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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*01:01 positive 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 major histocompatibility complex (pMHC) tetramers. Functionality was assessed by production of interferon gamma (IFN-γ) 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 TCRs of primary T cells (∆TCR) 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 pMHC tetramer binding and reactivity against EBV-LMP2–expressing cell lines. The mean fluorescence intensity of pMHC-tetramer binding was increased 1.5–2 fold when the endogenous TCRs of CD8+ T cells was knocked out. CD8+/∆TCR T cells modified to express EBV-LMP2–specific TCRs showed IFN-γ secretion and cytotoxicity toward EBV-LMP2–expressing malignant cell lines. Conclusions 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.

Reactivations of cytomegalovirus (CMV), Epstein Bar virus (EBV) and adenovirus (AdV) are frequently seen in immune compromised patients after allogeneic stem cell transplantation (alloSCT), and are associated with high morbidity and mortality. T cell immunity is essential for anti-viral protection, but a fully competent T cell repertoire generally does not develop until 3-6 months after transplantation. Especially patients transplanted with a virus non- experienced donor are at risk of developing severe complications. Adoptive transfer of partially HLA-matched virus specific T cells from healthy third party donors is a potential strategy to temporarily provide anti-viral immunity to these patients. However, these partially HLA-matched T cells harbor a risk of mediating allo-HLA cross-reactivity. Here, we investigated whether virus specificity and HLA restriction of the virus specific T cells influence the risk of allo-HLA cross-reactivity, and thus the development of GVHD. To determine the occurrence and diversity of allo-HLA cross-reactivity, virus specific CD8 T cells from homozygous HLA-A*01:01/B*08:01 and HLA-A*02:01/B*07:02 donors were isolated by cell sorting using tetramers for various peptides from CMV, EBV and AdV. Allo-HLA cross-reactivity was tested using an allogeneic EBV-LCL panel covering 116 different HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA alleles of interest. A significant proportion of the virus specific T cell populations (n=174; 20 specificities) isolated from 27 healthy donors exerted allo-HLA cross-reactivity, as measured by recognition of 1 or more HLA mismatched EBV-LCLs from the panel. Similar frequencies were found for the various viral specificities showing 30% of the CMV, 46% of the EBV and 36% of the AdV-specific T cell populations to be allo-HLA cross-reactive. However, for some specificities (e.g. HLA-A*0201-restricted EBV-LMP2-FLY) allo-HLA cross-reactivity was infrequent (n=1/11), whereas for other specificities (e.g. HLA-B*08:01-restricted EBV-BZLF1-RAK) the majority of the T cell populations (n=9/13) was allo-HLA reactive. Surprisingly, a much larger fraction of HLA-B*08:01 restricted virus specific T cell populations showed allo-HLA cross-reactivity (72%, 36 out of 50 T cell lines), compared to the other HLA restricted virus specific T cell populations (29% of HLA-A*01:01, 30% of HLA-A*02:01 and 26% of HLA-B*07:02 restricted virus specific T cell lines). HLA-B*08:01 restricted virus specific T cells also exhibited the broadest allo-HLA reactivity, reacting to a median of 5 allo EBV-LCLs (range 1-17). In contrast, HLA-A*01:01, HLA-A*02:01 and HLA-B*07:02 restricted virus specific T cells reacted to a median of 1, 2 and 3 (ranges 1-7) allo EBV-LCLs, respectively. Dissection of the diversity/specificity of the allo-HLA reactivity using the panel of 40 different single HLA transduced K562 cells further illustrated the extensive allo-HLA cross-reactivity for HLA-B*08:01 restricted T cells isolated from homozygous HLA-A*01/B*08 donors compared to virus specific T cells restricted by other HLA alleles. These data show that allo-HLA cross-reactivity by virus specific T cells is highly influenced by the HLA restriction and not by the viral specificity of the T cell populations. Of the HLA-A*01, A*02, B*07 and B*08-restricted virus specific T cell populations isolated from homozygous donors, HLA-B*08:01 restricted virus specific T cells showed the highest frequency and diversity of allo-HLA cross-reactivity. Our results indicate that selection of virus specific T cells with specific HLA restrictions may decrease the risk of developing GVHD after infusion of third-party virus specific T cells to patients with uncontrolled viral reactivation after alloSCT. Disclosures No relevant conflicts of interest to declare.

Reactivations of cytomegalovirus (CMV), Epstein Barr virus (EBV) and adenovirus (AdV) occur frequently in immune compromised patients after allogeneic stem cell transplantation (alloSCT) and cause high morbidity and mortality. T-cell immunity is essential for anti-viral protection, but a fully competent T-cell repertoire generally does not develop until 3-6 months after transplantation. Especially patients transplanted with a graft from a virus non-experienced donor are at risk. Adoptive transfer of partially HLA-matched virus-specific T cells from healthy third party donors is a potential strategy to temporarily provide anti-viral immunity to these patients. However, such T cells harbor a risk of mediating off-target toxicity due to allo-HLA cross-reactivity. It is not currently known whether the degree of allo-HLA cross-reactivity is random or whether rules exist that might allow prediction of specific T-cell populations. Here, we investigated whether virus specificity, HLA type of the donor or HLA restriction of the virus-specific T cells influence the risk of allo-HLA cross-reactivity. Through cell sorting using tetramers for various peptides from CMV, EBV and AdV, 164 CD8 T-cell populations (21 specificities) were isolated from peripheral blood of 24 healthy donors, homozygous for HLA-A*01:01/B*08:01 and HLA-A*02:01/B*07:02. Allo-HLA cross-reactivity was tested using an allogeneic EBV-LCL panel covering 116 different HLA molecules and confirmed using K562 cells retrovirally transduced with single HLA alleles of interest. Forty percent of all virus-specific T-cell populations exerted allo-HLA cross-reactivity. Similar frequencies were found for the various viral specificities showing 33% of the CMV, 43% of the EBV and 38% of the AdV-specific T-cell populations to be allo-HLA cross-reactive. Surprisingly, a much larger fraction of the HLA-B*08:01-restricted virus-specific T-cell populations exhibited allo-HLA cross-reactivity (77%) than from those restricted by the other HLAs (32% of HLA-A*01:01, 38% of HLA-A*02:01 and 26% of HLA-B*07:02-restricted virus-specific T-cell populations). HLA-B*08:01-restricted virus-specific T cells also exhibited the broadest allo-HLA reactivity, reacting to a median of 5 different allogeneic EBV-LCLs (range 1-17). In contrast, HLA-A*01:01, HLA-A*02:01 and HLA-B*07:02-restricted virus-specific T cells reacted to a median of 1, 2 and 3 (range 1-7) different allogeneic EBV-LCLs, respectively. Dissection of the diversity/specificity of the allo-HLA reactivities using a panel of 40 different single HLA-A, B, or C-transduced K562 cells further illustrated recurrent recognition of a restricted group of allogeneic HLA-B molecules by HLA-B*08:01-restricted T-cell populations, mediated by single T-cell clones. Heterozygosity for recurrently recognized allo-HLA-B molecules led to a significant decrease in the broadness of allo-HLA cross-reactivity by HLA-B*08:01-restricted T-cell populations, presumably due to negative thymic selection. In contrast, heterozygosity HLA-B molecules that were not part of the restricted group of cross-recognized alleles did not significantly decrease allo-HLA cross-reactivity. These data show that allo-HLA cross-reactivity by virus-specific T cells is highly influenced by their HLA restriction and the HLA background of the donors, but not by their virus specificity. Of the HLA-A*01, A*02, B*07 and B*08-restricted virus-specific T-cell populations isolated from homozygous donors, HLA-B*08:01-restricted virus-specific T cells showed the highest frequency and diversity of allo-HLA cross-reactivity with recurrent recognition of groups of specific mismatched allogeneic HLA-B alleles. Our results indicate that selection of virus-specific T cells with specific HLA restrictions and HLA backgrounds may decrease the risk of off-target toxicity after infusion of third-party virus-specific T cells to patients with uncontrolled viral reactivation after alloSCT. Disclosures No relevant conflicts of interest to declare.

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 viral infection. 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. Growth of such lymphomas can be curbed using adoptively transferred EBV-LMP1/2-specific T cells. Surprisingly, T cells recognizing EBV-derived peptides in the common HLA allele A*01:01 have not been found. In addition, an HLA-A*01:01-associated increased risk for EBV+ Hodgkin lymphomas and infectious mononucleosis has been reported, suggesting that HLA-A*01:01-restricted EBV-specific T cells may be absent or present at very low frequencies in these patients. A need thus exists for HLA-A*01:01-restricted EBV-specific T-cell products, especially directed against EBV-LMP1/2. Based on MHC class I peptide predictions, HLA-A*01:01-binding peptides derived from different immunogenic EBV antigens were identified and tetramer complexes were synthesized (EBNA3A-YTDHQTTPT, EBNA3A-FLQRTDLSY, BZLF1-FTPDPYQVPF, LMP2-ESEERPPTPY, LMP2-LTEWGSGNRTY). HLA-A*01:01-restricted EBV-specific T cells were present at very low frequencies in total PBMCs from all 6 donors. After sorting using flow cytometry, only EBV-LMP2-ESE specific T cells could be expanded (for 5/6 donors), yielding pure tetramer+ CD8 T-cell populations. Four out of 5 isolated T-cell populations exhibited intermediate to high avidity recognition of HLA-A*01:01-transduced TAP2-deficient T2 cells, loaded with EBV-LMP2-ESE peptide. This specific LMP2-derived peptide showed to be functionally processed, presented and recognized by EBV-LMP2-ESE-specific T cells when using HLA-A*01:01/LMP2-transduced K562 cells. To assess the suitability for TCR gene-therapy, the TCRs from the 4 functional T-cell populations were sequenced and cloned into a retroviral vector. Surprisingly, all 4 EBV-LMP2-ESE-specific T-cell populations used the TRBV6-2 gene for TCR beta-chain expression. Additionally, for TCRalpha-chain expression these populations used either TRAV12 or TRAV30. These TCRs contained small differences in the CDR3 region. Despite differences in tetramer binding, all TCRs were functional when transduced into primary CD8 T cells, CD4 T cells, and CD8 negative TCR knock-out Jurkat cells, implying CD8 independent recognition. Finally, recognition of HLA-A*01:01+ EBV-LCLs demonstrated the potential of these EBV-LMP2-ESE-specific TCRs to recognize naturally occurring endogenous LMP2. In conclusion, we isolated and validated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can be used for adoptive transfer or retro/lentiviral TCR gene therapy to treat EBV-associated type II/III lymphomas, EBV+ malignancies of epithelial origin and PTLDs. Disclosures No relevant conflicts of interest to declare.

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.