Identification of Functional HLA-A*01 :01-Restricted EBV-LMP2-Specific T-cell Receptors

BACKGROUND: Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs), is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated malignancies of latency type II/III that express EBV-antigens (LMP1/2). Patients who are HLA-A*01:01pos could benefit from such products, since no T cells recognizing any EBV-derived peptide in this common HLA allele have been found thus far. METHODS: HLA-A*01:01-restricted EBV-(LMP2)-specific T-cells were isolated using peptide-MHC-tetramers. Functionality was assessed by production of IFNγ and cytotoxicity when stimulated with EBV-LMP2-expressing cell-lines. Functionality of primary T cells transduced with HLA-A*01:01-restricted EBV-LMP2-specific TCRs was optimized by knocking out the endogenous TCR of primary T cells (ΔTCR) using CRISPR-Cas9 technology. RESULTS: EBV-LMP2-specific T cells were successfully isolated and their TCRs were characterized. TCR gene-transfer in primary T cells resulted in specific peptide-MHC-tetramer binding and reactivity against EBV-LMP2-expressing cell-lines. The mean-fluorescence intensity of peptide-MHC-tetramer binding was increased 1.5-2 fold when the endogenous TCR of CD8pos T cells was knocked out. CD8pos/ΔTCR T cells modified to express EBV-LMP2-specific TCRs showed IFNγ secretion and cytotoxicity towards EBV-LMP2-expressing malignant cell-lines. DISCUSSION: We isolated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can potentially be used in future TCR gene-therapy to treat EBV-associated latency type II/III malignancies