Structure-Guided Computational Engineering of pH-Selective B7-H3 CAR-T Cells to Mitigate On-Target/Off-Tumor Toxicity 2328499

Abstract Introduction Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy in treating hematological malignancies. However, its application in solid tumors is severely limited by on-target/off-tumor (OTOT) toxicity due to the recognition of shared antigens on healthy tissues. While strictly tumor-specific antigens are ideal, they are scarce, and clinically relevant targets like B7-H3 often exhibit basal expression in normal organs. Consequently, strategies to restrict CAR-T activation strictly to the tumor site are urgently needed. The acidic tumor microenvironment (TME), a hallmark of solid tumors distinct from physiological pH, presents a unique opportunity to improve specificity. Methods In this study, we addressed OTOT toxicity by engineering pH-dependent CAR-T cells that selectively engage B7-H3 under acidic conditions. Utilizing a structure-based computational approach, we performed in silico dual-pH scanning mutagenesis–systematically substituting residues within the complementarity-determining regions (CDRs) and paratopic interfaces with either Histidine (H) or Aspartic acid (D). This combinatorial strategy was designed to identify optimal electrostatic networks that destabilize binding at neutral pH (7.4) while preserving or enhancing affinity at acidic pH (6.5), thereby creating a precise molecular switch for tumor-specific activation. Results We identified 16 scFv variants exhibiting significant pH-selective binding profiles. Upon incorporation into CAR constructs, two lead variants demonstrated pH-dependent functional activity. These engineered CAR-T cells showed abolished activation against B7-H3 cells at neutral pH but exhibited robust activation and cytotoxicity in acidic environments. Conclusion This study validates the feasibility of using computational design to harness the acidic TME for safety tuning. Our findings establish a generalizable paradigm for developing “smart” CAR-T therapies that decouple potent anti-tumor activity from systemic toxicity. Funding Source n/a Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)