Isolation and Validation of the First Functional HLA-a*01:01 Restricted EBV-LMP2 Specific T Cells for Treatment of EBV Associated Type II/III Lymphomas
Authors: Huisman Wesley , van der Maarel Lieve E. , Hageman Lois , de Jong Rob C.M. , Amsen Derk , Falkenburg J.H. Frederik , Jedema Inge 
Published: October 2019

Abstract
Epstein Barr virus (EBV) is associated with the development of a broad range of malignancies, including Burkitt's lymphoma, Hodgkin and non-Hodgkin lymphomas, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma and gastric carcinoma. Differential expression of immunogenic antigens (e.g. EBV Nuclear Antigen (EBNA2-6) and Latent membrane proteins (LMPs)) is seen at the different latent phases of the virus. Although many EBV associated lymphomas only express weakly immunogenic EBV antigens (e.g. EBNA1 and BARF1), lymphomas with type II or III latency express LMP1 and LMP2. Different strategies have been developed to manufacture EBV LMP1/2 specific T cell products for clinical application. Surprisingly, to date, no EBV specific T cells recognizing a peptide in the common HLA allele A*01:01 have been found. Furthermore, an HLA-A*01:01 associated risk for EBV+ Hodgkin lymphomas and infectious mononucleosis has been reported. A need thus exists for HLA-A*01:01 restricted EBV specific T cell products, especially EBV-LMP1/2 specific T cells.
Based on MHC class I peptide predictions, HLA-A*01:01 binding peptides derived from different immunogenic EBV antigens were identified, and HLA-A*01:01/peptide tetramer complexes were synthesized (EBNA3A-YTDHQTTPT, EBNA3A-FLQRTDLSY, BZLF1-FTPDPYQVPF, LMP2-ESEERPPTPY, LMP2-LTEWGSGNRTY). For these 5 specificities, tetramer positive CD8 T cells were sorted by flow cytometry from peripheral blood mononuclear cells (PBMC) of 6 HLA-A*01:01 positive healthy donors and subsequently cultured with 1µM of specific peptide. Specificity of expanded T cells was confirmed by tetramer staining. Functional avidity was assessed using TAP2 deficient T2 cells transduced with HLA-A*01:01 and exogenously loaded with 10-12M to 10-6M of the respective specific peptide. The recognition of endogenously processed and presented antigen was analyzed using K562 cells transduced with HLA-A*01:01 and a retroviral vector encoding the full protein sequence of LMP2, HLA-A*01:01 positive EBV-LCLs and IFNy ELISA as read-out.
HLA-A*01:01 restricted EBV specific T cells were present at low frequencies in total PBMCs from all 6 donors (EBNA3A-YTD 0.008%; range 0.001-0.02%, EBNA3A-FLQ 0.01%; range 0.0016-0.012%, BZLF1-FTP 0.0033%; range 0.0025-0.0075%, LMP2-ESE 0.0049%; range 0.0015-0.15% and LMP2-LTE 0.003%; range 0.002-0.025%). After flow cytometric cell sorting, only EBV-LMP2-ESE specific T cells of 5/6 donors expanded, resulting in pure tetramer+ CD8 T cell lines. Analysis of the T cell receptor beta chain (TCR-Vβ) usage showed that these EBV-LMP2-ESE specific T cell populations were oligoclonal. To further analyze their functional avidity, EBV-LMP2-ESE specific T cells were tested against HLA-A*01:01 transduced TAP2 deficient T2 cells exogenously loaded with peptide. Four out of 5 isolated T cell populations recognized T2 cells loaded with 1-103nM peptide, indicating intermediate to high avidity. To investigate whether this specific LMP2-derived peptide can be functionally processed and presented and whether these T cells were able to recognize endogenously processed and presented antigen, EBV-LMP2-ESE specific T cells were tested against HLA-A*01:01/LMP2 transduced K562 cells. All functional EBV-LMP2-ESE specific T cell populations could recognize these transduced K562 cell lines, indicating that this LMP2-ESE peptide is successfully processed, presented and recognized by EBV-LMP2-ESE specific T cells. Recognition of EBV-LCLs illustrated the potential of the EBV-LMP2-ESE specific T cells to recognize endogenous LMP2.
We describe the isolation and validation of the first functional HLA-A*01:01 restricted EBV-LMP2 specific T-cell populations, which can be used for adoptive transfer to treat EBV associated type II/III lymphomas, malignancies of epithelial origin and PTLDs. Despite the very low frequency of these T cells, we were able to obtain these T cells from 5 out of 6 donors and showed their capacity to recognize both exogenously loaded as endogenously processed and presented EBV-LMP2-ESE peptide.

Disclosures
No relevant conflicts of interest to declare.