BCMA CAR-iNKTs demonstrate anti-myeloma activity in vitro and in vivo. (A) Constructs. Third-generation CARs comprised the single-chain variable fragment (scFv), a CD8 hinge, a CD28 transmembrane domain, CD28 and 4-1BB intracellular domains, and a CD3ζ chain. The extracellular domain of human CD34 protein (trCD34) after a P2A peptide was incorporated into the construct to enable detection of CAR⁺ T cells and purification of CAR⁺ cells for use in functional assays. (B) Flow cytometry showing iNKT purity, CAR⁺ cell levels, and CD4 distribution. Transduction efficiency before cell sorting was 10% BCMA CAR-iNKTs and 15% CD19 CAR-iNKTs. Data shown are the cells used in panels D to G. (C) BLI-based killing assay of MM.1S-CG and OPM2-CG targets by BCMA CAR-Ts and BCMA CAR-iNKTs 48 hours after coculture. (D) In vivo schema. (E) Kaplan-Meier survival analysis. (F) Normalized BLI images of mice. The asterisk denotes a mouse treated with BCMA CAR-iNKTs (10 × 10⁶) that had low tumor burden but was euthanized because of suffering from a likely neck injury/ataxia. (G) Quantitation of tumor burden over time using BLI. (H) Flow cytometry of mouse blood was used to quantitate the absolute number of CAR-iNKTs per μL blood on day 24 (day 6 after BCMA CAR-iNKT administration) and day 31 after MM.1S-CG engraftment (day 13 after BCMA CAR-iNKT treatment). (I) Kaplan-Meier survival curve of a repeat experiment with mice treated with 2 × 10⁶ BCMA CAR-iNKTs or controls. (J) Quantitation of tumor burden over time using BLI. P values for BLI were comparisons of either CD19 CAR-iNKTs or NTD iNKTs with BCMA CAR-iNKTs. TMD, transmembrane domain.