Human CAR T cells can be engineered to express CV1. (A) Vector encoding the CD19 second-generation CAR (19BBz) and CV1 were used to transduce primary human T cells. (B) Transduction efficiency was assessed via flow cytometry, using an anti-idiotype antibody against the anti-CD19 CAR, conjugated to Alexa647 fluorochrome reactive. (C) Supernatant of transduced T cells was subjected to immunoprecipitation and western blot analysis for expression and secretion of CV1. (D) Raji tumor cells were incubated with supernatant from transduced T cells and then subsequently analyzed via flow cytometry for CD47 expression. (E) Secreted CV1 after the coculture of 19BBz Orexi CAR T cells with CD19⁺ or CD19^– tumor cells, was measured via ELISA (3-4 independent donors). (F) NSG mice were injected IV with either PBS, WT CAR combined with recombinant CV1 at different doses, or OrexiCAR T cells; 1 hour postinjection (p.i.) peripheral blood was collected and analyzed for CV1 occupancy. (G) Quantification of geometric MFI of CV1 staining from panel F. Statistics were performed using student t test. ERss, endoplasmic reticulum signal peptide; HA, hemagglutinin epitope tag; MFI, mean fluorescence index; PBS, phosphate-buffered saline; TM, transmembrane domain.