Pooled Knock-In Screening for Enhanced Chimeric Antigen Receptor (CAR) Discovery

• Applicant: Dr. Jonas Kath
• Subject Area: Hematology, Oncology ; Immunology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 567650389

Chimeric Antigen Receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies but faces significant challenges in solid tumors, including insufficient tumor infiltration, cellular exhaustion, and inefficient antigen binding. A key issue lies in the design principles of conventional CARs, which differ from natural T-cell receptors. To accelerate the development of more effective CAR constructs, we employ a high-throughput approach for simultaneous screening of AI-generated and recombinantly assembled CAR libraries. Using CRISPR-Cas-mediated gene transfer, we optimize CAR-T cells for improved safety, cost-efficiency, and functional adaptability. A major focus is on CD3ε-fusion constructs (eTRuCs), which integrate CARs into the natural T-cell receptor complex, thereby enhancing sensitivity and efficacy. In an initial proof-of-concept study, a CAR library featuring Her2-specific nanobodies was generated and integrated into primary human T cells using pooled knock-in strategies. After stimulation with various cell models and subsequent long-read NGS analysis, specific CARs were identified that selectively target tumors while sparing healthy tissue. The project aims to develop highly specific CAR-T cells with improved antigen sensitivity and long-term functionality. The libraries are created in a stepwise manner with increasing complexity: Phase 1 focuses on small, AI-driven libraries targeting a single antigen. In Phase 2, highly diverse libraries with unknown specificity are introduced to identify novel target antigens. Phase 3 involves optimizing the entire CAR architecture through directed and partially directed recombinant assembly to generate innovative receptor structures. This strategy differs from traditional, hypothesis-driven CAR designs by enabling unbiased selection of the most effective receptors. The combination of CRISPR, AI-generated libraries, and high-throughput screening has the potential to significantly enhance CAR-T cell efficacy against solid tumors and unlock new therapeutic options.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professorin Dr. Cliona Rooney