Abstract 3158: CARP: a high-throughput platform for rapid VHH discovery of potent CAR-T therapy

Abstract Background: The conversion of suitable antibodies into functional CAR-T therapy presents challenges. Traditional methods for antibody discovery and screening face significant bottlenecks, including the difficulty of high-throughput identification of high-quality antibodies with diverse epitope and affinity coverage, also carry a high risk of missing functionally relevant candidates or falsely identify unsuitable candidates. Methods: Here, we introduce CARP (CAR-T AI-powered Rapid high-throughput screening Platform), a novel solution designed to rapidly identify VHHs with clinical potential in CAR structures. Our screening platform was applied for the development of CAR T-cell therapy targeting Cadherin 17 (CDH17). Following immunization of alpacas and llamas, hundreds of CDH17 VHH hits were analyzed using an AI-powered platform for lineage determination (epitope coverage), humanization, and immunogenicity profiling. A library of >400 CDH17 VHHs was screened in serial proliferation tumor cell line co-culture assays simulating long-term tumor-CAR-T interactions to evaluate their target-specific expansion capacity in CAR-T format. Results: After screening and validation of individual VHH candidates, CDH17 CAR hits consistently outperformed VHHs via traditional methods in functional assays, demonstrating superior activity and robustness. Moreover, CARP successfully identified multiple VHHs with identical target binding domain that demonstrated superior target-dependent expansion compared to clinical CDH17 CAR benchmarks and traditionally selected VHHs. Conclusion: Our CAR VHH platform not only accelerates the discovery process by enabling high-throughput screening to identify highly target-dependent and proliferative VHH leads, but also eliminates the need for additional rounds of immunization and immunogenicity assessments once lead VHHs are identified. CARP offers a powerful, efficient approach for uncovering VHHs that enhance CAR-T cell functionality, providing significant promise for advancing CAR-T therapy development. Citation Format: Weihong Wang, Huiyuan Tang, John Lee, Xinhao Wang, Leo Ng, Yong Wang, Toya N. Baral, Jia Yu, Jiaguo Li, Wenfeng Xu. CARP: a high-throughput platform for rapid VHH discovery of potent CAR-T therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3158.