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).Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3539-3) contains supplementary material, which is available to authorized users.

Summary: CD19- and B-cell maturation antigen (BCMA)–directed chimeric antigen receptor (CAR) T cells have enabled unprecedented responses in a subset of refractory patients with B-cell and plasma cell malignancies, leading to their approval by the FDA for the treatment of leukemia, lymphoma, and myeloma. These “living drugs” can become part of a synthetic immune system, persisting at least a decade in some patients. However, despite this tremendous impact, significant unmet treatment needs remain for patients with hematologic malignancies and solid cancers. In this perspective, we highlight recent innovations that advance the field toward production of a more potent and universal cellular immunotherapy of the future. Next-generation CAR T cells will incorporate advances in gene engineering and synthetic biology to enhance functionality and persistence, and reduce treatment-associated toxicities. The combination of autologous CAR T cells with various allogeneic cell treatment strategies designed to target the immunosuppressive tumor microenvironment will broaden the impact of future CAR T-cell therapies.

Natural killer T (NKT) cells represent a cell subpopulation that combines characteristics of natural killer (NK) cells and T cells. Through their endogenous T-cell receptors (TCRs), they reveal a pronounced intrinsic anti-tumor activity. Thus, a NKT cell transfected with a chimeric antigen receptor (CAR), which recognizes a tumor-specific surface antigen, could attack tumor cells antigen-specifically via the CAR and additionally through its endogenous TCR. NKT cells were isolated from peripheral blood mononuclear cells (PBMCs), expanded, and electroporated with mRNA encoding a chondroitin sulfate proteoglycan 4 (CSPG4)-specific CAR. The CAR expression on NKT cells and their in vitro functionality were analyzed. A transfection efficiency of more than 80% was achieved. Upon stimulation with melanoma cells, CAR-NKT cells produced cytokines antigen-specifically. Compared with conventional CAR-T cells, cytokine secretion of CAR-NKT cells was generally lower. Specific cytotoxicity, however, was similar with CAR-NKT cells showing a trend towards improved cytotoxicity. Additionally, CAR-NKT cells could kill target cells through their endogenous TCRs. In summary, it is feasible to generate CAR-NKT cells by using mRNA electroporation. Their CAR-mediated cytotoxicity is at least equal to that of conventional CAR-T cells, while their intrinsic cytotoxic activity is maintained. Thus, CAR-NKT cells may represent a valuable alternative to conventional CAR-T cells for cancer immunotherapy.

Chimeric antigen receptor (CAR)‐T cells are one of the impressive recent success stories of anti‐cancer immunotherapy. Especially in haematological malignancies, this treatment strategy has shown promising results leading to the recent approval of two CAR‐T cell constructs targeting CD19 in the United States and the European Union. After the huge success in haematological cancers, the next step will be the evaluation of its efficacy in different solid tumors, which is currently investigated in preclinical as well as clinical settings. A commonly examined tumor model in the context of immunotherapy is melanoma, since it is known for its immunogenic features. However, the first results of CAR‐T cell therapy in solid tumors did not reveal the same impressive outcomes that were observed in haematological malignancies, as engineered cells need to cope with several challenges. Obstacles include the lack of migration of CAR‐T cells from blood vessels to the tumor site as well as the immunosuppressive tumor microenvironment within solid tumors. Another hurdle is posed by the identification of an ideal target antigen to avoid on‐target/off‐tumor toxicities. Regarding immune escape mechanisms, which can be developed by tumor cells to bypass immune recognition, the observation of antigen loss should also be considered. This article gives an overview of the challenges displayed in CAR‐T cell therapy for the use in solid tumors and discusses different new strategies and approaches that deal with these problems in order to improve CAR‐T cell therapy, particularly for its use in melanoma.