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 γ/δ T cells with mRNA. PBMC 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. Using 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. We 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).

Adoptive cellular therapy has evolved into a powerful force in the battle against cancer, holding promise for curative responses in patients with advanced and refractory tumors. Autologous T cells, reprogrammed to target malignant cells via the expression of a chimeric antigen receptor (CAR) represent the frontrunner in this approach. Tremendous clinical regressions have been achieved using CAR-T cells against a variety of cancers both in numerous preclinical studies and in several clinical trials, most notably against acute lymphoblastic leukemia, and resulted in a very recent United States Food and Drug Administration approval of the first CAR-T-cell therapy. In most studies CARs are transferred to conventional αβT cells. Nevertheless, transferring a CAR into different cell types, such as γδT cells, natural killer cells, natural killer T cells, and myeloid cells has yet received relatively little attention, although these cell types possess unique features that may aid in surmounting some of the hurdles CAR-T-cell therapy currently faces. This review focuses on CAR therapy using effectors beyond conventional αβT cells and discusses those strategies against the backdrop of developing a safe, powerful, and durable cancer therapy.

Tumor cells can develop immune escape mechanisms to bypass T cell recognition, e.g., antigen loss or downregulation of the antigen presenting machinery, which represents a major challenge in adoptive T cell therapy. To counteract these mechanisms, we transferred not only one, but two receptors into the same T cell to generate T cells expressing two additional receptors (TETARs). We generated these TETARs by lentiviral transduction of a gp100-specific T cell receptor (TCR) and subsequent electroporation of mRNA encoding a second-generation CSPG4-specific chimeric antigen receptor (CAR). Following pilot experiments to optimize the combined DNA- and RNA-based receptor transfer, the functionality of TETARs was compared to T cells either transfected with the TCR only or the CAR only. After transfection, TETARs clearly expressed both introduced receptors on their cell surface. When stimulated with tumor cells expressing either one of the antigens or both, TETARs were able to secrete cytokines and showed cytotoxicity. The confirmation that two antigen-specific receptors can be functionally combined using two different methods to introduce each receptor into the same T cell opens new possibilities and opportunities in cancer immunotherapy. For further evaluation, the use of these TETARs in appropriate animal models will be the next step towards a potential clinical use in cancer patients.

The advent of CD19-specific chimeric antigen receptor (CAR) T cells has proven to be a powerful asset in the arsenal of cancer immunotherapy of acute lymphoblastic leukemia and certain B cell lymphomas. However, a sizable portion of patients treated with CD19-CAR T cells relapse with CD19-negative cancer cells, necessitating the quest for back-up antigens. Chondroitin sulfate proteoglycan 4 (CSPG4) expression has been reported on leukemic blasts bearing the ill-fated MLL 11q23 rearrangement. We aimed at exploring the use of CSPG4-specific CAR T cells against mixed-lineage leukemia (MLL)-rearranged leukemic blasts, using the precursor B cell leukemia cell line KOPN8 (MLL–MLLT1 translocation) as a model. First, we confirmed CSPG4 expression on KOPN8 cells. Bulk T cells electroporated with mRNA encoding a CSPG4-specific CAR upregulated activation markers and secreted the Th1 cytokines TNF and IFNγ in an antigen-specific manner upon co-culture with KOPN8 cells. More importantly, CSPG4-specific CAR T cells evinced specific degranulation towards KOPN8 cells and specifically lysed KOPN8 target cells in chromium lysis experiments. CSPG4 is a well-established CAR target in cutaneous melanoma. Here, we provide proof-of-principle data for the use of CSPG4-specific CAR T cells against MLL-translocated leukemias.