PS1314 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 W. , van der Maarel L. , Hageman L. , de Jong R. , Amsen D. , Falkenburg F. , Jedema I. 
Published: June 2019

Background:

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. Although many EBV‐associated lymphomas only express weak immunogenic EBV antigens (e.g. EBNA1 and BARF1), lymphomas with type II or III latency often express immunogenic LMP1 and LMP2. Different strategies have been developed to manufacture EBV‐LMP1/2‐specific T‐cell products for clinical application and potential effectivity of autologous and donor‐derived EBV‐LMP1/2‐specific T‐cells has been demonstrated in clinical trials. Surprisingly, to date, no EBV‐specific T‐cells recognizing a peptide in the common HLA‐A∗01:01 allele have been found, while an HLA‐A∗01:01‐associated risk for EBV+ Hodgkin lymphomas has been reported.

Aims:

A need thus exists for HLA‐A∗01:01‐restricted EBV‐specific T‐cell products, especially EBV‐LMP1/2 specific T‐cells.

Methods:

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‐YTD, EBNA3A‐FLQ, BZLF1‐FTP, LMP2‐ESE, LMP2‐LTE). 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. 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 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 transformed lymphoblastoid cell lines (EBV‐LCLs) and IFNy ELISA as read‐out.

Results:

HLA‐A∗01:01‐restricted EBV‐specific T‐cells were present at low frequencies in total PBMCs from all 6 donors. 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. EBV‐LMP2‐ESE‐specific T‐cells were subsequently 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 the EBV‐LMP2‐ESE peptide is successfully processed, presented and recognized by EBV‐LMP2‐ESE‐specific T‐cells. Recognition of HLA‐A∗01‐positive EBV‐LCLs further illustrated the potential of the EBV‐LMP2‐ESE‐specific T‐cells to recognize endogenous LMP2.

Summary/Conclusion:

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.