2026_Warmuth_Doetsch_affinity-combined-CAR-T_cell-products_scRNAseq

Adoptive cell therapy with genetically engineered T cells expressing Chimeric Antigen Receptors (CARs) of high binding affinity has proven to be remarkably successful in refractory and relapsed B-cell malignancies. However, its broader clinical potential remains underexploited by the development of side effects due to intense CAR-T cell activation, as well as tumor relapse due to antigen-escape and limited persistence of CAR-T cells during sustained activation via high-affinity receptors. These challenges highlight the critical need for strategies that maximize the safety and efficacy of CAR-T cell therapies. Recent studies suggest that optimal adjustments of the CAR binding affinity to its ligand offers promising opportunities. In this study, we characterized a broad range of CARs with varying affinities for the same target epitope down to physiological TCR-like binding interactions. While CAR affinity had minimal impact on in vitro functionality across a broad range, it strongly correlated with tumor control in vivo, which was significantly compromised for low-affinity binders. On the other hand, CARs with lower affinity induced only mild cytokine release syndrome (CRS) in humanized mouse models. Based on these findings, we implemented an affinity-combination approach to leverage the strong anti-tumor efficacy of high-affinity CARs alongside the enhanced safety profile of lower-affinity CARs. We observed strong synergistic effects in combined products, like lower monocyte-induced cytokine production as compared to high-affinity CAR-T cells alone, while preserving efficacy. High-affinity CARs maintained strong functionality in mixtures with low-affinity CARs going along with strongly reduced signs of exhaustion when applied in combination. In long-term in vitro and in vivo settings, low-affinity CAR-T cells dominated over time proving more resilience to chronic antigen exposure, comparable to physiological T cell responses. Overall, our findings demonstrate that affinity-combination might be an attractive strategy to generate more effective CAR-T cell products with an improved therapeutic index.