C₈Mab-21: A novel anti-human CCR8 monoclonal antibody for flow cytometry

Abstract

C-C motif chemokine receptor-8 (CCR8) belongs to class A of G protein-coupled receptors. CCR8 interacts with the specific chemokine ligand CCL1/I-309 in humans, which is produced by various cells, including tumor-associated macrophages and regulatory T cells (Treg). CCR8 is highly expressed on Treg and T-helper 2 cells recruited to the inflammation site and is implicated in allergy, asthma, and cancer progression. CCR8+Treg cells have been suggested an important regulator in the immunosuppressive tumor microenvironment. Therefore, it has been proposed for use in the development of sensitive monoclonal antibodies targeting CCR8. This study developed a specific mAb for human CCR8 (hCCR8), which is useful for flow cytometry by employing the Cell-Based Immunization and Screening (CBIS) method. The established anti-hCCR8 mAb (C₈Mab-21; mouse IgM, kappa) demonstrated reactivity with hCCR8-overexpressed Chinese hamster ovary-K1 (CHO/hCCR8) cells, TALL-1 (human adult acute T-lymphoblastic leukemia), CCRF-HSB2 (human T-lymphoblastic leukemia), and natural killer cells expressing endogenous hCCR8, as confirmed by flow cytometry. Furthermore, EC₅₀ values of C8Mab-21 for CHO/hCCR8 and TALL-1 were determined as 6.5 × 10⁻⁸ M and 2.0 × 10⁻⁸ M, respectively. C₈Mab-21, established by the CBIS method, provides a useful tool for analyzing the hCCR8-related biological response using flow cytometry.

Keywords

CCR8 / CBIS method / Monoclonal antibody / Flow cytometry

1 Funding

This research was supported, in part, by the Japan Agency for Medical Research and Development (AMED) under Grant Nos.: JP24am0521010 (to Y.K.), JP23ama121008 (to Y.K.), JP23bm1123027 (to Y.K.), and JP23ck0106730 (to Y.K.), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Grant Nos.: 22K06995 (to H.S.) and 22K07224 (to Y. K).

2 Conflict of interest

The authors declare no conflicts of interest.

References

[1] Bruni D, Angell HK, Galon J. The immune contexture and Immunoscore in cancer prognosis and therapeutic efficacy. Nat Rev Cancer. 2020; 20(11):662-680. doi: 10.1038/s41568-020-0285-7

[2] Demaria O, Cornen S, Daeron M, Morel Y, Medzhitov R, Vivier E. Harnessing innate immunity in cancer therapy. Nature. 2019; 574(7776):45-56. doi: 10.1038/s41586-019-1593-5

[3] Ribas A, Wolchok JD.Cancer immunotherapy using checkpoint blockade. Science. 2018; 359(6382):1350-1355. doi: 10.1126/science.aar4060

[4] Karin N. Chemokines and cancer: New immune checkpoints for cancer therapy. Curr Opin Immunol. 2018; 51:140-145. doi: 10.1016/j.coi.2018.03.004

[5] van den Bulk J, Verdegaal EM, de Miranda NF. Cancer immunotherapy: Broadening the scope of targetable tumours. Open Biol. 2018; 8(6):180037. a)doi: 10.1098/rsob.180037

[6] Morad G, Helmink BA, Sharma P, Wargo JA. Hallmarks of response, resistance, and toxicity to immune checkpoint blockade. Cell. 2021; 184(21):5309-5337. doi: 10.1016/j.cell.2021.09.020

[7] Lipson EJ, Forde PM, Hammers HJ, Emens LA, Taube JM, Topalian SL. Antagonists of PD-1 and PD-L1 in cancer treatment. Semin Oncol. 2015; 42(4):587-600. doi: 10.1053/j.seminoncol.2015.05.013

[8] Li C, Jiang P, Wei S, Xu X, Wang J. Regulatory T cells in tumor microenvironment: New mechanisms, potential therapeutic strategies and future prospects. Mol Cancer. 2020; 19(1):116. doi: 10.1186/s12943-020-01234-1

[9] Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol. 2007; 8(3): 239-245. doi: 10.1038/ni1443

[10] Yano H, Andrews LP, Workman CJ, Vignali DAA. Intratumoral regulatory T cells: Markers, subsets and their impact on anti-tumor immunity. Immunology. 2019; 157(3):232-247. doi: 10.1111/imm.13067

[11] Ohue Y, Nishikawa H. Regulatory T (Treg) cells in cancer: Can Treg cells be a new therapeutic target? Cancer Sci. 2019; 110(7):2080-2089. doi: 10.1111/cas.14069

[12] Tanaka A, Sakaguchi S. Regulatory T cells in cancer immunotherapy. Cell Res. 2017; 27(1):109-118. doi: 10.1038/cr.2016.151

[13] Fridman WH, Pagès F, Sautès-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012; 12(4):298-306. doi: 10.1038/nrc3245

[14] Sakaguchi S. Regulatory T cells: Key controllers of immunologic self-tolerance. Cell. 2000; 101(5):455-458. doi: 10.1016/s0092-8674(00)80856-9

[15] Brunkow ME, Jeffery EW, Hjerrild KA, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001; 27(1):68-73. doi: 10.1038/83784

[16] Takahashi T, Kuniyasu Y, Toda M, et al. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: Induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol. 1998; 10(12):1969-1980. doi: 10.1093/intimm/10.12.1969

[17] Tay C, Tanaka A, Sakaguchi S. Tumor-infiltrating regulatory T cells as targets of cancer immunotherapy. Cancer Cell. 2023; 41(3):450-465. doi: 10.1016/j.ccell.2023.02.014

[18] Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003; 4(4):330-336. doi: 10.1038/ni904

[19] Mortezaee K. Selective targeting or reprogramming of intra-tumoral Tregs. Med Oncol. 2024; 41(3):71. doi: 10.1007/s12032-024-02300-0

[20] Wakiyama H, Kato T, Furusawa A, et al. Treg-dominant tumor microenvironment is responsible for hyperprogressive disease after PD-1 blockade therapy. Cancer Immunol Res. 2022; 10(11):1386-1397. doi: 10.1158/2326-6066.Cir-22-0041

[21] Guan X, Hu R, Choi Y, et al. Anti-TIGIT antibody improves PD-L1 blockade through myeloid and T(reg) cells. Nature. 2024;627:646-655. doi: 10.1038/s41586-024-07121-9

[22] Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell. 2017; 168(4):707-723. doi: 10.1016/j.cell.2017.01.017

[23] Wang L, Simons DL, Lu X, et al. Connecting blood and intratumoral Treg cell activity in predicting future relapse in breast cancer. Nat Immunol. 2019; 20(9):1220-1230. doi: 10.1038/s41590-019-0429-7

[24] De Simone M, Arrigoni A, Rossetti G, et al. Transcriptional landscape of human tissue lymphocytes unveils uniqueness of tumor-infiltrating T regulatory cells. Immunity. 2016; 45(5):1135-1147. doi: 10.1016/j.immuni.2016.10.021

[25] Plitas G, Konopacki C, Wu K, et al. Regulatory T cells exhibit distinct features in human breast cancer. Immunity. 2016; 45(5):1122-1134. doi: 10.1016/j.immuni.2016.10.032

[26] Barsheshet Y, Wildbaum G, Levy E, et al. CCR8(+)FOXp3(+) Treg cells as master drivers of immune regulation. Proc Natl Acad Sci U S A. 2017; 114(23):6086-6091. doi: 10.1073/pnas.1621280114

[27] Nagira Y, Nagira M, Nagai R, et al. S-531011, a novel anti-human CCR8 antibody, induces potent antitumor responses through depletion of tumor-infiltrating CCR8-expressing regulatory T cells. Mol Cancer Ther. 2023; 22(9):1063-1072. doi: 10.1158/1535-7163.Mct-22-0570

[28] Wu Y, Xi J, Li Y, et al. Discovery of a potent and selective CCR8 small molecular antagonist IPG7236 for the treatment of cancer. J Med Chem. 2023; 66(7):4548-4564. doi: 10.1021/acs.jmedchem.3c00030

[29] Kim N, Kim MH, Pyo J, et al. CCR8 as a therapeutic novel target: Omics-integrated comprehensive analysis for systematically prioritizing indications. Biomedicines. 2023; 11(11):2910. doi: 10.3390/biomedicines11112910

[30] Märkl F, Huynh D, Endres S, Kobold S.Utilizing chemokines in cancer immunotherapy. Trends Cancer. 2022; 8(8): 670-682. doi: 10.1016/j.trecan.2022.04.001

[31] Bernardini G, Spinetti G, Ribatti D, et al. I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo. Blood. 2000; 96(13):4039-4045.

[32] Goya I, Gutiérrez J, Varona R, et al. Identification of CCR8 as the specific receptor for the human beta-chemokine I-309: Cloning and molecular characterization of murine CCR8 as the receptor for TCA-3. J Immunol. 1998; 160(4):1975-1981.

[33] Liu X, Xu X, Deng W, et al. CCL18 enhances migration, invasion and EMT by binding CCR8 in bladder cancer cells. Mol Med Rep. 2019; 19(3):1678-1686. doi: 10.3892/mmr.2018.9791

[34] Spinetti G, Bernardini G, Camarda G, et al. The chemokine receptor CCR8 mediates rescue from dexamethasone-induced apoptosis via an ERK-dependent pathway. J Leukoc Biol. 2003; 73(1):201-207. doi: 10.1189/jlb.0302105

[35] Denis C, Deiteren K, Mortier A, et al. C-terminal clipping of chemokine CCL1/I-309 enhances CCR8-mediated intracellular calcium release and anti-apoptotic activity. PLoS One. 2012; 7(3):e34199. doi: 10.1371/journal.pone.0034199

[36] Campbell JR, McDonald BR, Mesko PB, et al. Fc-optimized anti-CCR8 antibody depletes regulatory T cells in human tumor models. Cancer Res. 2021; 81(11):2983-2994. doi: 10.1158/0008-5472.Can-20-3585

[37] Saito M, Harigae Y, Li G, et al. C(3)Mab-2: An anti-mouse CCR3 monoclonal antibody for immunocytochemistry. Monoclon Antib Immunodiagn Immunother. 2022; 41(1): 45-49. doi: 10.1089/mab.2021.0050

[38] Saito M, Suzuki H, Tanaka T, et al. Development of an anti-mouse CCR8 monoclonal antibody (C(8)Mab-1) for flow cytometry and immunocytochemistry. Monoclon Antib Immunodiagn Immunother. 2022; 41(6):333-338. doi: 10.1089/mab.2021.0069

[39] Nanamiya R, Takei J, Asano T, et al. Development of anti-human CC chemokine receptor 9 monoclonal antibodies for flow cytometry. Monoclon Antib Immunodiagn Immunother. 2021; 40(3):101-106. doi: 10.1089/mab.2021.0007

[40] Ouchida T, Suzuki H, Tanaka T, Kaneko MK, Kato Y. Cx(4)Mab-1: A novel anti-mouse CXCR4 monoclonal antibody for flow cytometry. Monoclon Antib Immunodiagn Immunother. 2024; 43(1):10-16. doi: 10.1089/mab.2023.0023

[41] Schöneberg T, Liebscher I. Mutations in G protein-coupled receptors: Mechanisms, pathophysiology and potential therapeutic approaches. Pharmacol Rev. 2021; 73(1):89-119. doi: 10.1124/pharmrev.120.000011

[42] Hauser AS, Attwood MM, Rask-Andersen M, Schiöth HB, Gloriam DE. Trends in GPCR drug discovery: New agents, targets and indications. Nat Rev Drug Discov. 2017; 16(12):829-842. doi: 10.1038/nrd.2017.178

[43] Proudfoot AE.Chemokine receptors: Multifaceted therapeutic targets. Nat Rev Immunol. 2002; 2(2):106-115. doi: 10.1038/nri722

[44] Jo M, Jung ST. Engineering therapeutic antibodies targeting G-protein-coupled receptors. Exp Mol Med. 2016; 48(2):e207. doi: 10.1038/emm.2015.105

[45] Itai S, Yamada S, Kaneko MK, et al. Establishment of EMab- 134, a sensitive and specific anti-epidermal growth factor receptor monoclonal antibody for detecting squamous cell carcinoma cells of the oral cavity. Monoclon Antib Immunodiagn Immunother. 2017; 36(6):272-281. doi: 10.1089/mab.2017.0042

[46] Asano T, Ohishi T, Takei J, et al. AntiHER3 monoclonal antibody exerts antitumor activity in a mouse model of colorectal adenocarcinoma. Oncol Rep. 2021; 46(2):173. doi: 10.3892/or.2021.8124

[47] Tanaka T, Ohishi T, Asano T, et al. An antiTROP2 monoclonal antibody TrMab6 exerts antitumor activity in breast cancer mouse xenograft models. Oncol Rep. 2021; 46(1):132. doi: 10.3892/or.2021.8083

[48] Kudo Y, Suzuki H, Tanaka T, Kaneko MK, Kato Y. Development of a novel anti-CD44 variant 5 monoclonal antibody C(44)Mab-3 for multiple applications against pancreatic carcinomas. Antibodies (Basel). 2023; 12(2):31. doi: 10.3390/antib12020031

[49] Suzuki H, Kaneko MK, Kato Y. Roles of podoplanin in malignant progression of tumor. Cells. 2022; 11(3):575. doi: 10.3390/cells11030575

[50] Kaneko MK, Suzuki H, Kato Y. Establishment of a novel cancer-specific anti-HER2 monoclonal antibody H(2)Mab- 250/H(2)CasMab-2 for breast cancers. Monoclon Antib Immunodiagn Immunother. 2024; 43(2):35-43. doi: 10.1089/mab.2023.0033

[51] Arimori T, Mihara E, Suzuki H, et al. Locally misfolded HER2 expressed on cancer cells is a promising target for development of cancer-specific antibodies. Structure. 2024; 32:536-549.e5. doi: 10.1016/j.str.2024.02.007

[52] Ueyama A, Nogami W, Nashiki K, et al. Immunotherapy targeting CCR8+ regulatory T cells induces antitumor effects via dramatic changes to the intratumor CD8+ T cell profile. J Immunol. 2023; 211(4):673-682. doi: 10.4049/jimmunol.2300067

[53] Kidani Y, Nogami W, Yasumizu Y, et al. CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory. Proc Natl Acad Sci U S A. 2022; 119(7):e2114282119. doi: 10.1073/pnas.2114282119

[54] Zheng D, Wang X, Cheng L, et al. The chemokine receptor CCR8 is a target of chimeric antigen T cells for treating T cell malignancies. Front Immunol. 2022;13:808347. doi: 10.3389/fimmu.2022.808347

[55] Li G, Suzuki H, Ohishi T, et al. Antitumor activities of a defucosylated antiEpCAM monoclonal antibody in colorectal carcinoma xenograft models. Int J Mol Med. 2023; 51(2):18. a)doi: 10.3892/ijmm.2023.5221

[56] Niwa R, Satoh M. The current status and prospects of antibody engineering for therapeutic use: focus on glycoengineering technology. J Pharm Sci. 2015; 104(3): 930-941. doi: 10.1002/jps.24316

[57] Kalluri R. The biology and function of fibroblasts in cancer. Nat Rev Cancer. 2016; 16(9):582-598.doi: 10.1038/nrc.2016.73

[58] Santi A, Kay EJ, Neilson LJ, et al. Cancer-associated fibroblasts produce matrix-bound vesicles that influence endothelial cell function. Sci Signal. 2024; 17(827):eade0580. doi: 10.1126/scisignal.ade0580

[59] Eskandari-Malayeri F, Rezaei M. Immune checkpoint inhibitors as mediators for immunosuppression by cancer-associated fibroblasts: A comprehensive review. Front Immunol. 2022;13:996145. doi: 10.3389/fimmu.2022.996145

[60] Cords L, Engler S, Haberecker M, et al. Cancer-associated fibroblast phenotypes are associated with patient outcome in non-small cell lung cancer. Cancer Cell. 2024; 42(3):396-412.e395. doi: 10.1016/j.ccell.2023.12.021