Solid organ transplantation is the treatment of choice for various end-stage diseases, but requires the continuous need for immunosuppression to prevent allograft rejection. This comes with serious side effects including increased infection rates and development of malignancies. Thus, there is a clinical need to promote transplantation tolerance to prevent organ rejection with minimal or no immunosuppressive treatment. Polyclonal regulatory T-cells (Tregs) are a potential tool to induce transplantation tolerance, but lack specificity and therefore require administration of high doses. Redirecting Tregs towards mismatched donor HLA molecules by modifying these cells with chimeric antigen receptors (CAR) would render Tregs far more effective at preventing allograft rejection. Several studies on HLA-A2 specific CAR Tregs have demonstrated that these cells are highly antigen-specific and show a superior homing capacity to HLA-A2+ allografts compared to polyclonal Tregs. HLA-A2 CAR Tregs have been shown to prolong survival of HLA-A2+ allografts in several pre-clinical humanized mouse models. Although promising, concerns about safety and stability need to be addressed. In this review the current research, obstacles of CAR Treg therapy, and its potential future in solid organ transplantation will be discussed.

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.

HLA‐sensitized patients on the transplant waiting list harbor antibodies and memory B cells directed against allogeneic HLA molecules, which decreases the chance to receive a compatible donor organ. Current desensitization strategies non‐specifically target circulating antibodies and B cells, warranting the development of therapies that specifically affect HLA‐directed humoral immune responses. We developed Chimeric HLA Antibody Receptor (CHAR) constructs comprising the extracellular part of HLA‐A2 or HLA‐A3 coupled to CD28‐CD3ζ domains. CHAR‐transduced cells expressing reporter constructs encoding T‐cell activation markers, and CHAR‐transduced CD8+ T cells from healthy donors were stimulated with HLA‐specific monoclonal antibody‐coated microbeads, and HLA‐specific B cell hybridomas. CHAR T cell activation was measured by upregulation of T cell activation markers and IFNγ secretion, whereas CHAR T cell killing of B cell hybridomas was assessed in chromium release assays and by IgG ELISpot. HLA‐A2‐ and HLA‐A3‐CHAR expressing cells were specifically activated by HLA‐A2‐ and HLA‐A3‐specific monoclonal antibodies, either soluble or coated on microbeads, as shown by CHAR‐induced transcription factors. HLA‐A2 and HLA‐A3 CHAR T cells efficiently produced IFNγ with exquisite specificity and were capable of specifically lysing hybridoma cells expressing HLA‐A2‐ or HLA‐A3‐specific B‐cell receptors, respectively. Finally, we mutated the α3 domain of the CHAR molecules to minimize any alloreactive T‐cell reactivity against CHAR T cells, while retaining CHAR activity. These data show proof of principle for CHAR T cells to serve as precision immunotherapy to specifically desensitize (highly) sensitized solid organ transplant candidates and to treat antibody‐mediated rejection after solid organ transplantation.

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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.