Fast receptor-antigen dissociation kinetics promote CAR-T cell tumor infiltration, functional fitness, and antitumor efficacy in solid tumors

Background: CAR-T cells have shown limited efficacy against solid tumors, where they face two major challenges: restricted tumor infiltration and sustained intensive engagement that drive them to exhaustion. Conventional CAR design has largely emphasized high-affinity interactions with slow dissociation, further limiting tumor infiltration and exacerbating exhaustion and dysfunction. We hypothesize that most current CARs are kinetically over-stabilized for solid tumors and that a fast-on/fast-off, cheek-kiss mode of engagement permits transient CAR-antigen disengagement, thereby facilitating tumor infiltration and preserving CAR-T cell fitness in tumor mass. Methods: We identified two monoclonal antibodies targeting the same epitope of epidermal growth factor receptor with comparable kon but distinct koff rates: cetuximab (Cet) and its humanized derivative H1. CAR-T cells incorporating these binders were evaluated in vitro and in vivo. Large, established human tumor xenografts were treated with low-dose CAR-T cells in NSG mice to model conditions of limited T cell availability and sustained tumor antigen exposure. Results: Cet and H1 exhibited comparable kon but markedly different koff rates, with H1 displaying fast-off kinetics with approximately 18-fold shorter binding dwell time. As CARs, H1 showed reduced antigen occupancy and less stable tumor-T cell engagement than Cet. Despite comparable in vitro cytotoxicity, H1 CAR-T cells accumulated more efficiently following tumor stimulation. At high doses, both CAR-T cell products eradicated established tumors. Under low-dose conditions, however, H1 CAR-T cells exhibited superior expansion, persistence, tumor infiltration, and antitumor efficacy, whereas Cet CAR-T cells showed limited expansion and only transient tumor control. Cet CAR-T cells progressively downregulated surface CAR expression, increased PD-1 expression, and exhibited reduced tumor infiltration and impaired functional recovery. In contrast, H1 CAR-T cells preserved surface CAR expression, exhibited greater tumor infiltration, maintained a less exhausted, more central memory-like phenotype, and retained effector function. Conclusion: Fast-on/fast-off binding kinetics enhance CAR-T tumor infiltration, expansion, persistence, and functional fitness, resulting in improved control of established solid tumors. The data provides a rationale for shifting CAR design from affinity optimization toward kinetic optimization to improve the efficacy and safety of CAR-T therapy for solid tumors.