RNA-transfection of ?/d T cells with a chimeric antigen receptor or an a/ß T-cell receptor: a safer alternative to genetically engineered a/ß T cells for the immunotherapy of melanoma.

BMC cancer

PubMedID: 28818060

Harrer DC, Simon B, Fujii SI, Shimizu K, Uslu U, Schuler G, Gerer KF, Hoyer S, Dörrie J, Schaft N. RNA-transfection of ?/d T cells with a chimeric antigen receptor or an a/ß T-cell receptor: a safer alternative to genetically engineered a/ß T cells for the immunotherapy of melanoma. BMC Cancer. 2017;17(1):551.
BACKGROUND
Adoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect ?/d T cells with mRNA.

METHODS
PBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand ?/d T cells and bulk T cells, respectively. Additionally, CD8(+) T cells and ?/d T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous ?/d T cell-target Daudi were analyzed.

RESULTS
Using zoledronic-acid in average 6 million of ?/d T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of ?/d T cells yielded approximately ten times less cells. OKT3-expanded and CD8(+) MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, ?/d T cells produced IFN? and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-?/d T cells in antigen-specific cytokine secretion. While the cytokine production of ?/d T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8(+) T cells, mock-electroporated ?/d T cells also lysed tumor cells reflecting the ?/d T cell-intrinsic anti-tumor activity. After transfection, ?/d T cells were still able to kill MHC-deficient Daudi cells.

CONCLUSION
We present a protocol adaptable to GMP for the expansion of ?/d T cells and their subsequent RNA-transfection with tumor-specific TCRs or CARs. Given the transient receptor expression, the reduced cytokine release, and the equivalent cytotoxicity, these ?/d T cells may represent a safer complementation to genetically engineered conventional T cells in the immunotherapy of melanoma (Exper Dermatol 26: 157, 2017, J Investig Dermatol 136: A173, 2016).