure, "complementarity determining region" or "CDR region" or "CDR" refers to a region of an antibody variable region that is hypervariable in sequence, forms structurally defined loops ("hypervariable loops"), and / or contains residues that contact an antigen ("antigen contact residues"). CDRs are primarily involved in binding to an antigen epitope. Heavy and light chain CDRs are typically numbered CDR1, CDR2, and CDR3, starting from the N-terminus. The CDRs in an antibody heavy chain variable region are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs in an antibody light chain variable region are referred to as LCDR1, LCDR2, and LCDR3. For a given light or heavy chain variable region amino acid sequence, the precise boundaries of the amino acid sequences of the individual CDRs can be determined using any one or a combination of many well-known antibody CDR assignment systems. Examples of assignment systems include Chothia, which is based on the three-dimensional structure of an antibody 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, which is based on the diversity of antibody sequences (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)), AbM System (University of Bath), Contact System (University College London), International ImMunoGeneTics Database (IMGT) (website: imgt.cines.fr / superior), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures.

[0098] For example, according to different rules for identifying CDRs, the residues of each CDR are described as follows:

[0099] Below are the CDR ranges defined in the Kabat, AbM, Chothia, Contact, and IMGT schemes. [Table 4]

[0100] Unless otherwise specified, the term "CDR" or "CDR sequence" as used herein includes CDR sequences determined by any of the above methods. A CDR may also be identified based on the position having the same Kabat number as a reference CDR sequence (e.g., one of the exemplary CDRs of the present invention). Unless otherwise specified, when referring to the position of a residue in an antibody variable region (including heavy chain variable region residues and light chain variable region residues) in the present invention, this refers to the position numbered according to the Kabat numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). According to one embodiment, the HCDRs and LCDRs in the antibodies of the present invention are identified according to the Kabat scheme.

[0101] Unless otherwise specified, the boundaries of the CDRs of the antibodies of the present invention can be determined by one of ordinary skill in the art according to any method known in the art (e.g., different assignment systems, or a combination thereof).

[0102] It should be noted that the CDR boundaries of the variable regions of the same antibody obtained based on different assignment systems may be different. Accordingly, the CDR sequences of the variable regions of the same antibody defined by different assignment systems may be different. Therefore, with respect to the definition of an antibody having a specific CDR sequence defined in the present invention, an antibody whose variable region sequence contains the specific CDR sequence described above, but whose claimed CDR boundaries differ from the specific CDR boundaries defined in the present invention due to the application of a different scheme (e.g., a different assignment system or a combination thereof), is also included within the scope of that antibody.

[0103] Antibodies with different specificities (i.e., antibodies with different binding sites for different antigens) have different CDRs. However, despite the differences in CDRs between antibodies, the amino acid positions within the CDRs that are directly involved in antigen binding are limited. The minimum overlapping region can be determined using at least two of the Kabat, Chothia, AbM, Contact, and North schemes, thereby obtaining the "minimum binding unit" for antigen binding. The minimum binding unit can be a subsection of the CDR. As will be apparent to those skilled in the art, the residues in the remaining portions of the CDR sequence can be determined by the folding structure of the antibody and protein. Therefore, the present invention also contemplates variants of a given CDR. For example, in a CDR variant, the amino acid residues of the minimum binding unit remain unchanged, and the remaining CDR residues defined according to the Kabat or Chothia scheme are substituted with conservative amino acid residues.

[0104] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" includes CDR sequences determined by any of the above methods.

[0105] In the present invention, expression plasmids for various chimeric heavy and light chains are mixed and combined and then introduced into expression cells to produce anti-CCR8 chimeric antibodies.

[0106] For humanized antibodies according to the invention, murine CDR regions can be inserted into human germline framework regions using methods known in the art (see also U.S. Patent No. 5,225,539 to Winter et al., and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[0107] According to certain aspects, the amino acid changes include amino acid deletions, additions, substitutions, etc. According to certain aspects, the anti-CCR8 antibodies or antigen-binding fragments thereof of the present invention include those having amino acid sequences mutated by amino acid deletions, additions, or substitutions, but still having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity (particularly in the CDR regions shown in the sequences) with the above antibodies (particularly in the CDR regions shown in the sequences). According to certain aspects, the antibodies of the present invention have at least one, e.g., 1, 2, 3, 4, or 5, amino acid mutations by deletion, addition, or substitution in their CDR regions compared to the CDR regions shown in a particular sequence. According to certain aspects, the antibodies of the present invention have at least one, e.g., 1, 2, 3, 4, or 5 or more, amino acid mutations by deletion, addition, or substitution in their CDR regions compared to the CDR regions shown in a particular sequence.

[0108] "Percent (%) amino acid sequence identity" simply refers to the percentage of amino acid residues in a candidate amino acid sequence that are identical to those in a reference amino acid sequence after the amino acid sequences have been aligned to achieve maximum sequence identity (by introducing gaps, if necessary), without considering conservative substitutions as part of the sequence identity. Alignment to determine amino acid sequence identity can be performed using various methods in the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software / algorithms. Those skilled in the art can determine appropriate parameters for measuring alignment, including the algorithms needed to achieve maximum alignment over the entire length of the sequences being compared.

[0109] According to one aspect, the present invention provides an antibody or antigen-binding fragment thereof, which comprises a heavy chain constant region and / or a light chain constant region. Preferably, the light chain constant region is a λ or κ chain constant region, and the heavy chain constant region is selected from mouse mIgG2a, human IgG1, human IgG2, human IgG3, or IgG4 subclasses, or modified forms thereof. According to a preferred aspect, the heavy chain constant region is a human IgG1 subclass or a human IgG4 subclass having an S228P mutation. According to a preferred aspect, the modified form of the constant region comprises an amino acid sequence modification and / or glycosylation.

[0110] According to certain aspects, one or more amino acid modifications are introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) sequence containing an amino acid modification (e.g., an addition, deletion, or substitution, preferably a substitution) at one or more amino acid positions.

[0111] In some embodiments, the antibody comprises at least one modification that enhances cell-killing ability. In some embodiments, the enhanced cell-killing ability is 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 least one of the following residues in the heavy chain constant region according to the EU numbering system: 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, 309, 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, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, and 439. As used herein, unless the context dictates otherwise, reference is made to the EU numbering system for the numbering of amino acid positions in the constant region. In the specification, amino acid sequence mutations may be designated by the single letter code of the parent amino acid, followed by the position number, and then the single letter code of the mutated amino acid. For example, a mutation of leucine (L) at position 234 to alanine (A) is designated "L234A." Sometimes, a slash ( / ) is used to list multiple options. For example, a deletion of cysteine ​​(C) at position 236 can be designated "C236 deletion." In a preferred embodiment, the modifications are in the heavy chain constant region at 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 one embodiment, the one or more mutations in the heavy chain constant region are one or more mutations selected from N297A substitution, N297Q substitution, L235A and L237A substitution, L234A and L235A substitution, E233P substitution, L234V substitution, L235A substitution, C236 deletion, P238A substitution, D265A substitution, A327Q substitution, and P329A substitution. In one embodiment, the modification is one or more mutations selected from G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L. In one embodiment, the one or more mutations in the heavy chain constant region are one or more mutations selected from L235V, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330L, A330M, I332E, E333A, K334A, K334E, and P396L. In one embodiment, the one or more mutations in the heavy chain constant region are one or more mutations selected from M252Y, S254T, and T256E according to the EU numbering system in the heavy chain constant region.

[0112] According to one embodiment, the heavy chain constant region comprises any of the following combinations of mutations: (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) has one or more sets of mutations occurring simultaneously at combinations of positions selected from L234 / L235 / G236 / S239 / H268 / D270 / S298, (7) M252 / S254 / T256, (8) L234 / L235 / D265, (9) G236 / S239 / I332, and (10) S239 / I332.

[0113] According to one embodiment, the heavy chain constant region comprises any of the following combinations of mutations: (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) S298A / E333A / K334A, (7) S298B / E333B / K334B, (8) S298C / E333C / K334C, (9) S298D / E333C / K334C, (10) S298E / E333C / K334C, (11) S298C / E333C / K334C, (12) S298D / E333C / K334C, (13) S298E / E333C / K334C, (14) S298C / E333C / K334C, (15) S298C / E333C / K334C, (16) S298D / E333C / K334C, (17) S298E / E333C / K334C, (18) S298E / E333C / K334C, (19) S298E / E333C / K334C, (20) S298E / E333C / K334C, (21) S298E / E333C / K334C, (22) S29 It has one or more combinations of mutations selected from 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.

[0114] In certain 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 certain embodiments, the number of cysteine ​​residues in the hinge region (CH1 domain) can be altered, e.g., increased or decreased, by introducing one, two, or more mutations (e.g., amino acid substitutions) into the hinge region.

[0115] In certain aspects, the antibodies provided in this disclosure can be further modified to contain other nonproteinaceous moieties that are known in the art and readily available.

[0116] Antibody expression In yet another aspect, the present invention provides a polynucleotide molecule encoding an anti-CCR8 antibody or any fragment thereof described in the present disclosure. Such a nucleic acid molecule may comprise a polynucleotide molecule encoding the amino acid sequence of the light chain and / or heavy chain variable region of such an antibody or at least a portion thereof, or a polynucleotide molecule encoding the amino acid sequence of the light chain and / or heavy chain of such an antibody or at least a portion thereof.

[0117] For example, the polynucleotide molecule of the present invention includes a nucleic acid encoding any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 95, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 95.

[0118] In one aspect, polynucleotide molecules encoding antibodies of the present invention include polynucleotide molecules that have mutations due to nucleotide deletions, additions, or substitutions and that have 60%, 70%, 80%, 90%, 95%, or 100% or more identity to the coding regions corresponding to the CDR regions shown in the above sequences.

[0119] In another aspect, the present invention provides expression vectors, preferably eukaryotic expression vectors, comprising a polynucleotide molecule described herein. In one embodiment, a polynucleotide molecule described herein is comprised in one or more expression vectors.

[0120] In another aspect, the present invention provides a host cell comprising a polynucleotide molecule described herein or an expression vector described herein. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0121] According to yet another aspect, the present invention provides a method for preparing an anti-CCR8 antibody or antigen-binding fragment thereof described herein, comprising expressing the antibody or antigen-binding fragment thereof in a host cell described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0122] The present invention provides mammalian host cells for expressing the recombinant antibodies of the invention or any fragment thereof, many of which are immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells, A549 cells, 293T cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, sheep, bovine, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.

[0123] In one aspect, the present invention provides a method for preparing an anti-CCR8 antibody, comprising introducing an expression vector into a mammalian host cell, and producing the antibody by culturing the host cell for a period of time sufficient to express the antibody in the host cell, and more preferably, to secrete the antibody into the culture medium in which the host cell is cultured.

[0124] Standard protein purification methods can be used to recover antibodies from the culture medium. Antibody molecules prepared by the methods described in this disclosure can be purified by known techniques such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and size-exclusion chromatography. The conditions actually used to purify a particular protein will depend on factors such as positive charge, hydrophobicity, and hydrophilicity, and 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, such as size-exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography.

[0125] Antibodies expressed by different cell lines or transgenic animals will likely have different glycosylation profiles. However, all antibodies encoded by the nucleic acid molecules provided in this disclosure or comprising the amino acid sequences provided in this disclosure are part of the present invention, regardless of how glycosylated they are. Similarly, according to certain aspects, non-fucosylated antibodies generally have greater potency in vitro and in vivo than fucosylated antibodies, and are advantageous because they are less immunogenic since their carbohydrate structure is a normal component of native human IgG in serum.

[0126] Pharmaceutical compositions and pharmaceutical preparations According to yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-CCR8 antibody or antigen-binding fragment thereof described in the present disclosure, a polynucleotide molecule described in the present disclosure, a vector described in the present disclosure, a host cell described in the present disclosure, or an immunoconjugate described in the present disclosure, and a pharmaceutically acceptable carrier or excipient. Naturally, the anti-CCR8 antibody or pharmaceutical composition thereof provided by the present invention can be formulated with a carrier, excipient, and other drugs to form a formulation suitable for combined administration, thereby improving its transportability, deliverability, tolerability, etc.

[0127] The term "pharmaceutical composition" refers to a formulation containing an active ingredient, adjusted so that the biological activity of the active ingredient is present in an effective form, and containing no additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0128] Pharmaceutical formulations comprising the anti-CCR8 antibodies of the present invention described in the present disclosure can be prepared by mixing the anti-CCR8 antibodies of the present invention having the desired purity with one or more optional pharmaceutical additives (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)), preferably in the form of an aqueous solution or a lyophilized formulation.

[0129] The pharmaceutical compositions or formulations of the present invention may further comprise one or more additional therapeutic agents as necessary for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. According to one embodiment, the pharmaceutical compositions of the present invention comprise a composition of polynucleotide molecules encoding anti-CCR8 antibodies.

[0130] The pharmaceutical compositions of the present invention may further contain one or more additional therapeutic agents necessary for the specific indication being treated, i.e., substances effective in the prevention or treatment of tumors (e.g., cancer), preferably substances with activities that do not adversely affect each other. Examples include chemotherapeutic agents, hormonal agents, etc. The therapeutic agents are contained in an appropriate combination in amounts effective for the intended use. In one embodiment, the additional therapeutic agent is a chemotherapeutic agent. In one embodiment, the additional therapeutic agent is another antibody. In one embodiment, the additional therapeutic agent is another monoclonal antibody. In one preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets an immune checkpoint. In a more preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets PD-1. In a more preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets PD-L1. In a more preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets CTLA4.

[0131] According to 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, a 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.

[0132] According to yet another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, a vector described herein, a host cell described herein, an immunoconjugate described herein, a pharmaceutical composition described herein, or a pharmaceutical combination described herein.

[0133] Combination Products or Kits In certain aspects, the present invention also provides combination products 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 active agents, small molecule drugs, immunomodulatory agents, etc.).

[0134] In one embodiment, the combination product is used for the prevention or treatment of CCR8 / immune checkpoint-associated diseases and / or diseases mediated by CCR8 and / or immune checkpoints. In one embodiment, the additional therapeutic agent is an established standard of care. In one embodiment, the additional therapeutic agent is a chemotherapeutic agent. In one embodiment, the additional therapeutic agent is another antibody. In one embodiment, the additional therapeutic agent is another monoclonal antibody. In one preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets an immune checkpoint. In a more preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets PD-1. In a more preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets PD-L1. In a more preferred embodiment, the additional therapeutic agent is a monoclonal antibody that targets CTLA4.

[0135] In some embodiments, two or more components of the combination product may be administered to a subject sequentially, separately, or simultaneously.

[0136] According to one aspect, 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 indicating the method of administration.

[0137] According to one aspect, the present invention also provides a pharmaceutical preparation comprising an anti-CCR8 antibody or fragment thereof, pharmaceutical composition, or combination product of the present invention, and optionally a package insert indicating the method of administration.

[0138] Medical Uses and Treatment Methods Any of the anti-CCR8 antibodies or corresponding immune complexes provided in the present disclosure can be used in therapeutic methods. It should be understood that the term "antibody" as used herein also includes compositions containing the antibody. The anti-CCR8 antibodies of the present invention can be used in a therapeutically or prophylactically effective amount in the therapeutic or prophylactic methods described in any aspect of the present invention.

[0139] In yet another aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof, polynucleotide, expression vector, host cell, immunoconjugate, or pharmaceutical composition described herein in the preparation of a medicament. Furthermore, the present invention provides the use of a combination pharmaceutical described herein in the preparation of a medicament. That is, the present invention substantially provides the use of an effective amount of an antibody or antigen-binding fragment thereof, polynucleotide, expression vector, host cell, immunoconjugate, or pharmaceutical composition described herein in combination with another therapeutic agent in the preparation of a medicament. Among other uses, such a medicament is used for the prevention and / or treatment of tumors, autoimmunity, or infectious diseases. Here, preferred tumors include 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, or metastatic cancers thereof.

[0140] According to yet another aspect, the present invention provides use of an antibody or antigen-binding fragment thereof, polynucleotide, expression vector, host cell, immunoconjugate, pharmaceutical composition, or pharmacological combination described herein for the treatment and / or prevention of tumor, autoimmune, 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, or metastatic cancer thereof.

[0141] In yet another aspect, the present invention provides a method for treating and / or preventing a tumor, autoimmune disease, or infectious disease, 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 pharmacological 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, or metastatic cancer thereof.

[0142] In certain aspects, methods of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intra-arterial, intra-articular (e.g., intra-articular in arthritic joints), inhalation, aerosol delivery, or topical administration to the affected area.

[0143] The term "treatment" refers to clinical intervention aimed at altering the natural course of a disorder in the individual receiving treatment. Goals of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing adverse experiences or direct or indirect pathological consequences of disease, slowing the rate of disease progression, improving or mitigating the disease state, achieving remission, or improving prognosis. The antibodies of the present invention can reduce the severity of disease in one or more aspects, i.e., exert a therapeutic effect. In certain embodiments, such a therapeutic effect is manifested in one or more of the following ways: extending the patient's life expectancy (survival), slowing disease progression, and reducing the need for medical care.

[0144] The present invention also provides for the co-administration of therapeutically effective amounts of one or more therapeutic treatments (e.g., therapeutic modalities and / or other therapeutic agents) to a subject. The antibodies of the present invention can be used in therapy alone or in combination with other therapeutic agents. In certain embodiments, the antibodies of the present invention are co-administered with at least one additional therapeutic agent.

[0145] Methods for diagnosis and detection In another aspect, the present invention provides a method for detecting the presence of CCR8 in a sample using the antibody or antigen-binding fragment thereof described in the present disclosure. In the present disclosure, the term "detection" includes quantitative detection or qualitative detection. In certain embodiments, the sample is a biological sample. In certain embodiments, the biological sample is blood, serum, or other biologically derived liquid sample. In certain embodiments, the biological sample contains cells or tissues. In certain embodiments, the CCR8 is human CCR8 or cynomolgus monkey CCR8. The method of the present invention includes contacting the sample with the antibody or antigen-binding fragment thereof described in the present disclosure, or a detection composition comprising the antibody or antigen-binding fragment thereof, and detecting the presence of a binding substance resulting from binding of the antibody or antigen-binding fragment thereof to CCR8. When used for detection purposes, the antibody or antigen-binding fragment thereof described in the present disclosure may be labeled to indicate whether a conjugate has formed. In certain embodiments, the method may be an in vitro method or an in vivo method.

[0146] In certain embodiments, CCR8 is detected pre-treatment, e.g., before treatment initiation, or pre-treatment of a particular treatment after a treatment interval. In one embodiment, an anti-CCR8 antibody or antigen-binding fragment for use in a method of diagnosis or detection is provided.

[0147] The present invention includes all combinations of the specific aspects described. Furthermore, further aspects and the full scope of applicability of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples are intended to illustrate preferred embodiments of the invention and are not intended to limit the scope of the invention, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited in this disclosure, including those cited by reference, are incorporated herein by reference in their entirety for all purposes.

[0148] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments combining specific embodiments with other methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments are not limited to those listed in the examples of the present invention. [Example]

[0149] The present invention is illustrated by the following examples, but is not limited thereto. The present invention has been disclosed in detail in this disclosure, and specific embodiments thereof have been disclosed. Those skilled in the art will recognize that various changes and modifications can be made to specific embodiments of the invention without departing from the spirit and scope of the invention.

[0150] Example 1. Animal immunization and preparation of anti-human CCR8 mouse hybridoma antibodies Mice were immunized using the full-length human CCR8 gene (DNA or mRNA) and a modified cell line overexpressing human CCR8. Furthermore, the full-length monkey CCR8 gene (DNA or mRNA) and a modified cell line overexpressing monkey CCR8 were used for immunization or boosting. SP2 / 0 cell fusion was performed, and positive clones were selected by flow cytometry. The detailed procedures are as follows.

[0151] Animal immunization: The gene encoding full-length human CCR8 (UniProtKB / Swiss-Prot: P51685) was inserted into the plasmid PCDNA3.1 or PCDNA3.4 (established by Shanghai Ruizhi Chemical Research Co., Ltd.). Female Balb / c mice were immunized with this DNA at 4 μg / mouse. DNA immunization was repeated 6–7 times at 2-week intervals. Ten days after the final immunization, 293F cells expressing human CCR8 (established by Shanghai Ruizhi Chemical Research Co., Ltd., 293F-hCCR8) were injected intraperitoneally for boosting. Three days later, the mice's spleens were removed and subjected to cell fusion.

[0152] Female Balb / c mice were inoculated with 2–5 × 10 293F cells expressing human CCR8 (293F-hCCR8) as an immunogen. 6 The mice were immunized intraperitoneally with 293F-hCCR8 cells (2–5 × 10 cells / mouse). Cell immunization was repeated three times at 2-week intervals. Ten days after the last immunization, 293F-hCCR8 cells (2–5 × 10 cells / mouse) were administered intraperitoneally. 6 The mice were reinjected intraperitoneally with 1000 cells / mouse, and 3 days later, the spleens of the mice were removed and subjected to cell fusion.

[0153] Female Balb / c mice were immunized with mRNA encoding full-length human CCR8 (UniProtKB / Swiss-Prot: P51685) (prepared by Shanghai Hongcheng Bio Pharmaceutical Co., Ltd., 50 μg / mouse). mRNA immunization was repeated three or four times at three-week intervals. Ten days after the final immunization, 293F-hCCR8 cells were intraperitoneally injected for boosting. Three days later, the mouse spleens were removed and subjected to cell fusion.

[0154] Cell fusion: Mouse spleen cells were electrofused with SP2 / 0 cells (ATCC No. CRL-1581) at a ratio of 2:1 (BTX Electrofusion equipment: ECM2001). + ) and cultured in HAT medium (GIBCO, Catalog No. H0262) in a 96-well culture plate, and after 10 days, antibodies were screened from the hybridoma cell supernatant.

[0155] Screening for human CCR8- and cynomolgus monkey CCR8-specific positive clones: Human CCR8-expressing cells (CHOK1-hCCR8, established by Shanghai ChemPartner Co., Ltd.) and cynomolgus monkey CCR8-expressing cells (CHOK1-cynoCCR8, established by Shanghai ChemPartner Co., Ltd.) were placed in a 96-well U-bottom assay plate at 5 × 10 in 100 μL / well. 7The cells were seeded at a density of 1000 cells / mL. 100 μL of hybridoma cell culture supernatant was added and incubated for 1 hour at 4°C. The cells were washed twice with FACS buffer (PBS containing 1% FBS) and centrifuged at 300 g. Alexa488-labeled anti-mouse IgG antibody was added and incubated for 1 hour at 4°C. The cells were washed three times with FACS buffer (PBS containing 1% FBS) and centrifuged at 300 g. The mean fluorescence intensity (MFI) was measured using a flow cytometer (Beckman Coulter CytoFLEX). Positive clones were selected by comparison with the negative control (CHOK1 cells, which do not express CCR8). As shown in Table 1, the mean fluorescence intensity of clones binding to human CCR8 (CHOK1-hCCR8) and cynomolgus monkey CCR8 (CHOK1-cynoCCR8) was higher than that of the blank control cells (CHOK1). This indicates that these clones specifically bind to human and cynomolgus monkey CCR8.

[0156] [Table 1]

[0157] Example 2. Preparation and identification of anti-human CCR8 mouse monoclonal antibodies Hybridoma cells that exhibited binding activity to human CCR8 and cynomolgus monkey CCR8, as shown 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., part number AA0272) to prepare purified monoclonal antibodies. The binding activity of the anti-human CCR8 antibodies and their inhibitory activity against CCL1 binding were detected using flow cytometry.

[0158] 2.1 Binding activity of anti-human CCR8 antibodies to human CCR8 and cynomolgus monkey CCR8 Binding assay using anti-CCR8 antibodies and cells expressing human CCR8 or cynomolgus CCR8: 1 × 10 cells of the 293F cell line expressing human CCR8 (293F-hCCR8, established by Shanghai ChemPartner Co., Ltd.) or the 293T cell line expressing cynomolgus monkey CCR8 (293T-cyno CCR8, established by Kyinno Biotechnology Co., Ltd.) were used. 6 The solution was adjusted to 100 cells / mL and seeded at 100 μL / well in a 96-well U-bottom plate. The mixture was centrifuged and the supernatant discarded. Anti-human CCR8 antibody, control antibody (anti-human CCR8 433H, clone number: 433H, BD Pharmingen, catalog number 624092; 10A11, the sequences of which are set forth in International Publication WO2020138489 (light chain variable region: SEQ ID NO: 59, heavy chain variable region: SEQ ID NO: 41)) were fused with the human and mouse constant region sequences listed in Table 5 of the present disclosure, respectively. An IgG1 isotype antibody (Biointron Biotechnology Co., Ltd., catalog number B117901) was diluted to an initial working concentration in FACS buffer (PBS solution containing 1% FBS) and serially diluted with FACS buffer. The cells were resuspended in serially diluted antibodies at 100 μL / well, mixed thoroughly by pipetting, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. The cell pellet was then incubated with diluted secondary antibodies (goat anti-human IgG (H+L) cross-adsorbed secondary antibody, AlexaFluor (登録商標) 100 μL of 647 (Invitrogen, Cat. No. A21445) was added to each well, mixed well with a pipette, and resuspended by incubating at 4°C for 45 minutes. After incubation, the cells were centrifuged, washed three times, and resuspended in 100 μL / well of FACS buffer. Median fluorescence intensity (MFI) was measured using a flow cytometer (BECKMAN COULTER CytoFLEX), and experimental data were analyzed using Graphpad Prism 8.0 software. EC 50 was calculated by plotting the logarithm of the antibody on the X-axis and the corresponding MFI value on the Y-axis, and fitting the dose-effect curve of the antibody with a selected four-parameter regression model.

[0159] As shown in Figures 1 and 2 and Table 2, for example, antibody clones 27B9-1G3, 559E1B10, 563E10E12, 569D11B5, and 589D7C7 exhibited strong binding activity to 293F-hCCR8 and were significantly associated with EC 50 The EC values ​​ranged from 107.0 to 360.6 ng / mL. The EC values ​​for the binding of the controls 433H and 10A11_mIgG2a to 293F-hCCR8 were 50 The EC values ​​were 206.0 ng / mL and 185.1 ng / mL, respectively. Antibody clones 27B9-1G3, 559E1B10, 563E10E12, 569D11B5, and 589D7C7 all showed stronger binding activity to cynomolgus monkey CCR8, and the EC values ​​were 206.0 ng / mL and 185.1 ng / mL, respectively. 50 On the other hand, the binding activity of the control antibodies 433H and 10A11_mIgG2a to cynomolgus monkey CCR8 was very weak, and the EC 50 The values ​​were 15312.5 ng / mL and 5770.5 ng / mL, respectively.

[0160] [Table 2]

[0161] 2.2 Receptor ligand inhibition assay of anti-CCR8 antibodies against CCL1 CCL1 is a ligand for CCR8, and CCR8 is the only known receptor for CCL1. High CCL1 expression in tumor tissues is negatively correlated with cancer prognosis. CCL1 induces Treg migration by binding to CCR8, and inhibiting CCL1-CCR8 binding can suppress Treg migration 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 CCL1 to Promote the Stemness Property of Breast Cancer Cells. Stem Cells. 2017 Dec;35(12): 2351-2365). Anti-CCR8 antibodies that inhibit CCL1-CCR8 binding may have stronger antitumor activity. A competitive binding assay was used to measure the activity of anti-CCR8 antibodies in inhibiting the binding of AlexaFluor-647-labeled human CCL1 to 293F-hCCR8. The detailed procedure is as follows.

[0162] The 293F cell line expressing human CCR8 (293F-hCCR8, established by Shanghai ChemPartner Co., Ltd.) was cultured at 1 × 10 6The cells were adjusted to 100 cells / mL and seeded into a 96-well U-bottom plate at 100 μL per well. The plate was centrifuged and the supernatant discarded. Anti-CCR8 antibody, control antibody (anti-human CCR8, clone number: 433H, BD Pharmingen, catalog number 624092), and hIgG1 isotype antibody (Biointron Biotechnology Co., Ltd., catalog number B117901) were diluted to 2x initial concentrations in FACS buffer (PBS solution containing 1% FBS) and then serially diluted in FACS buffer. Cells were resuspended in 50 μL / well of the serially diluted antibody and mixed thoroughly with a pipette. Then, 50 μL / well of 20 nM human CCL1-AlexaFluor-647 (Almac, CAF-7) solution was added, mixed thoroughly with a pipette, and incubated at 4°C for 1 hour. After incubation, cells were centrifuged, washed three times, and resuspended in FACS buffer at 100 μL / well. Median fluorescence intensity (MFI) was measured using a flow cytometer (BECKMAN COULTER CytoFLEX), and the experimental data were analyzed using Graphpad Prism 8.0 software. The logarithm of antibody concentration was plotted on the X axis and the corresponding MFI value on the Y axis. The dose-effect curve of the antibody was fitted with a selected four-parameter regression model to determine the IC. 50 was calculated.

[0163] As shown in Figure 3 and Table 3, all of the antibody clones 27B9-1G3, 559E1B10, 563E10E12, 569D11B5, and 589D7C7 had the ability to inhibit the binding of CCL1 to CCR8, with IC50 values ​​of inhibitory activity ranging from 75.0 ng / mL to 131.7 ng / mL. On the other hand, the IC50 values ​​of inhibitory activity against the control 433H were 50 The value was 94.3ng / mL.

[0164] [Table 3]

[0165] Example 3. Sequencing of anti-CCR8 antibodies and functional characterization of chimeric antibodies 3.1 Sequencing of anti-CCR8 antibodies and construction, expression, and purification of the chimeric antibodies Hybridoma clones that showed excellent performance in the activity characterization were sequenced, and the resulting sequences are shown in Table 4.

[0166] The sequenced light and heavy chain variable regions (see Table 4) were incorporated into a human Fc constant region (IgG1 / K, see Table 5) or a mouse Fc constant region (mIgG2a), respectively, for in vitro functional characterization or in vivo pharmacodynamic testing. Sequencing confirmed that the constructed hIgG1 and mIgG2a subclass chimeric antibodies matched the sequences shown in Tables 4 and 5.

[0167] Expression was carried out using Expi293F cells (Thermofisher, catalog number A1452), and purification was carried out using a protein A column. The detailed procedure is as follows.

[0168] Expression of antibodies in Expi293 cells: One day before transfection, Expi293 cells were cultured at 1.5 × 10 6 The cells were diluted to 3 × 10 cells / mL and incubated at 37°C, 8% CO2, and 120 rpm on a shaker. On the second day, the density and viability of Expi293 cells were measured. The cell density for transfection was 3 × 10 cells / mL. 6Cells / mL, cell viability should be 95% or higher. Preparation of PEI / plasmid complexes: PEI (1 mg / mL, Polysciences, catalog no. 24765-1) was mixed thoroughly by inversion. Plasmids were diluted with OPM-293CD05 medium (OPM Biosciences, catalog no. 81075-001) to a transfection volume of 1 μg / mL plasmid, with the volume of medium used for diluting the plasmid at 1 / 20 of the transfection volume. The heavy and light chain plasmids were gently mixed at a ratio of 1:1.5. The PEI reagent was diluted with OPM-293CD05 medium, with the volume of PEI used being twice the volume of the plasmid and 1 / 20 of the volume of medium used for diluting the PEI. The resulting mixture was mixed thoroughly by gentle inversion and incubated at room temperature for 5 minutes. The diluted PEI reagent was added to the diluted plasmid and mixed thoroughly by gentle inversion. The PEI / plasmid complex was incubated at room temperature for 15 minutes, and the resulting solution was added dropwise to a shaker flask while gently rotating the flask. After transfection, the flask was incubated at 37°C, 8% CO2, and 120 rpm in a shaker. On the second day after transfection (24 hours after transfection), 10% OPM-293ProFeed (OPM Biosciences, catalog number F081918) was added to the flask while gently rotating the flask. The flask was then returned to the shaker and incubation continued for 5–7 days. The supernatant was collected.

[0169] Antibody purification using a Protein A column: A gravity chromatography column was prepared. The top cover of the gravity chromatography column was removed. A gasket was placed on the bottom of the gravity column and pressed firmly. Protein A (Cytiva, catalog number 17549801) was prepared. The required volume of packing suspension was accurately calculated based on the target packing volume and suspension ratio. Required packing suspension volume = target packing volume / packing suspension ratio. The packing was thoroughly stirred to ensure complete suspension. The packing suspension was added to the bottom of the gravity chromatography column. At least 10 CV of equilibration buffer (PBS) was added to the gravity chromatography column. After equilibration was complete, the pH was monitored at the outlet. If the target pH was not reached, the addition of equilibration buffer was continued until the desired pH equilibration was achieved. A fixed amount of sample was slowly added to the gravity chromatography column. At least 10 CV of wash buffer was added to the gravity chromatography column. Five CV of elution buffer (10-50 mM NaAc, pH 3.0-3.5) was slowly added to the gravity chromatography column and incubated for 3-5 minutes before collecting the eluate. The elution step was repeated if necessary. Neutralization: The pH was adjusted to the target value using neutralization buffer (1 M Tris). Protein concentration was measured using a Nanodrop. The antibody storage buffer was exchanged into PBS by ultrafiltration.

[0170] [Table 4]

[0171] [Table 5]

[0172] 3.2 Binding activity of anti-CCR8 chimeric antibodies to human CCR8 and cynomolgus monkey CCR8 The binding activity of anti-CCR8 chimeric antibodies to human and cynomolgus monkey CCR8 was detected using the binding assay approach described in Example 2-2.1. The results are shown in Figures 4 and 5 and Table 6. The EC values ​​of the binding activity of hIgG1- and mIgG2a-subclass chimeric antibodies 27B9-1G3, 559E1B10, 563E10E12, 569D11B5, and 589D7C7 to 293F-hCCR8 cells were 50 The EC value was 27.3 ng / mL to 273.4 ng / mL, and the binding activity of 433H to 293F-hCCR8 cells was 50 The EC value was 153.4 ng / mL. The hIgG1-subclass chimeric antibody 27B9-1G3 and the mIgG2a-subclass chimeric antibodies 563E10E12, 569D11B, 589D7C7, and 559E1B10 exhibited strong binding activity to cynomolgus monkey CCR8. 50 On the other hand, the binding activity of 433H and 10A11_hIgG1 to cynomolgus monkey CCR8 was weaker, and the EC 50 The values ​​were 14580.5 and 6309.0 ng / ml.

[0173] [Table 6]

[0174] 3.3 Receptor-ligand inhibition assay of anti-CCR8 chimeric antibodies against CCL1 The receptor ligand inhibitory activity of anti-CCR8 chimeric antibodies against CCL1 was detected using the approach described in Example 2-2.2. The results are shown in Figure 6 and Table 7. The hIgG1- or mIgG2a-subclass chimeric antibodies dose-dependently inhibited the binding of CCL1 to 293F-hCCR8, and the IC 50 The IC50 values ​​for the inhibitory activity against 433H and 10A11_hIgG1 were 70.5 ng / mL and 54.6 ng / mL, respectively.

[0175] [Table 7]

[0176] 3.4 Inhibition of CCL1-induced β-arrestin recruitment by anti-CCR8 chimeric antibodies Upon binding to CCR8, CCL1 induces chemotaxis and receptor endocytosis, the latter dependent on the expression and recruitment of β-arrestins. The β-arrestin assay has been used to detect the role of CCL1 in CCR8 activation (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). Inhibition of CCL1-induced β-arrestin recruitment by anti-CCR8 antibody was detected using Tango-H_CCR8-CHO-K1 cells (Genomeditech (Shanghai) Co., Ltd., GM-C09028). The experimental procedures provided by Genomeditech (Shanghai) Co., Ltd. were used to detect inhibition of CCL1-induced β-arrestin recruitment by anti-CCR8 antibody. The detailed procedures are as follows.

[0177] Tango-H_CCR8-CHO-K1 cells were seeded into cell culture flasks and doxycycline (Selleck, S4163-100mg) was added to the culture medium at a final concentration of 10 μg / mL. The cells were then continuously cultured for approximately 48 hours to induce doxycycline-induced human CCR8 expression. After induction, 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 discarded. The cells were resuspended in fresh F12K complete medium (Gibco, 21127022), tested for cell viability, counted, and then resuspended in fresh medium at 5 × 10 5 The solution was adjusted to cells / mL and seeded at 100 μL per well into an opaque white plate. The plate was placed in a 37°C, 5% CO2 incubator and cultured overnight to allow cell attachment. Anti-CCR8 antibody, positive control antibody, and hIgG1 isotype antibody (Biointron Biotechnology Co., Ltd., B117901) were diluted 2-fold in complete F12K medium and serially diluted in complete F12K medium. The culture medium in the Tango-H_CCR8-CHO-K1 cell plate was carefully discarded, and 50 μL of the serially diluted antibodies were added to each well. Next, 50 μL of human CCL1 (R&D, 272-I) solution, diluted to a final concentration of 20 nM in complete F12K medium, was added to each well. After 6 hours of incubation at 37°C, 5% CO2, the plate was allowed to equilibrate for at least 15 minutes. 100 μL of luciferase substrate solution (Vazyme, DD1203) was added to each well, mixed, and incubated in the dark at room temperature for 5 minutes. Relative light units (RLU) were measured using a microplate reader. Experimental data were analyzed using Graphpad Prism 8.0 software. Antibody dose-effect curves were plotted against log-transformed antibody concentrations on the x-axis and corresponding RLU values ​​on the y-axis and fitted with a selected four-parameter regression model.

[0178] As shown in Figure 7 and Table 8, the hIgG1- or mIgG2a-subclass chimeric antibodies 27B9-1G3, 563E10E12, 569D11B5, 589D7C7, 559C12G12, and 559E1B10 inhibited CCL1-induced β-arrestin recruitment and IC50 The IC values ​​of 433H, 10A11_hIgG1, and 10A11_mIgG2a were 260.2 ng / mL to 1757.0 ng / mL. 50 The values ​​were 1291.5, 3268.0 ng / mL, and 3250.0 ng / mL.

[0179] [Table 8]

[0180] Example 4. Humanization of anti-CCR8 antibodies CDRs were determined using Kabat numbering. The human germline gene with the highest homology to the mouse sequence was selected as the antibody framework, and the mouse CDRs were grafted onto this human antibody framework. Backmutations were introduced depending on the importance of amino acid residues. That is, several important amino acids in the grafted framework region were backmutated to the corresponding mouse-derived amino acids. Multiple mutants were designed for both the heavy and light chains.

[0181] 4.1 Humanization of anti-CCR8 antibody 27B9-1G3 The sequences of the murine variable regions and the amino acid sequences of selected human germline genes are shown below in Table 9, and the sequences of the humanized heavy and light chains designed from these are shown in Table 10.

[0182] The humanized heavy and light chains were combined to obtain the following humanized antibodies: [Table A]

[0183] 4.2 Humanization of anti-CCR8 antibody 559E1B10 The sequences of the murine variable regions and the amino acid sequences of selected human germline genes are shown in Table 11 below, and the sequences of the humanized heavy and light chains designed from these are shown in Table 12.

[0184] The humanized heavy and light chains were combined to obtain the following humanized antibodies: [Table B]

[0185] 4.3 Humanization of anti-CCR8 antibodies 563E10E12, 569D11B5, and 589D7C7 The sequences of the three antibody clones, 563E10E12, 569D11B5, and 589D7C7, are similar to each other and are considered to belong to the same sequence family. Therefore, it is possible to humanize the three antibodies together. The sequences of the mouse variable regions and the amino acid sequences of selected human germline genes are shown in Table 13 below, and the sequences of the humanized heavy and light chains designed based on these sequences are shown in Table 14.

[0186] The humanized heavy and light chains were combined to obtain the following humanized antibodies: [Table C]

[0187] [Table 9]

[0188] [Table 10]

[0189] [Table 11]

[0190] [Table 12]

[0191] [Table 13]

[0192] [Table 14]

[0193] Example 5. Functional characterization of humanized anti-CCR8 antibodies The light and heavy chain variable regions of humanized antibodies 27B9-1G3, 559E1B10, and 563E10E12 were each integrated into a human Fc constant region (hIgG1 / K, the sequences of which are shown in Table 5). The corresponding genes were synthesized and their sequences were confirmed to match the designed sequences. The antibodies were expressed in Expi293 cells using the method described in Example 3 and purified on a Protein A column. To investigate the functionality of the resulting antibodies, experiments were performed to examine their binding to human and cynomolgus CCR8, inhibition of CCL1 binding, and inhibition of CCL1-mediated β-arrestin recruitment.

[0194] 5.1 Binding assays of humanized anti-CCR8 antibodies The binding activity of humanized anti-CCR8 antibodies to human CCR8 and cynomolgus monkey CCR8 was detected using the approach described in Example 2-2.1. The results are shown in Figures 8 and 9 and Table 15. The binding activity of the 27B9-1G3 humanized antibody to 293F-hCCR8 is comparable to that of its chimeric parent antibody. Most of the 559E1B10-humanized antibodies binding to 293F-hCCR8 showed binding activity comparable to that of their chimeric parent antibody. Most of the 563E10E12-humanized antibodies binding to 293F-hCCR8 showed binding activity comparable to that of their chimeric parent antibody. The humanized antibodies 559E1B10_hzH1L1, 563E10E12_hzH1L0, and 563E10E12_hzH1L1 show strong binding activity to 293T-cynoCCR8, but 433H and 10A11_hIgG1 show no binding activity to cynomolgus monkey CCR8.

[0195] [Table 15-1] [Table 15-2]

[0196] 5.2 Receptor ligand inhibitory activity of humanized anti-CCR8 antibodies against CCL1 The receptor ligand inhibitory activity of anti-CCR8 humanized antibodies against CCL1 was detected using the receptor ligand inhibition assay approach described in Example 2-2.2. The results are shown in Figure 10 and Table 16. Some of the 27B9-1G3 humanized antibodies, such as 27B9-1G3_hzH3L1 and 27B9-1G3_hzH3L2, had inhibitory activity comparable to that of their chimeric parent antibodies. Most of the 559E1B10 humanized antibodies, such as 559E1B10_hzH2L1, 559E1B10_hzH3L1, 559E1B10_hzH1L2, 559E1B10_hzH3L2, 559E1B10_hzH0L1, and 559E1B10_hzH0L2, had inhibitory activity comparable to that of their chimeric parent antibodies. All of the 563E10E12 humanized antibodies had inhibitory activity comparable to that of their chimeric parent antibodies. The 559E1B10 humanized antibodies 559E1B10_hzH3L1, 559E1B10_hzH1L2, 559E1B10_hzH0L1, and 559E1B10_hzH0L2, and the 563E10E12 humanized antibody all had inhibitory activity superior to or comparable to that of 433H.

[0197] [Table 16-1] [Table 16-2]

[0198] 5.3. Experiment on inhibition of CCL1-induced β-arrestin recruitment by humanized anti-CCR8 antibody Inhibition of CCL1-induced β-arrestin recruitment by humanized anti-CCR8 antibodies was determined using the approach described in Example 3-3.4. The results are shown in Figure 11 and Table 17. Some 27B9-1G3 humanized antibodies, such as 27B9-1G3_hzH3L1 and 27B9-1G3_hzH3L2, had inhibitory activity comparable to their chimeric parent antibodies. Most of the 559E1B10 humanized antibodies, such as 559E1B10_hzH2L1, 559E1B10_hzH3L1, 559E1B10_hzH1L2, 559E1B10_hzH3L2, 559E1B10_hzH0L1, and 559E1B10_hzH0L2, had inhibitory activity comparable to their chimeric parent antibodies. All of the 563E10E12 humanized antibodies had inhibitory activity comparable to that of their chimeric parent antibodies. The 559E1B10 humanized antibodies 559E1B10_hzH3L1, 559E1B10_hzH1L2, 559E1B10_hzH0L1, and 559E1B10_hzH0L2 and the 563E10E12 humanized antibodies all had inhibitory activity superior to or comparable to that of 433H.

[0199] [Table 17]

[0200] 5.4 Measurement of humanized anti-CCR8 antibody-dependent FcγRIIIa activation using Jurkat-human FcγRIIIa(158V)-NFAT In mouse models, anti-CCR8 antibodies have been shown to suppress tumor growth primarily by eliminating Treg cells within tumor tissue through the ADCC effect (Helena Van Damme et al., Therapeutic depletion of CCR8 +Tumor-infiltrating regulatory T cells elicit antitumor immunity and synergize with anti-PD-1 therapy. J Immunother Cancer. 2021 Feb;9(2):e001749. doi: 10.1136 / jitc-2020-001749). Anti-CCR8 antibody-dependent FcγRIIIa activation was measured by incubating Jurkat-human FcγRIIIa(158V)-NFAT with CCR8-expressing cells. The Fab portion of the CCR8 antibody binds to the target site on target cells, while its Fc portion binds to the Fcγ receptor on effector cells, activating the NFAT signaling pathway in the effector cells. The ADCC activity of the antibody was reflected by quantifying luciferase activity induced by activation of the NFAT signaling pathway. The detailed procedure is as follows.

[0201] 293F-human CCR8 cells (Shanghai ChemPartner Co., Ltd., 293F-hCCR8) were digested with trypsin containing 0.25% EDTA (Gibco, 25200072) and centrifuged at 300 g for 5 minutes. The resulting cell suspension was diluted to 3 × 10 in DMEM medium. 5 The solution was adjusted to 100 cells / mL, and 100 μL / well was seeded onto an opaque white plate and cultured overnight in a 37°C, 5% CO2 incubator to allow cell adhesion. Anti-CCR8 antibody, positive control antibody, and hIgG1 isotype antibody (Biointron Biotechnology Co., Ltd., B117901) were diluted 2-fold with 1640 medium and serially diluted 5-fold with culture medium. Recovered Jurkat-human FcγRIIIa(158V)-NFAT cells (Jiman Biotechnology (Shanghai) Co., Ltd., GM-C05619) were centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 1640 medium was added to adjust the cell concentration to 3 × 10 6The concentration was adjusted to cells / mL. The 293F-hCCR8 cell culture plate was removed, the supernatant was aspirated, and 50 μL of Jurkat-human FcγRIIIa(158V)-NFAT cells and 50 μL of serially diluted anti-CCR8 antibodies were added. After 6 hours of induction at 37°C and 5% CO2, the plates were allowed to equilibrate at room temperature for at least 15 minutes. 100 μL of luciferase substrate solution (Vazyme, DD1203) was added to each well, mixed, and incubated in the dark at room temperature for 5 minutes. Relative light units (RLU) were measured using a microplate reader. Experimental data were analyzed using Graphpad Prism 8.0 software. The dose-effect curves of the anti-CCR8 antibodies were plotted against the logarithm of the anti-CCR8 antibody concentration on the x-axis and the corresponding RLU values ​​on the y-axis and fitted with a selected four-parameter regression model.

[0202] As shown in Figures 12 and 13 and Tables 18 and 19, the chimeric and humanized antibodies 559E1B10 and 563E10E12 had the ability to activate Jurkat-human FcγRIIIa(158V)-NFAT in a dose-dependent manner. The humanized antibodies 559E1B10_hzH0L1, 559E1B10_hzH1L1, 559E1B10_hzH2L1, 559E1B10_hzH3L1, 559E1B10_hzH0L2, 559E1B10_hzH1L2, 559E1B10_hzH2L2, and 559E1B10_hzH3L2 have activity comparable to that of the chimeric parent antibody 559E1B10_hIgG1. All of the humanized antibodies of 563E10E12, such as 563E10E12_hzH0L0, 563E10E12_hzH1L0, and 563E10E12_hzH0L1, had activity comparable to that of their chimeric parent antibodies.

[0203] [Table 18]

[0204] [Table 19]

[0205] Example 6. Binding activity of anti-CCR8 antibodies to HuT78 cells endogenously expressing CCR8 Both the human T lymphoma cell line HuT78 and activated human Treg cells express CCR8 (James M Fox et al. and Yiftah Barsheshet et al., CCR8 + FOXp3 + Treg cells as master drivers of immune regulation. Proc Natl Acad Sci USA. 2017 Jun 6;114(23):6086-6091. doi: 10.1073 / pnas.1621280114). The binding activity of anti-CCR8 antibodies to endogenous CCR8 was measured by FACS. HuT78 cells (Cobioer, CBP60267) were cultured at 1 × 10 6 The antibody solution was adjusted to 100 cells / mL and placed in a 96-well U-bottom plate at 100 μL per well. The supernatant was discarded after centrifugation. Anti-CCR8 antibodies, control antibodies 433H, 10A11_hIgG1, and B16_hIgG1:7-B16.001 (light chain variable region SEQ ID NO: 81, heavy chain variable region SEQ ID NO: 80, as described in U.S. Patent Publication No. 2021 / 0277129A1), and hIgG1 isotype (Niointron Biotechnology Co., Ltd., B117901) were serially diluted 4-fold in 2% FBS-containing PBS (FACS buffer) to an initial concentration of 10 μg / mL. Cells were resuspended in the serially diluted antibodies at 100 μL per well, mixed thoroughly with a pipette, and incubated at 4°C for 1 hour, followed by three washes with FACS buffer. Cells were incubated with secondary antibodies AF647-goat anti-human IgG (H+L) (Invitrogen, A21445) or AF647-donkey anti-mouse IgG (H+L) (Invitrogen, Cat. No. A31571, detecting mouse-derived antibody 433H) diluted 1:1000 in FACS buffer, plated at 100 μL / well, mixed well with a pipette, and incubated at 4°C for approximately 45 minutes. After incubation, cells were centrifuged and washed three times with FACS buffer. Cells were resuspended in 100 μL / cell of FACS buffer. Median fluorescence intensity (MFI) was measured using a flow cytometer, and experimental data were analyzed using Graphpad Prism 8.0 software. EC50 was determined by plotting the anti-CCR8 antibody concentration on the x-axis and the corresponding MFI value on the y-axis and fitting the dose-effect curve of the anti-CCR8 antibody with a four-parameter regression model.

[0206] As shown in Figure 14 and Table 20, the humanized antibodies 563E10E12_hzH1L0 and 563E10E12_hzH1L1 bound to HuT78 cells that endogenously express CCR8 in a dose-dependent manner, and the EC 50 The binding activity EC was equivalent to that of B16_hIgG1 and 10A11_hIgG1. 50 The maximum binding MFI was higher than that of B16_hIgG1 and 10A11_hIgG1. However, 559E1B10_hzH1L1 showed almost no binding activity to HuT78.

[0207] [Table 20]

[0208] Example 7. Specific binding of anti-CCR8 antibodies to human CCR8 7.1 Specific binding of anti-CCR8 antibodies to CHO-K1 cells expressing human CCR8 CHO-K1 cells overexpressing CCR8 were used for mouse immunization and screening of CCR8 antibodies. Binding of anti-CCR8 antibodies to cell surface CCR8 was detected using a CHO-K1 cell line overexpressing human CCR8 (CHOK1-hCCR8, Shanghai Chem Partner Co., Ltd.) and a wild-type CHO-K1 cell line (GM-15570, Genomeditech Biotechnology) using the method described in Assay Example 2-2.1.

[0209] As shown in Figure 15 and Table 21, all of 559E1B10_hzH1L1, 563E10E12_hzH1L0 and 563E10E12_hzH1L1 bound to CHOK1-hCCR8 in a dose-dependent manner, but did not bind to CHOK1 blank cells.

[0210] [Table 21]

[0211] 7.2 CCR8-dependent Jurkat-human FcγRIIIa(158V)-NFAT activation mediated by anti-CCR8 antibodies Using a CHO-K1 cell line overexpressing human CCR8 (CHOK1-hCCR8, established by Shanghai ChemPartner Co., Ltd.) and a blank CHO-K1 cell line (Genomeditech Biotechnology, GM-15570), we confirmed that the activation of Jurkat-human FcγRIIIa(158V)-NFAT by anti-CCR8 antibodies is dependent on CCR8. The concentrations of CHOK1-hCCR8 cells and blank CHOK1 cells were adjusted to 3 × 10 in RPMI-1640 complete medium. 5 The concentration was adjusted to cells / mL. Using the procedure described in Example 5-5.4, activation of Jurkat-human FcγRIIIa(158V)-NFAT was detected by anti-CCR8 antibodies. 4A19 is the sequence in patent WO2021194942A1, with SEQ ID NO: 18 for the light chain mutant region and SEQ ID NO: 6 for the heavy chain mutant region. As shown in Figure 16 and Table 22, the anti-CCR8 antibodies activated the activity of the Jurkat-human FcγRIIIa(158V)-NFAT reporter, but were unable to activate Jurkat-human FcγRIIIa(158V)-NFAT in the presence of blank CHO-K1 cells. This indicates that the anti-CCR8 antibodies specifically recognize CCR8 and mediate CCR8-dependent ADCC effects.

[0212] [Table 22]

[0213] 7.3 Anti-CCR8 antibodies do not bind to human CCR4 CCR4 is the gene with the highest homology to human CCR8 (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.), but its homology with CCR8 is only 46% (NCBI blast results). The binding activity of anti-CCR8 antibodies to human CCR4 was measured as follows: 293F cells were incubated with ExpiFectamine (登録商標) Transient expression of hCCR4-GFP (Sino, HG13064-ACG) was achieved by following the instructions of the 293 transfection kit (Gibco, A14524). 1 × 10 cells were transfected. 6 The antibody was adjusted to 30 μg / mL and placed in a 96-well U-bottom plate at 100 μL / well. The cells were then centrifuged to recover the cells. Anti-CCR8 antibody was diluted to an initial concentration of 30 μg / mL in PBS containing 2% FBS (FACS buffer) and serially diluted 4-fold in FACS buffer. The cells were resuspended at 100 μL / well, mixed thoroughly with a pipette, and incubated at 4°C for 1 hour, followed by washing three times with FACS buffer. The cells were resuspended in 100 μL / well of the secondary antibody AF647-goat anti-human IgG (H+L) (Invitrogen, A21445) diluted 1:1000 in FACS buffer, mixed thoroughly with a pipette, and incubated at 4°C for approximately 40 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer. LIVE / DEAD (登録商標)After labeling dead cells according to the instructions in the Fixable Near IR (780) Viability Kit (Invitrogen, L34992), the cells were resuspended in 100 μL / cell of FACS buffer and the median fluorescence intensity (MFI) was measured using a flow cytometer. CCR4 expression in hCCR4-GFP-transfected cells was measured using an anti-CCR4 antibody (Biolegend, 359412). Binding of anti-CCR8 antibodies to GFP-positive cells transfected with hCCR4-GFP (CCR4-expressing cells) and live cells transfected with a blank plasmid was analyzed. As shown in Figure 17, the 563E10E12 chimeric and humanized antibodies did not bind to hCCR4-GFP-transfected cells (GFP-positive cells) or 293F cells, whereas the 559E1B10 chimeric and humanized antibodies showed weak binding to hCCR4 at high concentrations.

[0214] 7.4 Binding of anti-CCR8 antibodies to immune cells in PBMCs CCR8 is expressed at low levels on Treg cells in the peripheral blood of healthy donors, but not on other immune cells (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). We used PBMCs from healthy donors to confirm whether anti-CCR8 antibodies bind nonspecifically to peripheral immune cells. Cryopreserved PBMCs from healthy donors (Allcells, catalog numbers Z0060 and Y1446) were resuscitated and 2 × 10 6 The solution was adjusted to 100 cells / mL and placed in a 96-well U-bottom plate at 100 μL / well. The plate was centrifuged and the supernatant was discarded. Dead cells were classified as LIVE / DEAD. (登録商標)Labeling was performed according to the instructions of the Fixable Near IR(780) Viability Kit (Invitrogen, L34992). FcX reagent (Biolegend, 422302) diluted 1:25 in FACS buffer was added at 50 μL / well to block Fc receptor staining by antibodies, and the cells were incubated for 15 minutes at room temperature. PE anti-human CD3 antibody (Biolegend-317308, 1:50 dilution), FITC-anti-human CD8 antibody (Biolegend-301006, 1:50 dilution), and Pacific Blue were used. (登録商標) Anti-human CD19 (Biolegend-363036, 1:50 dilution) and Brilliant Violet 605 (登録商標) Anti-human CD14 antibody (Biolegend-367126, 1:50 dilution) was prepared in FACS buffer and added to the cells at 40 μL / well and mixed thoroughly. Anti-CCR8 antibody and hIgG1 isotype (Biointron Biotechnology Co., Ltd., B117901) were diluted to an initial concentration of 100 μg / mL in FACS buffer, serially diluted 5-fold in FACS buffer, added to the cells at 10 μL / well, and incubated at 4°C for 1 hour. After incubation, the cells were washed three times with FACS buffer and resuspended in FACS buffer at 100 μL / well. Median fluorescence intensity (MFI) was measured by flow cytometry.

[0215] As shown in Figure 23, the anti-CCR8 antibodies 559E1B10_hzH1L1_hIgG1, 563E10E12_hzH1L0_hIgG1, and 563E10E12_hzH1L1_hIgG1 inhibited CD8 + and CD4 + (CD3 + CD8 - )T cells, CD14 + Monocytes, CD19 + B cells and other CD3 - CD14 - CD19 - Did not specifically bind to immune cells

[0216] [Table 23-1] [Table 23-1]

[0217] Example 8. Fc mutation and defucosylation enhance ADCC mediated by anti-CCR8 antibodies 8.1 Mutations S239D / I332E, S239D / A330L / I332E, L235V / F243L / R292P / Y300L / P396L and defucosylation enhance activation of anti-CCR8 antibodies and 293F-human CCR8 Jurkat-human FcγRIIIa(158V)-NFAT Mutations S239D / I332E, S239D / A330L / I332E, or L235V / F243L / R292P / Y300L / P396L in the antibody constant region (Fc) may increase affinity for FcγRIIIa and enhance the ADCC effect of antibodies (Greg A Lazar et al., Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci U S 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 Fcγ receptor binding properties. Breast Cancer Res. 2011;13(6):R123. doi: 10.1186 / bcr3069). These sets 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).

[0218] The Fc region of an anti-CCR8 humanized antibody was modified with S239D / I332E (DE), S239D / I332E (L235V / F243L / R292P / Y300L / P396L / A330L / IS239D / A330L / I332E (DLE), or L235V / F243L / R292P / Y300L / P396L (VLPLL) mutations to enhance the ADCC effect of the antibody. Abfucosylation or hypofucosylation can also enhance the ADCC effect of antibodies. Several afucosylated or hypofucosylated antibodies have received marketing approval or are in late-stage clinical studies (Natasha A Pereira et al., The "less-is-more" in therapeutic antibodies: Afucosylated 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 in growth medium ExpiCHO Expression Medium (Gibco, Cat. No. A2910001) containing 100 μM 2F-peracetyl-fucose (MILLIPORE, Cat. No. 344827-10MGCN) prior to transient transfection. On the day of transient transfection, cells were cultured at 6 × 10 6 The plasmid was diluted to 1000 cells / mL and transiently transfected using the ExpiCHO transfection kit (Gibco, catalog no. A29129) at a concentration of 1 μg plasmid / mL. 18-22 hours after transient transfection, nutrient supplements (Gibco, catalog no. A29129) were added to the transiently transfected cells and cultured at 37°C, 8% CO2, and 100 rpm in a shaking incubator. Seven days after transient transfection, the cell culture medium was harvested, centrifuged, and filtered through a 0.45 μm filter to recover the supernatant. The resulting supernatant was purified using Protein A resin to recover the antibody.

[0219] Using the procedure described in Example 5-5.4, the activity of Jurkat-human FcγRIIIa(158V)-NFAT was measured by incubating anti-CCR8 antibodies and 293F-hCCR8 cells. As shown in Figure 18 and Table 24, compared to the 559E1B10_hzH1L1_hIgG1 antibody, the Fc variant antibodies 559E1B10_hzH1L1_DE and 559E1B10_hzH1L1_VLPLL enhanced the activation of Jurkat-human FcγRIIIa(158V)-NFAT and increased EC 50 Compared with the 563E10E12_hzH1L0_hIgG1 antibody, the Fc mutant antibodies 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE, and 563E10E12_hzH1L0_VLPLL, and the defucosylated antibody 563E10E12_hzH1L0_AF enhanced the activity of Jurkat-human FcγRIIIa(158V)-NFAT, resulting in a 9- to 21-fold increase in EC 50 Compared with the 563E10E12_hzH1L1_hIgG1 antibody, the Fc mutant antibodies 563E10E12_hzH1L1_DE and 563E10E12_hzH1L1_VLPLL enhanced the activation of Jurkat-human FcγRIIIa(158V)-NFAT, and increased EC 50 The activity was improved by 6-14 fold. The Fc variant humanized antibodies derived from 559E1B10 and 563E10E12 or the defucosylated antibody 563E10E12_hzH1L0 showed activity comparable to that of defucosylated B16 (B16_AF).

[0220] [Table 24]

[0221] 8.2. Fc mutation and defucosylation enhance the activity of anti-CCR8 antibodies and HuT78 cells Jurkat-human FcγRIIIa(158V)-NFAT Anti-CCR8 antibodies with different Fc mutations or defucosylation were incubated with HuT78 cells and Jurkat-human FcγRIIIa(158V)-NFAT cells to examine the effects of the S239D / I332E, S239D / A330L / I332E, L235V / F243L / R292P / Y300L / P396L mutations, or defucosylation on the activity of anti-CCR8 antibodies and HuT78 cells in Jurkat-human FcγRIIIa(158V)-NFAT. HuT78 cells were cultured at 3 x 10 in DMEM culture medium as target cells. 5 The concentration was adjusted to cells / mL. Activation of Jurkat-human FcγRIIIa(158V)-NFAT by anti-CCR8 antibodies was measured using the procedure in Example 5-5.4.

[0222] As shown in Figure 19 and Table 25, incubation of HuT78 cells with the Fc variant antibodies 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE, 563E10E12_hzH1L0_VLPLL and the defucosylated antibody 563E10E12_hzH1L0_AF enhanced the activity of the 563E10E12_hzH1L0 antibody against Jurkat-human FcγRIIIa(158V)-NFAT, resulting in increased EC 50 The activation of Jurkat-human FcγRIIIa(158V)-NFAT by the Fc variant or defucosylated 563E10E12_hzH1L0 antibody was comparable to that of the defucosylated B16 antibody.

[0223] [Table 25]

[0224] Example 9. ADCC effect induced by healthy human PBMCs and anti-CCR8 antibodies The ADCC effect of anti-CCR8 antibodies was measured using PBMCs as effector cells and CHOK1-hCCR8 as target cells. Cryopreserved PBMCs (Allcells) collected from healthy donors were cultured at 2 × 10 in RPMI-1640 complete culture medium containing 200 IU / mL of IL-2 (Peprotech, Cat. No. 200-02). 6 The cells were adjusted to 100 cells / mL and incubated overnight in a 37°C, 5% CO2 incubator. The next day, unbound PBMCs were removed with a pipette, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. 1640 medium was then added to the cells at a concentration of 3 × 10 6 Anti-CCR8 antibody and hIgG1 isotype (Biointron Biotechnology Co., Ltd., Catalog No. B117901) were diluted 2-fold with RPMI-1640 medium to an initial concentration of 0.8 μg / mL, followed by five-fold serial dilutions. The collected CHOK1-hCCR8 cells were centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the cells were diluted to 2 × 10 cells / mL with DPBS. 6 Adjusted to cells / mL. CellTrace (登録商標) Label CHOK1-hCCR8 cells by adding Violet (Invitrogen, Cat. No. C34557) according to the instructions, and then use CellTrace (登録商標) Violet-labeled CHO-hCCR8 cells were cultured in RPMI-1640 medium at approximately 1.5 × 10 5 Adjusted to cells / mL. CellTrace (登録商標) Add 50 μL of Violet-labeled CHOK1-hCCR8, 50 μL of PBMC, and 100 μL of diluted anti-CCR8 antibody to a U-bottom 96-well plate, mix, and then place in an incubator at 37°C and 5% CO2 for approximately 5 hours. Add 2 μL of propidium iodide (PI) solution (Invitrogen, catalog number 006990-50) to each well, mix, and incubate at room temperature for 5 minutes in the dark to label dead cells. CellTrace (登録商標) The percentage of PI-positive cells among Violet-positive cells was measured using a flow cytometer. Antibody-induced ADCC was measured using Cell Trace. (登録商標)The lysis rate was calculated as the percentage of PI-positive cells labeled with Violet. The experimental data were analyzed using Graphpad Prism 8.0 software. The dose-effect curve of the anti-CCR8 antibody was plotted by plotting the logarithmically transformed anti-CCR8 antibody concentration on the x-axis and the corresponding lysis rate (%) on the y-axis and fitting it with a selected four-parameter regression model.

[0225] As shown in Figure 20 and Table 26, compared with 563E10E12_hzH1L0_hIgG1, the defucosylated humanized antibody 563E10E12_hzH1L0_AF and the Fc variant antibodies 563E10E12_hzH1L0_DE and 563E10E12_hzH1L0_DLE all enhanced the ADCC activity induced by these antibodies and PBMCs. The humanized antibody 563E10E12_hzH1L0_hIgG had ADCC activity comparable to that induced by B16_hIgG1. The ADCC activity induced by the combination of the defucosylated and Fc variant humanized antibody 563E10E12_hzH1L0 and PBMCs was comparable to that induced by the defucosylated B16 antibody.

[0226] [Table 26]

[0227] Example 10. Specific removal of CCR8-positive Treg cells from peripheral PBMCs by anti-CCR8 antibodies The specific depletion of CCR8-expressing Treg cells in PBMCs by anti-CCR8 antibodies was measured using PBMCs from healthy humans. Because the percentage of CCR8-expressing Treg cells in healthy human PBMCs is relatively low, IL-2 was used to promote Treg cell expansion and activation in this experiment. Cryopreserved PBMCs from a healthy donor (Allcells) were revived and adjusted to 2 x 106 cells / mL in RPMI-1640 complete medium containing 200 IU / mL IL-2 (Peprotech, catalog no. 200-02). 100 μL / well of the solution was added to a 96-well U-bottom plate and incubated at 37°C and 5% CO2 for 48 hours. Anti-CCR8 antibodies and hIgG1 isotype antibodies (Biointron Biotechnology Co., Ltd., catalog no. B117901) were diluted in RPMI-1640 complete medium to an initial concentration of 20 μg / mL, followed by four-fold serial dilutions. 100 μL of diluted antibody was added to the 96-well plate, mixed thoroughly with a pipette, and incubated at 37°C and 5% CO2 for 96 hours. Cells were collected by centrifugation. Dead cells were classified as LIVE / DEAD. (登録商標) Labeling was performed according to the instructions in the Fixable NearIR (780) Viability Kit (Invitrogen, L34992). 50 μL / well of a 1:25 diluted FcX reagent (Biolegend, Catalog No. 422302) was added and incubated for 15 minutes at room temperature. 50 μL / well of a staining mixture of AF700 anti-human CD3 (Biolegend, Catalog No. 317340) and Percpcy5.5 anti-human CD4 (Biolegend, Catalog No. 300530) diluted 1:50 was added and incubated for 1 hour at 4°C, followed by washing three times with FACS buffer. Cells were fixed and labeled with PE anti-human Foxp3 (Biolegend, Catalog No. 320208) according to the instructions in the Foxp3 / Transcription Factor Staining Kit (Invitrogen, Catalog No. 00-5523-00) and washed three times with FACS buffer. The cells were resuspended in 100 μL / cell of FACS buffer and analyzed using a flow cytometer. The experimental data were analyzed using Graphpad Prism 8.0 software. At the end of the experiment, Treg cells (Foxp3 + CD4+ ) CCR8 expression was detected, and the proportion of Treg cells among CCR8-positive cells was 31.5%.

[0228] As shown in Figure 21, the defucosylated 563E10E12_hzH1L0_AF antibody, the Fc mutated 563E10E12_hzH1L0_DE, 563E10E12_hzH1L0_DLE, and 563E10E12_hzH1L0_VLPLL antibodies, and the defucosylated B16_AF antibody dose-dependently and partially reduced Treg cells in PBMCs, but not CD8 cells that do not express CCR8. + T(CD3 + CD4 - ) had no effect on the Treg cell count. The 563E10E12_hzH1L0_hIgG1 and B16_hIgG1 antibodies only reduced a small proportion of Treg cells at high concentrations. This is because, after activation of peripheral PBMCs with IL-2, Treg cells express a certain level of CCR8, whereas CD8 cells that do not express CCR8 are depleted. + This indicates that T cells are not depleted, but CCR8-expressing Treg cells are depleted by anti-CCR8 antibodies.

[0229] Example 11. Anti-CCR8 antibodies inhibit tumor growth in tumor-bearing mouse models 11.1 Anti-CCR8 antibodies suppress growth of MC38 colon carcinoma in CCR8-humanized mice The MC38 colon cancer animal model (Biocytogen (Beijing) Pharmaceuticals Co., Ltd.) was developed using human CCR8-humanized mice (C57BL / 6-Ccr8 tm1(CCR8) Tumors were established using Bcgen, B-hCCR8, Biocytogen (Beijing) Pharmaceuticals Co., Ltd., Catalog No. 110096) and used for in vivo efficacy testing of anti-CCR8 antibodies. MC38 cells (5 × 106 cells / mL) resuspended in PBS were subcutaneously inoculated into the right flank of B-hCCR8 mice at 100 μL / animal. The average tumor volume was approximately 100 mm. 3When the tumor size reached 100 mg / kg, the mice were divided into groups of 6–8 and subcutaneously administered 10 mg / kg of anti-CCR8 antibody or negative control (hIgG1) (Biointron Biotechnology Co., Ltd., catalog number B117901) or an equivalent volume of vehicle control (PBS) twice a week. After administration, the mice were fed a normal diet and their health status was monitored. Body weight and tumor volume were recorded.

[0230] Compared with the vehicle control and negative control (hIgG1), the anti-CCR8 antibodies 559E1B10_hzH1L1_mIgG2a, 563E10E12_hzH1L0_mIgG2a, 563E10E12_hzH1L1_mIgG2a, and 10A11_mIgG2a inhibited the growth of MC38 colon cancer cells (Figure 22). Twenty days after administration, the anti-CCR8 antibodies 559E1B10_hzH1L1_mIgG2a, 563E10E12_hzH1L0_mIgG2a, 563E10E12_hzH1L1_mIgG2a, and 10A11_mIgG2a demonstrated tumor growth inhibition (TGI) rates of 40.1%, 54.0%, 22.5%, and 53%, respectively. In the experiment, no adverse events were observed in any of the animals during the administration period, the animals maintained good feeding status, and their body weight increased to a certain extent, demonstrating that the animals tolerated the anti-CCR8 antibody treatment well (Figure 23).

[0231] 11.2 Fc-variant and afrucosylated anti-CCR8 antibodies inhibit the growth of MC38 colon carcinoma in CCR8-humanized mice The MC38 colon cancer model was established in CCR8-humanized B-hCCR8 mice using the method described in Example 11-11.1. The average tumor volume was approximately 100 mm 3 At the time of tumor growth, mice were divided into groups of five and subcutaneously injected twice weekly with 10 mg / kg of anti-CCR8 antibody or the same volume of vehicle control (PBS). After administration, mice were maintained as usual and their survival status was monitored, and their body weight and tumor volume were recorded.

[0232] As shown in Figure 24, compared to vehicle, the anti-CCR8 antibody 563E10E12_hzH1L0_hIgG1, its Fc variants 563E10E12_hzH1L0_DE and 563E10E12_hzH1L0_DLE antibodies, and the defucosylated 563E10E12_hzH1L0_AF antibody all inhibited the growth of MC38 colon cancer cells. On day 25 after administration, the 563E10E12_hzH1L0_hIgG1 antibody, its Fc variants 563E10E12_hzH1L0_DE and 563E10E12_hzH1L0_DLE antibodies, and the defucosylated 563E10E12_hzH1L0_AF antibody exhibited tumor growth inhibition (TGI) rates (%) of 60.1%, 57.8%, 60.2%, and 52.9%, respectively. No behavioral abnormalities or weight loss were observed in any of the mice in these treatment groups, indicating that the administered doses of anti-CCR8 antibodies were well tolerated by the mice (Figure 25).

[0233] 11.3 Anti-CCR8 antibodies target human CD34 + Inhibits growth of human non-small cell lung cancer cells in HSC and PBMC-humanized mice 11.3.1 Anti-CCR8 antibodies target human CD34 + Inhibits growth of non-small cell lung cancer cell line A549 in HSC-humanized mice Human CD34 + HSC-humanized mice express human hematopoietic cells, hCD34 + The cells were established by immune reconstitution in irradiated NCG mice transplanted with HSCs (Gem Pharmatech LLC.). Human non-small cell lung cancer A549 cells were inoculated with human CD34+ cells in 30% Matrigel at an inoculum volume of 0.2 mL / animal. + HSC-humanized mice were inoculated subcutaneously into the right flank. The average tumor volume was approximately 100 mm 3At this time, mice were randomly assigned to groups and administered anti-CCR8 antibodies (1–20 mg / kg), pembrolizumab (1–20 mg / kg), a combination of anti-CCR8 antibodies (1–20 mg / kg) and pembrolizumab (1–20 mg / kg), or a negative control (PBS) twice weekly for 4 weeks. After administration, mice were fed a normal diet and tumor volume was recorded. At the end of the experiment, tumor samples were collected and minced with dissecting scissors. Tumor tissue was then isolated using a human tumor isolation kit (Miltenyi Biotech) combined with a gentle MACS isolation device (Miltenyi Biotech). The resulting cell suspension was filtered through 70 μm MACS Smart Strainers to create a single-cell suspension. Immune cell subpopulations (Treg cells and CD8 + T cells) were analyzed by flow cytometer.

[0234] The 563E10E12_hzH1L0_hIgG1 antibody, the Fc mutant 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, and 563E10E12_hzH1L0_DLE antibodies, and the defucosylated 563E10E12_hzH1L0_AF antibody effectively inhibited tumor growth compared to the negative control group.

[0235] 11.3.2 CCR8 antibodies inhibit the growth of the human non-small cell lung cancer cell line NCI-H1299 in a human PBMC-humanized mouse model Human non-small cell lung cancer NCI-H1299 cells were inoculated subcutaneously into the right flank of human PBMC-humanized mice (Gem Pharmatech LLC.) with 30% Matrigel at an inoculation volume of 0.2 mL / animal. The mean tumor volume was approximately 100 mm. 3At this time, mice were randomly divided into groups and administered anti-CCR8 antibody (1-20 mg / kg), pembrolizumab (1-20 mg / kg), a combination of anti-CCR8 antibody (1-20 mg / kg) and pembrolizumab (1-20 mg / kg), or vehicle control (PBS) twice weekly for 4 weeks. After administration, mice were fed a normal diet, and tumor volume was recorded. At the end of the experiment, tumor samples were collected and minced with dissection scissors. Tumor tissue was dissociated using a human tumor isolation kit (Miltenyi Biotech) and a gentleMACS separator (Miltenyi Biotech), and the resulting cell suspension was filtered through 70 μm MACS Smart Strainers to prepare a single-cell suspension. Immune cell subpopulations (Treg cells and CD8 + T cells) were analyzed by flow cytometer.

[0236] The 563E10E12_hzH1L0_hIgG1 antibody, the Fc variants 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, and 563E10E12_hzH1L0_DLE antibodies, and the defucosylated 563E10E12_hzH1L0_AF antibody effectively suppressed tumor growth compared to the vehicle control group.

[0237] 11.3.3 CCR8 antibodies inhibit the growth of human non-small cell lung cancer NCI-H292 in a human peripheral blood mononuclear cell (PBMC)-humanized mouse model Human non-small cell lung cancer NCI-H292 cells were inoculated subcutaneously into the right flank of human PBMC-humanized mice (Gem Pharmatech LLC.) at an inoculation volume of 0.2 mL / animal with 30% Matrigel. The mean tumor volume was approximately 100 mm. 3At this time, mice were randomly divided into groups and administered anti-CCR8 antibody (1–20 mg / kg), pembrolizumab (1–20 mg / kg), a combination of anti-CCR8 antibody (1–20 mg / kg) and pembrolizumab (1–20 mg / kg), or a negative control (PBS) every two weeks for three weeks. After administration, mice were fed a normal diet, and tumor volume was recorded. At the end of the experiment, tumor samples were collected and minced with dissecting scissors. Tumor tissue was dissociated using a human tumor isolation kit (Miltenyi Biotech) and a gentleMACS separator (Miltenyi Biotech), and the resulting cell suspension was filtered through 70 μm MACS Smart Strainers to obtain a single-cell suspension. Immune cell subpopulations and functional biomarkers were analyzed by flow cytometry. Immune cell subpopulations (Treg cells and CD8+ T cells) were analyzed by flow cytometry.

[0238] The 563E10E12_hzH1L0_hIgG1 antibody, its Fc variants 563E10E12_hzH1L0_DE and 563E10E12_hzH1L0_DLE antibodies, and the defucosylated 563E10E12_hzH1L0_AF antibody effectively suppressed tumor growth compared to the vehicle control group.

[0239] 11.4 Combination of CCR8 antibody and pembrolizumab inhibits growth of human colon cancer HT29 in a human PBMC-humanized mouse model Human rectal cancer HT29 cells were subcutaneously inoculated into the right flank of human PBMC-humanized mice (Shanghai Model Organisms Center, Inc.) with 30% Matrigel at an inoculation volume of 0.1 mL / animal. The average tumor volume was approximately 100 mm. 3At this time, mice were randomly assigned to groups and administered anti-CCR8 antibody (10 mg / kg), pembrolizumab (3.25 mg / kg), a combination of anti-CCR8 antibody (10 mg / kg) and pembrolizumab (3.25 mg / kg), or a negative control (PBS) every two weeks for four weeks. After administration, mice were fed a normal diet and tumor volume was recorded. At the end of the experiment, tumor samples were collected and finely minced with dissection scissors. Tumor tissue was isolated using a human tumor isolation kit (Miltenyi Biotech) combined with a gentle MACS isolation device (Miltenyi Biotec), and the resulting cell suspension was filtered through 70 μm MACS Smart Strainers to obtain a single-cell suspension. Immune cell subpopulations (Treg cells and CD8 + T cells) were analyzed by flow cytometry.

[0240] The 563E10E12_hzH1L0_hIgG1 antibody, its Fc variants 563E10E12_hzH1L0_VLPLL and 563E10E12_hzH1L0_DE and 563E10E12_hzH1L0_DLE antibodies, and the reduced fucosylation antibody 563E10E12_hzH1L0_AF effectively inhibited tumor growth compared to the vehicle control group.

[0241] 11.5 Efficacy of anti-CCR8 antibodies against the EMT-6 tumor model of breast cancer in CCR8-humanized mice Murine EMT-6 breast cancer cells were subcutaneously inoculated into the right flank of human CCR8-humanized mice (Shanghai Model Organisms Center, Inc.) at an inoculum volume of 0.1 mL / animal (in PBS). The average tumor volume was approximately 50–100 mm. 3At this time, mice were randomly assigned to groups and administered anti-CCR8 antibodies (1-20 mg / kg) or vehicle control (PBS) twice weekly for 3 weeks. After administration, mice were fed with normal chow, and tumor volume was recorded. At the end of the experiment, tumor samples were collected and minced with dissecting scissors. Tumor tissue was dissociated using a human tumor disaggregation kit (Miltenyi Biotech) in combination with a gentle MACS disaggregator (Miltenyi Biotec), and the resulting cell suspension was filtered through 70 μm MACS Smart Strainers to create a single cell suspension. Immune cell subpopulations (Treg cells and CD8 + T cells) were analyzed by flow cytometer.

[0242] The 563E10E12_hzH1L0_hIgG1 antibody, the Fc variants 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, and 563E10E12_hzH1L0_DLE antibodies, and the defucosylated 563E10E12_hzH1L0_AF antibody effectively suppressed tumor growth compared to the vehicle control group.

[0243] Subsequently, mouse EMT-6 breast cancer cells were subcutaneously inoculated into the right flank of human CCR8-humanized mice (Shanghai Model Organisms Center, Inc.) at an inoculum volume of 0.1 mL / animal (in PBS). The average tumor volume was approximately 50–100 mm. 3At this time, mice were randomly assigned to groups and administered anti-CCR8 antibody (1–20 mg / kg), anti-mouse PD-1 (mPD-1) (Leinco Technologies, P372), or vehicle control (PBS) twice weekly. After administration, mice were fed a normal diet, and tumor volume was recorded. At the end of the experiment, tumor samples were collected and finely minced with dissecting scissors. Tumor tissue was dissociated using a human tumor isolation kit (Miltenyi Biotech) and a gentle MACS separator (Miltenyi Biotec). The resulting cell suspension was filtered through 70 μm MACS Smart Strainers to prepare single-cell suspensions. Immune cell subpopulations (Treg cells and CD8 + T cells) were analyzed by flow cytometry.

[0244] The results are shown in Figure 26. When 563E10E12_hzH1L0_DLE was administered alone at doses of 5 mg / kg and 10 mg / kg, significant antitumor activity was observed in humanized mice (B-hCCR8) implanted with EMT-6 cells compared to the vehicle control group. Tumor volume increase was significantly suppressed (p<0.05) (Figure 26). Furthermore, when 563E10E12_hzH1L0_DLE was administered in combination with an anti-mPD-1 antibody (5 mg / kg) at a dose of 5 mg / kg, the tumor growth inhibition rate (TGI=94.2%) was superior to that of 563E10E12_hzH1L0_DLE alone (TGI=53.8%) and the anti-mPD-1 antibody alone (TGI=76.4%).

[0245] No obvious adverse events were observed in any of the treatment groups during the experiment. Similar to the vehicle control group, the body weight of the animals in the treatment groups gradually increased, indicating that the test substance was well tolerated by the animals (Figure 27). At the end of the experiment, the tumor Treg depletion rates in the 5 mg / kg 563E10E12_hzH1L0_DLE antibody, anti-mPD-1 antibody, and 10 mg / kg 563E10E12_hzH1L0_DLE antibody combination treatment groups were 26.3%, 24.9%, 23.4%, and 27.3%, respectively, compared to the vehicle control group, and CD8 +The ratios of T / Treg cells increased by 32.5%, 22.6%, 46.8%, and 11.5%, respectively.

[0246] 11.6 Efficacy of anti-CCR8 antibodies against the melanoma B16F10 tumor model in human CCR8-humanized mice Murine B16F10 melanoma cells were inoculated subcutaneously into the right flank of human CCR8-humanized mice (Biocytogen) at an inoculum volume of 0.1 mL / animal (in PBS). The mean tumor volume was approximately 50–100 mm. 3 At this time, mice were randomly assigned to groups and administered 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), or vehicle control (PBS) twice weekly for 3 weeks. After administration, mice were fed a normal diet, and tumor volume was recorded. At the end of the experiment, tumor samples were collected and minced with dissecting scissors. Tumor tissue was dissociated using a human tumor isolation kit (Miltenyi Biotech) and a gentle MACS separator (Miltenyi Biotech). The resulting cell suspension was filtered through 70 μm MACS Smart Strainers to create a single-cell suspension. Immune cell subpopulations (Treg cells and CD8 + T cells) were analyzed by flow cytometry.

[0247] The 563E10E12_hzH1L0_hIgG1 antibody, the Fc variants 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, and 563E10E12_hzH1L0_DLE antibodies, and the defucosylated 563E10E12_hzH1L0_AF antibody effectively suppressed tumor growth compared to the vehicle control group.

[0248] 11.7 Efficacy of anti-CCR8 antibodies against the CT26 colon cancer tumor model in CCR8-humanized mice Mouse CT26 colon cancer cells were subcutaneously inoculated into the right flank of human CCR8-humanized mice (Shanghai Model Organisms Center, Inc.) at an inoculum volume of 0.1 mL / animal (in PBS). The average tumor volume was approximately 50–100 mm. 3At this time, mice were randomly assigned to groups and administered 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), or vehicle control (PBS) twice weekly for 3 weeks. After administration, mice were fed a normal diet, and tumor volume was recorded. At the end of the experiment, tumor samples were collected and minced with dissecting scissors. Tumor tissue was isolated using a human tumor isolation kit (Miltenyi Biotech) combined with a gentleMACS separator (Miltenyi Biotech), and the resulting cell suspension was filtered through 70 μm MACS Smart Strainers to create a single-cell suspension. Immune cell subpopulations (Treg cells and CD8+ T cells) were analyzed by flow cytometry.

[0249] The 563E10E12_hzH1L0_hIgG1 antibody, the Fc variants 563E10E12_hzH1L0_VLPLL, 563E10E12_hzH1L0_DE, and 563E10E12_hzH1L0_DLE antibodies, and the defucosylated 563E10E12_hzH1L0_AF antibody effectively suppressed tumor growth compared to the vehicle control group.

[0250] It will be apparent to those skilled in the art that various modifications and variations can be made to the described methods and systems of the present invention without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology, immunology, or related fields are intended to be within the scope of the following claims. [Sequence List Free Text]

[0251] [Table SL1]

Table SL2

Table SL3

Table SL4

Table SL5

Table SL6

Table SL7

Table SL8

Table SL9

Table SL10

Table SL11

Claims

1. A CCR8-binding antibody or antigen-binding fragment thereof, having the following characteristics: (1) an EC200 / mL or less for human CCR8 and / or cynomolgus monkey CCR8 ５０ specifically binds to (2) inhibiting the binding of human and / or cynomolgus monkey CCR8 to the ligand CCL1; (3) inhibiting CCL1-induced β-arrestin recruitment; (4) CCR8 ＋ depleting Treg cells; (5) inhibiting tumor growth, preferably without affecting body weight; An antibody or antigen-binding fragment thereof having one or more of the following:

2. A CCR8-binding antibody or antigen-binding fragment thereof, (i) three complementarity-determining regions HCDR1, HCDR2, and HCDR3 of a heavy chain variable region set forth in any of SEQ ID NOs: 21 to 40, and / or three complementarity-determining regions LCDR1, LCDR2, and LCDR3 of a light chain variable region set forth in any of SEQ ID NOs: 42 to 54; (ii) A sequence having the CDR combination described in (i) above, which further includes 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), preferably amino acid substitutions, and preferably conservative amino acid substitutions, compared to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3, while maintaining affinity to CCR8. An antibody or antigen-binding fragment thereof comprising:

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

4. CCR8-binding antibodies or antigen-binding fragments thereof, as shown in the following table: Table 1 An antibody or antigen-binding fragment thereof comprising any one of the combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 shown in

5. A CCR8-binding antibody or antigen-binding fragment thereof, comprising a heavy chain variable region VH and / or a light chain variable region VL, (a) the heavy chain variable region VH is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the amino acid sequences selected from SEQ ID NOs: 21 to 40; or (ii) comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 21 to 40; or (iii) 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 any one of the amino acid sequences selected from SEQ ID NOs: 21 to 40, wherein preferably, the amino acid changes do not occur within a CDR, and preferably, the amino acid changes do not occur within a framework region (FR); and / or (b) the light chain variable region VL is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the amino acid sequences selected from SEQ ID NOs: 42-54; or (ii) comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 42 to 54; or (iii) 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 any one of the amino acid sequences selected from SEQ ID NOs: 42 to 54, wherein preferably, the amino acid changes do not occur in the CDRs, and preferably, the amino acid changes do not occur in the FRs; An antibody or antigen-binding fragment thereof.

6. CCR8-binding antibodies or antigen-binding fragments thereof, as shown in the following table: Table 2 An antibody or antigen-binding fragment thereof comprising any one combination of a heavy chain variable region VH and a light chain variable region VL shown in

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

8. 8. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain constant region and / or a light chain constant region, wherein the light chain constant region is preferably a λ or κ chain constant region; and the heavy chain constant region is selected from mouse mIgG2a, human IgG1, human IgG2, human IgG3, and IgG4 subclasses, more preferably a constant region of the human IgG1 subclass or of the human IgG4 subclass having an S228P mutation.

9. comprising a heavy chain and / or a light chain, (a) the heavy chain is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the amino acid sequences selected from SEQ ID NOs: 55 to 74; (ii) comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 55 to 74; or (iii) 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 any one of the amino acid sequences selected from SEQ ID NOs: 55 to 74, wherein preferably, the amino acid changes do not occur within the CDRs of the heavy chain variable region, more preferably, the amino acid changes do not occur within the heavy chain variable region, and most preferably, the amino acid changes occur within the heavy chain constant region; and / or (b) the light chain is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the amino acid sequences selected from SEQ ID NOs: 75 to 82; (ii) comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 75 to 82; or (iii) 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 any one of the amino acid sequences selected from SEQ ID NOs: 75 to 82, wherein preferably, the amino acid changes do not occur within the CDRs of the light chain variable region, preferably, the amino acid changes do not occur within the light chain variable region, and most preferably, the amino acid changes occur within the light chain constant region. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

10. The antibody or antigen-binding fragment thereof comprises a heavy chain constant region, the sequence of which has one or more amino acid substitutions compared to the sequence of a native human heavy chain constant region, and preferably, the one or more amino acid substitutions enhance the ADCC activity of the antibody; more preferably, the one or more amino acid substitutions are at positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 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, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 352, 354, 355, 356, 358, 359, 360, 361, 3 10. The antibody or antigen-binding fragment thereof of claim 9, wherein the antibody or antigen-binding fragment thereof occurs in one or more of: 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. 11. The antibody or antigen-binding fragment thereof of claim 10, wherein the one or more amino acid substitutions occur within the sequence of the heavy chain constant region at one or more of the following positions: L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339, and P396, as numbered according to the EU numbering system.

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

13. The antibody or antigen-binding fragment thereof according to claim 10, wherein the one or more amino acid substitutions are one or more selected from an N297A substitution, an N297Q substitution, an L235A and L237A substitution, an L234A and 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, as numbered 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 within the sequence of the heavy chain constant region at one or more of positions 235, 239, 243, 292, 300, 330, 332, and 396, as numbered by the EU numbering system.

15. 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 the substitutions S239D, L235V, F243L, R292P, Y300L, A330L, I332E, and P396L as numbered according to the EU numbering system.

16. The heavy chain constant region contains a set of simultaneous mutations at the following positions numbered according to the EU numbering system: (1) L235 / F243 / R292 / Y300 / P396, (2) F243 / R292 / Y300 / V305 / P396, (3) D270 / K326 / A330 / K334, (4) S239 / A330 / I332, and (5) S298 / E3 11. The antibody or antigen-binding fragment thereof according to claim 10, having one or more of the following residues selected from the group consisting of (1) L234 / L235 / G236 / S239 / H268 / D270 / S298, (2) M252 / S254 / T256, (3) L234 / L235 / D265, (4) G236 / S239 / I332, and (5) S239 / I332.

17. The heavy chain constant region comprises a set of mutations at the following positions numbered 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, and (6). The antibody or antigen-binding fragment thereof according to claim 10, having one or more of the following residues selected from the group consisting of (1) L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, (2) M252Y / S254T / T256E, (3) L234A / L235A / D265A, (4) L234F / L235E / D265A, (5) G236A / S239D / I332E, and (6) S239D / I332E.

18. CCR8-binding antibodies or antigen-binding fragments thereof, as shown in the following table: Table 3 An antibody or antigen-binding fragment thereof comprising any one of the combinations of heavy chain HC and light chain LC shown in

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 defucosylated or hypofucosylated.

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

21. The antigen-binding fragment may be a Fab, Fab', Fd, Fab'-SH, Fv, a single chain antibody (e.g., scFv), or (Fab'). ２ 20. The antibody or antigen-binding fragment thereof of any one of claims 1 to 19, which is an antibody fragment selected from a single domain antibody, a diabody (dAb), or a linear antibody.

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

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

24. 24. A host cell comprising a polynucleotide according to claim 22 or a vector according to claim 23, wherein preferably the host cell is a prokaryotic or eukaryotic cell, more preferably selected from a yeast cell, a mammalian cell (for example wherein the host cell is a CHO cell, such as a CHO-K1 cell or an expiCHO cell, or wherein the host cell is a 293 cell, such as a HEK293 cell), or other cell suitable for the preparation of an antibody or antigen-binding fragment thereof.

25. 22. A method for preparing the CCR8-binding antibody or antigen-binding fragment thereof, comprising culturing a host cell comprising nucleic acid encoding the antibody or antigen-binding fragment thereof of 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 another substance, such as a therapeutic agent or a label.

27. 27. A pharmaceutical composition comprising a CCR8-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, or an immunoconjugate according to claim 26, optionally together with one or more other therapeutic agents, and optionally a pharmaceutically acceptable excipient.

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 26, and optionally one or more other therapeutic agents, wherein the other therapeutic agents are preferably monoclonal antibodies, more preferably monoclonal antibodies targeting immune checkpoints, and most preferably monoclonal antibodies targeting PD-1.

29. A method for 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 described in any one of claims 1 to 21, or the polynucleotide described in claim 22, or the vector described in claim 23, or the host cell described in claim 24, or the immunoconjugate described in claim 26, or the pharmaceutical composition described in claim 27, or the combination pharmaceutical described in claim 28.

30. 20. A method for preventing and / or treating a tumor, autoimmunity, or infectious disease in a subject, comprising administering to a 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 pharmaceutical combination according to claim 28, wherein 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, or a metastatic cancer thereof.

31. 31. The method of claim 30, further comprising co-administering one or more therapeutic treatments to the subject, such as therapeutic modalities and / or other therapeutic agents, preferably comprising 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 combined medicine according to claim 28, in the manufacture of a medicament for preventing and / or treating a tumor, autoimmunity, or an infectious disease.

33. 28. 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 another therapeutic agent, in the manufacture of a medicament for preventing and / or treating a tumor, autoimmunity, or infectious disease, wherein the other therapeutic agent is preferably a monoclonal antibody, more preferably a monoclonal antibody targeting an immune checkpoint, and most preferably a monoclonal antibody targeting PD-1.

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

35. 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, renal cancer, bladder cancer, hepatocellular carcinoma, or thymic cancer, or a metastatic cancer thereof.

36. The use of any one of claims 32 to 35, further comprising administering to a subject in combination with one or more other therapeutic treatments.

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

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