Abstract LB368: A first-in-class CTLA-4×TIGIT BsAb selectively targets tumor-associated Treg cells to improve the safety profile of CTLA-4 targeting therapy

Abstract Background: Ipilimumab, an immune-checkpoint inhibitor, has demonstrated clinical benefits but is also associated with adverse events due to its effects on peripheral regulatory T cells (Tregs). Thus, selective targeting tumor-associated Tregs could improve the safety profile of ipilimumab. Based on this hypothesis, we developed a CTLA-4×TIGIT bispecific antibody (BsAb). By targeting CTLA-4 and TIGIT, it could selectively engage tumor-associated Tregs that co-express both markers, while minimizing its impact on peripheral Tregs, thereby reducing the toxicity associated with ipilimumab. Method: Bioinformatics was applied to analyze the expression profile of CTLA-4 and TIGIT on immune cells including Tregs derived from human peripheral blood mononuclear cells and tumor infiltrating lymphocytes. In vitro, we established CHO-K1-CTLA4 cells representing peripheral Tregs and CHO-K1-CTLA-4/TIGIT cells representing tumor-associated Tregs. CTLA-4/CD80 blockage and ADCC activity of CTLA-4×TIGIT BsAb were determined on these cells. Functionally, we evaluated T cell activation with CTLA-4×TIGIT BsAb treatment. ADCC activity of BsAb towards ex vivo expanded Tregs was also determined. In vivo, the MC38 syngeneic model in hCTLA-4/hTIGIT C57BL/6 mice was applied to validate the anti-tumor activity of the BsAb. The safety profile was evaluated in cynomolgus monkeys through a 4-week Good Laboratory Practice (GLP) toxicity study. Results: Single-cell sequencing analysis revealed the selective co-expression of CTLA-4 and TIGIT on tumor-associated Tregs compared to peripheral Tregs. CTLA-4×TIGIT BsAb was designed with one arm targeting CTLA-4 and another arm targeting TIGIT. Compared to the parental CTLA-4 antibody, the monovalent binding to CTLA-4 reduced the binding and blocking activity in CHO-K-1-CTLA4 cells. But due to its co-targeting to CTLA-4 and TIGIT, CTLA-4×TIGIT BsAb exerted good binding avidity and CTLA-4/CD80 blockage activity in CHO-K1-CTLA-4/TIGIT cells. Moreover, it exhibited potent ADCC activity towards CHO-K1-CTLA-4/TIGIT cells and ex vivo expanded Tregs. In the MC38 syngeneic model, CTLA-4×TIGIT BsAb exhibited potent anti-tumor activity and selective depletion of tumor-associated Tregs without affecting peripheral Tregs. In the GLP toxicity study, the CTLA-4×TIGIT BsAb exhibited a more favorable safety profile than CTLA-4 mAbs such as ipilimumab and tremelimumab. Conclusion: Our preclinical study thoroughly evaluated the in vitro and in vivo activity of the CTLA-4×TIGIT BsAb and demonstrated its mechanism of action in targeting tumor-associated Tregs, which confirms that the specificity towards tumor-associated Treg cells preserves efficient anti-tumor activity while enhancing the safety profile of CTLA-4 targeting therapy. The GLP toxicity study demonstrated that CTLA-4×TIGIT BsAb is expected to reduce peripheral adverse reactions and improve tolerance. The promising preclinical data support its advancing into clinical testing, and an Investigational New Drug (IND) application to NMPA has been submitted in Dec 2024. Citation Format: Hong Yan, Luyao Bao, Chenglong Liu, Simeng Chen, Wei Zhang, Xing Sun, Wenjing Cheng, Zeyun Xue, Wei Wang, Jimin Yuan, Cheng Liao. A first-in-class CTLA-4×TIGIT BsAb selectively targets tumor-associated Treg cells to improve the safety profile of CTLA-4 targeting therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB368.