符合 GMP 的 TRUCKs 生产：靶向 GD2 并释放诱导型 IL-18 的 CAR-T 细胞

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors.

Thus, approaches using 4 th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs (" T cells redirected for universal cytokine-mediated killing "), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs.

We generated IL-18-secreting TRUCKs targeting the tumor antigen GD 2 using the CliniMACS Prodigy system using a recently described "all-in-one" lentiviral vector combining constitutive anti-GD 2 CAR expression and inducible IL-18. Starting with 0. 84 x 10 8 and 0. 91 x 10 8 T cells after enrichment of CD4 + and CD8 + we reached 68. 3-fold and 71. 4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77. 7% and 55. 1% was obtained, and yields of 4. 5 x 10 9 and 3. 6 x 10 9 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD 2 -CAR mediated activation after co-cultivation with GD 2 -expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation.

IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD 2 -expressing target cells indicated by an increased expression of activation markers (e. g. CD25, CD69) on both CD4 + and CD8 + T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e. g. IL-2, granzyme B, IFN- , perforin, TNF- ).

Manufactured TRUCKs showed a specific cytotoxicity towards GD 2 -expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence).

Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD 2 -expressing target cells.

In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products.