RNA-transfection of γ/δ T cells with a chimeric antigen receptor or an α/β T-cell receptor: a safer alternative to genetically engineered α/β T cells for the immunotherapy of melanoma

BMC Cancer

Research Article

Ugur Uslu¹  Stefanie Hoyer¹  Dennis C. Harrer²  Niels Schaft²  Gerold Schuler²  Jan Dörrie²  Bianca Simon³  Kerstin F. Gerer³  Kanako Shimizu⁴  Shin-ichiro Fujii⁴ 

[1] Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany;Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany;Department of Dermatology, Faculty of Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany;Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstraße 14, D-91052, Erlangen, Germany;Department of Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg Erlangen-Nürnberg, Erlangen, Germany;Laboratory for Immunotherapy, RIKEN Center for Integrative Medical Sciences (IMS), 1-7-22 Suehiro-cho, Tsurumi-ku, 230-0045, Yokohama, Kanagawa, Japan;

关键词: Immune evasion;    Cross-reaction;    MHC-downregulation;    γ/δ T cell;    Adoptive T-cell therapy;    Chimeric antigen receptor;    Melanoma;    mRNA-electroporation;    Zoledronate;   

DOI  :  10.1186/s12885-017-3539-3

 received in 2017-03-06, accepted in 2017-08-10,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundAdoptive 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 γ/δ T cells with mRNA.MethodsPBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8+ T cells and γ/δ 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 γ/δ T cell-target Daudi were analyzed.ResultsUsing zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ 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, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ 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 γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells.ConclusionWe present a protocol adaptable to GMP for the expansion of γ/δ 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 γ/δ 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).

【 授权许可】

CC BY   
© The Author(s). 2017