The cells were stained with antibodies for cell-surface antigens and treated with Transcription Factor Buffer Set (BD Biosciences) for intracellular staining. cells that are essential for maintaining immune homeostasis, Tregs that infiltrate tumor tissue promote tumor growth by suppressing antitumor immunity. Selective reduction of tumor-infiltrating Tregs is, therefore, expected to activate antitumor immunity without affecting immune homeostasis. We previously reported that selective Treg depletion targeted by a C-C motif chemokine receptor 8 (CCR8) resulted in induction of strong antitumor immunity without any obvious autoimmunity in mouse models. Thus, herein, we developed a novel humanized anti-CCR8 monoclonal antibody, S-531011, aimed as a cancer immunotherapy strategy for patients with cancer. S-531011 exclusively recognized human CCR8 among all chemokine receptors and showed β3-AR agonist 1 potent antibody-dependent cell-mediated cytotoxicity activity toward CCR8+ cells and neutralization activity against CCR8-mediated signaling. We observed that S-531011 reduced tumor-infiltrating CCR8+ Tregs and induced potent antitumor activity in a tumor-bearing human-CCR8 knock-in mouse model. Moreover, combination therapy with S-531011 and anti-mouse programmed cell death 1 (PD-1) antibody strongly suppressed tumor growth compared with antiCPD-1 antibody alone with no observable adverse effects. S-531011 also depleted human tumor-infiltrating Tregs, but not Tregs derived from human peripheral blood mononuclear cells. These results suggest that S-531011 is a promising drug for inducing antitumor immunity without severe side effects in the clinical setting. Introduction Immunotherapy is becoming a standard treatment for various types of cancers. Although immune-checkpoint inhibitors (ICI), such as anti-programmed cell death 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) antibodies, have shown clinical benefits in patients with cancer, most patients are not cured (1, 2). One of the proposed mechanisms of resistance to ICIs involves immunosuppressive Rabbit polyclonal to Smac cells, such as tumor-associated macrophage, myeloid-derived suppresser cells, and regulatory T cells (Treg), which are present in the tumor microenvironment and inhibit the function of effector T cells (3). Tregs are inhibitory immune cells essential for maintaining immune homeostasis. Indeed, Tregs dysfunction caused by mutations or Tregs depletion results in the onset of severe autoimmune diseases in both humans and mice (4, 5). β3-AR agonist 1 On the contrary, it has been reported that tumor-infiltrating Tregs assist tumor growth by suppressing antitumor immunity (6). These findings suggest that targeting tumor-infiltrating Tregs without affecting normal tissue-resident Tregs may be safer compared with targeting systemic Tregs. Therefore, a molecule that is selectively expressed in tumor-infiltrating Tregs can be considered a suitable target molecule for Treg-targeting cancer immunotherapy. Based on this rationale, we identified a C-C motif chemokine receptor 8 (CCR8), which is expressed mainly on tumor-infiltrating Tregs, as a target molecule (7). CCR8 is a seven-transmembrane chemokine receptor (8, 9). C-C chemokine ligand (CCL) 1 is one of the ligands of CCR8 and has been reported to play a major role in potentiating Treg-suppressive activity (10). On the other hand, the functional involvement of CCR8 signaling in tumor-infiltrating Tregs is still unclear. Although some reports show that CCR8 signaling is not necessary for suppressing tumor immunity (11, 12), CCR8 blockade prevents the induction of Tregs and β3-AR agonist 1 inhibits their suppressive function, which likely attenuates immunosuppression, reinvigorating antitumor immunity (13). Previously, we reported that an anti-mouse CCR8 antibody showed strong antitumor effects, without causing autoimmunity, in a tumor-bearing mouse model (7). Other studies have also reported antitumor effects of anti-mouse CCR8 antibodies in mouse models (11, 14, 15). In humans, high CCR8 expression on tumor-infiltrating Tregs has been reported to be correlated with poor prognosis in several types of cancers (16, 17). These findings suggest that selective depletion of CCR8+ Tregs using an anti-human CCR8 antibody can potentially restore antitumor immunity, thereby exerting antitumor effects in patients with cancer. In this study, we developed a novel humanized anti-human CCR8 antibody, S-531011, which has antibody-dependent cell-mediated cytotoxicity (ADCC) activity against tumor-infiltrating CCR8+ Tregs and neutralization activity against the receptor signaling. We.