Abstract

Adoptive T-cell therapies engineered with T-cell receptors (TCRs) or TCR-like antibodies have shown considerable promise in cancer immunotherapy. However, identifying tumor antigen-specific TCR-like antibodies, particularly against human leukocyte antigen-presented neoantigens, remains challenging. Here, we present a function-based, rather than affinity-based, antibody screening platform utilizing Synthetic T-cell receptor and Antigen Receptor (STAR)-T cell libraries. We found that antigen engagement in STAR-T cells triggers synchronous receptor endocytosis and T-cell activation, and we integrated these paired processes into an Endocytosis-Activation (E-A) functional readout for antibody screening. Applying E-A functional screening, we rapidly identified multiple nanobodies targeting the cell-surface antigen CD22 as well as the intracellular neoantigen P53 R175H . STAR-T cells engineered with these nanobodies mediated potent anti-tumor efficacy both in vitro and in vivo. Furthermore, this platform yielded nanobodies that can be directly reformatted into other therapeutic modalities, including chimeric antigen receptors and bispecific antibodies, while maintaining cytotoxic function. Overall, the E-A screening platform links antibody discovery directly to T-cell function, providing a robust approach for identifying therapeutic antibodies, especially neoantigen-specific nanobodies, for T cell-based cancer immunotherapy.