Abstract 6350: 2MW4691, a novel CCR8/CTLA-4 bispecific antibody, displays potent anti-tumor efficacy by specifically depeleting tumor-infiltrating Tregs and blocking CTLA-4 signaling on CD8+ T cells

Abstract Background: Regulatory T cells (Tregs) are one of the representative immunosuppressive cells playing a critical role in tumor microenvironment by suppressing anti-tumor immune responses. A great many Treg-depleting therapeutics targeting CTLA-4, CCR8, and CD25 are currently being investigated in clinical trials, and the overwhelming majority of these studies showed systemic toxicity or limited efficacy. Novel modalities favoring tumor-infiltrating Tregs depletion are urgently needed. Here, a CCR8/CTLA-4 bispecific antibody 2MW4691 with inequivalent target arm affinities was developed to specifically deplete tumor-infiltrating Tregs and block CTLA-4 signaling on CD8+ T cells. Methods: Single-cell RNA sequencing data collection and computational analysis were applied to investigate CCR8 and CTLA-4 gene expression profiles in tumor-infiltrating Tregs. 2MW4691 was generated by combining parental bivalent anti-CCR8 and anti-CTLA-4 antibodies with different target arm affinities. CCR8 and CTLA-4 single and double-positive cells were used to evaluate the binding and blocking abilities of 2MW4691. Human FcγRIIIa receptor system, NK cells, and hPBMCs were used to assess the ADCC activity of 2MW4691. hCCR8/hCTLA-4 double transgenic mouse syngeneic models were used to evaluate the anti-tumor efficacy of 2MW4691 in vivo. A repeat dose administration study was performed in cynomolgus monkeys to evaluate the pharmacokinetic and toxicity profiles of 2MW6471. Results: Single-cell RNA sequencing analysis confirmed the co-expression of CCR8 and CTLA-4 in tumor-infiltrating Tregs of NSCLC patients. 2MW4691 was designed as a tetravalency symmetric structure with high binding affinity to CCR8 and low binding affinity to CTLA-4, which resulted in strong ADCC activity towards CCR8 single and CCR8/CTLA4 double-positive cells, but limited ADCC activity towards CTLA-4 single positive cells in cell-based assays. In the staphylococcal enterotoxin B stimulation assay, 2MW4691 promoted T-cell activation, suggesting that it maintained the ability to block CTLA-4-mediated downstream signaling from its parental anti-CTLA-4 antibody. In vivo, 2MW4691 exhibited potent anti-tumor efficacy in hCCR8 or hCTLA4 humanized mice bearing MC38 tumors. Further, the advantage in tumor inhibition of 2MW4691 compared with its parental antibodies was proved in the hCCR8/hCTLA-4 double knock-in mouse model. In the NHP study, 2MW4691 exhibited good pharmacokinetic and safety profiles with negligible impact on peripheral T cells. Conclusion: 2MW4691 demonstrated superior anti-tumor efficacy compared with its parental antibodies in CDX models and favorable tolerability in NHPs. The encouraging preclinical results support further evaluation of 2MW4691 in clinical trials. Citation Format: Cuicui Guo, Xiaodong Dai, Xiumei Xiong, Hongqin Liu, Xiaowei Cen, Hai Wu, Jinchao Zhang, Xun Gui. 2MW4691, a novel CCR8/CTLA-4 bispecific antibody, displays potent anti-tumor efficacy by specifically depeleting tumor-infiltrating Tregs and blocking CTLA-4 signaling on CD8+ T cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6350.