Comparison of FR-specific CARs with CD27, CD28, and 4-1BB costimulatory endodomain. (A) FR-specific CAR expression on primary human T cells. As shown, CD8⁺ T cells can efficiently express FR-specific CARs with or without CD27, CD28, and 4-1BB costimulatory endodomains as measured by flow cytometry. (B) CAR-transduced T cells showed lytic function in a bioluminescent killing assay. FR CAR-T cells killed FR⁺ SKOV3 but did not kill FR⁻ C30 cells at the indicated E/T ratio more than ∼ 20 hours. Untransduced T cells (UNT) served as negative controls. Mean and SD of triplicate wells from 1 of at least 3 independent experiments is shown. (C) Bioluminescence images show fLuc⁺ SKOV3 tumors in NSG mice immediately before and 3 weeks after first intravenous injection of 10 × 10⁶ CAR-T cells on days 40 and 45 after tumor inoculation. (D) Tumors in mice treated with FR-27z, -28z, and -BBz CAR-T cells regressed; tumors treated with untransduced T cells (UNT) did not regress 3 weeks after first T-cell dose (P < .0001). Arrows indicate timing of T-cell infusion. FR-z CAR-transduced T cells only slowed tumor growth (P = .015). Before infusion, CAR⁺ T-cell frequency (∼ 50%) and surface scFv expression level (range, 315-398 mean fluorescence intensity) was equalized. (E) Circulating human CD4⁺ and CD8⁺ T-cell counts. In contrast to CD28, CD27 and 4-1BB signaling enhances the survival of circulating human CD4⁺ and CD8⁺ T cells in vivo 3 weeks after T-cell infusion (27z vs z, P = .029; BBz vs z, P = .024; 28z vs z, P = .20). Mean cell concentration (cells/μL) ± SD for all evaluable mice in each treatment group is shown. (F) Surface CAR expression on persisting FR-specific human CD3⁺ T cells from blood of treated mice measured by flow cytometry. Mean CAR⁺ expression frequency per CD3⁺ T cells and SD per group is shown.