Preclinical evaluation of SLC35D3-targeted CAR-T cells for tumor-selective immunotherapy.

Chimeric antigen receptor (CAR)-T therapy has transformed management of hematological malignancies, yet efficacy in solid tumors is limited by scarcity of tumor-restricted antigens. Here we characterize SLC35D3, a nucleotide-sugar transporter that we previously identified through integrated transcriptomic and protein-level analyses, as a novel therapeutic target and provide the first preclinical evaluation of SLC35D3-targeted CAR-T cells. We generated rat monoclonal antibodies against human SLC35D3, selected clone #021, humanized it to produce h021-12, and incorporated its single-chain variable fragment into a second-generation CD8α/4-1BB/CD3ζ CAR. For functional assays, we established isogenic tumor-cell models-knockout (KO), wild-type (WT), and overexpressing (OE)-and confirmed graded SLC35D3 surface expression by flow cytometry. In vitro functional assays revealed that h021-12 CAR-T cells exhibited antigen-density-dependent cytotoxicity and interferon-γ release. In tumor engraftment-inhibition xenograft studies, a single intravenous dose of h021-12 CAR-T cells profoundly suppressed OE tumor growth while sparing KO tumors and causing no discernible body weight loss, indicating favorable safety and strict antigen dependence. Activity against WT tumors with physiological SLC35D3 levels remained modest, highlighting antigen-density dependence and avenues for improvement. Overall, our findings provide compelling preclinical proof of concept for targeting SLC35D3 and underscore the need for further optimization to enable future translation into clinical studies in precision oncology.