Controlling TCR and CAR activation by targeting LCK recruitment with a first-in-class small-molecule inhibitor

T-cell activation is driven by the recruitment of lymphocyte-specific protein tyrosine kinase (LCK) to the T-cell receptor (TCR), a critical step in initiating immune responses. Existing LCK inhibitors lack specificity because they target the conserved kinase domain shared by Src family kinases, resulting in off-target effects. Here, we introduce a novel strategy to selectively modulate T-cell activation by disrupting the interaction between the SH3 domain of LCK and the receptor kinase (RK) motif of CD3{varepsilon}. Using computational modeling and high-throughput virtual screening, we identified candidate compounds targeting the SH3(LCK) domain. Functional validation revealed that one compound, C10, selectively disrupted the SH3-RK interaction, leading to reduced TCR-driven activation and proliferation, while sparing activation via alternative receptors and B-cell responses. Moreover, C10 modulated the activity of CD3{varepsilon}-containing CAR and TRuC T cells, attenuating cytokine production and promoting a central-memory-like phenotype associated with enhanced persistence. These findings establish targeted disruption of LCK recruitment as a viable strategy for fine-tuning T-cell responses and propose SH3(LCK) as a druggable domain with therapeutic potential for autoimmune diseases, graft-versus-host disease, and optimizing CAR T-cell therapies.