Generation of CD8(+) T cells expressing two additional T-cell receptors (TETARs) for personalised melanoma therapy.

Adoptive T-cell therapy of cancer often fails due to the tumor cells' immune escape mechanisms, like antigen loss or down-regulation. To anticipate immune escape by loss of a single antigen, it would be advantageous to equip T cells with multiple specificities. To study the possible interference of 2 T-cell receptors (TCRs) in one cell, and to examine how to counteract competing effects, we generated TETARs, CD8(+) T cells expressing two additional T-cell receptors by simultaneous transient transfection with 2 TCRs using RNA electroporation. The TETARs were equipped with one TCR specific for the common melanoma antigen gp100 and one TCR recognizing a patient-specific, individual mutation of CCT6A (chaperonin containing TCP1, subunit 6A) termed "CCT6A(m) TCR." These CD8(+) T cells proved functional in cytokine secretion and lytic activity upon stimulation with each of their cognate antigens, although some reciprocal inhibition was observed. Murinisation of the CCT6A(m) TCR increased and prolonged its expression and increased the lytic capacity of the dual-specific T cells. Taken together, we generated functional, dual-specific CD8(+) T cells directed against a common melanoma-antigen and an individually mutated antigen for the use in personalised adoptive T-cell therapy of melanoma. The intended therapy would involve repetitive injections of the RNA-transfected cells to overcome the transiency of TCR expression. In case of autoimmunity-related side effects, a cessation of treatment would result in a disappearance of the introduced receptors, which increases the safety of this approach.

Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells.

For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs) is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs). However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs' immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1), and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

Human T cells expressing two additional receptors (TETARs) specific for HIV-1 recognize both epitopes.

Adoptive TCR transfer against rapidly mutating targets, such as HIV-1 or cancer, must counteract corresponding immune escape. Hence, we generated T cells expressing two additional receptors (TETARs) specific for HIV-1 by TCR mRNA electroporation. An HLA-A2-restricted gag-specific TCR and an HLA-B13-restricted nef-specific TCR were chosen. When both TCRs were transfected simultaneously, strong competitive effects occurred that were overcome by replacing the human constant domains of one TCR with murine counterparts and adapting the amounts of TCR-RNA used for transfection. The resulting TETAR responded to both epitopes with cytokine secretion and cytotoxic function. Cell sorting revealed that one individual cell indeed recognized both epitopes. The T cells diminished their reactivity to each epitope after stimulation but sequentially killed targets that presented the gag epitope and then targets that presented the nef epitope, or vice versa. Taken together, TETARs represent a sophisticated tool to study TCR functionality and might be a useful strategy in immunotherapy.