Preclinical development and selection of nanobody-based CAR-T cells targeting HER2-positive solid tumors

Chimeric antigen receptor (CAR)-T cell therapy revolutionized treatment of hematological malignancies, but translation to solid tumors remains difficult. Therefore, different avenues are explored to develop novel CAR-T cell products. Nanobodies have advantageous features for CAR design, such as ease of engineering and low immunogenicity, but predictive values on nanobody characteristics for optimal CAR-T cell function remain unclear. We performed a side-by-side evaluation of 12 nanobody-based (nano)CARs against human epidermal growth factor receptor 2 (HER2). All nanoCARs showed the ability to activate a reporter T cell line upon recognition of HER2^pos cells. When evaluated in primary T cells, 6/12 nanoCARs showed the strongest reactivity and cytotoxicity against HER2^pos glioblastoma and breast cancer cells. Moreover, only 1/12 was able to confer potent cytotoxicity against HER2^pos melanoma cells. These data add to the current view that nanobody selection for CAR design remains subject to extensive side-by-side screening. Still, superior cytotoxicity across multiple solid tumor cell lines-determined using <i>in vitro</i> assays evaluating activation, cytokine secretion, and target cell-specific killing-led to selection and further characterization of a lead nanoCAR 1R59b, showing tumor control in an <i>in vivo</i> xenograft model, providing a promising HER2 nanoCAR for further (pre)clinical investigation.