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

Abstract

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.

Keywords:

• Adoptive T-cell therapy
• anti-tumor activity
• immune escape
• immunotherapy
• melanoma
• neo-antigen
• TCR transfer

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Acknowledgments

We thank Wolfgang Uckert for the murine TCR constant domains, Kris Thielemans for the pGEM4Z-5_UTR-sig-huSurvivin-DC.LAMP-3_UTR vector, and Christian Hofmann for preparatory work. We also thank Stefanie Baumann and Verena Wellner for excellent technical assistance, and the medical staff for acquisition of donor material.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.

Funding

This work was partially financed by the BMBF (project DCmutaVacc, Förderkennzeichen 01GU1107A to GS) and by the IZKF of the Medical Faculty of the FAU Erlangen-Nürnberg.