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 (GPCRs). 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 (TH2) cells recruited to the inflammation site and is implicated in allergy, asthma, and cancer progression. The CCR8+Treg has been suggested to be an important regulator in the immunosuppressive tumor microenvironment (TME); therefore, it has been desired to develop sensitive monoclonal antibodies (mAbs) for 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) reacted with hCCR8-overexpressed Chinese hamster ovary-K1 (CHO/hCCR8) cells, TALL-1 (acute T lymphoblastic leukemia), CCRF-HSB2 (human T-lymphoblastic leukemia), and natural killer cells, which express endogenous hCCR8 by flow cytometry. Furthermore, C8Mab-21 demonstrated a moderate binding affinity for CHO/hCCR8 and TALL-1 with a dissociation constant of 6.5×10-8 M and 2.0×10-8 M, respectively. C8Mab-21, which was established by the CBIS method, could be a useful tool for analyzing the hCCR8-related biological response using flow cytometry.