10.04 A library of novel cancer testis specific T-cell receptors for T-cell receptor gene therapy

BackgroundThe positive clinical effect of T-cell receptor(TCR) gene therapy on tumor regression has previously been demonstrated by NY-ESO-1 TCR-gene therapy. To seriously increase the number of cancer patients that can be treated with TCR-gene therapy we aim to identify a novel set of high-affinity Cancer Testis (CT) specific TCRs targeting different CT-antigens in a variety of prevalent HLA-class I alleles.Materials and MethodsIn this study, we selected by bioinformatic tools the most promising CT-genes to target, and from these genes we identified by HLA-peptidomics the naturally processed and presented HLA-class I peptides. With these peptides HLA-tetramers were generated, and by MACS enrichment and single cell sorting CT-specific CD8+ T-cell clones were selected from the allo-HLA repertoire of healthy donors. By performing several different functional assays the high function avidity CT-clones with a safe recognition pattern were selected. To evaluate the potential for clinical application in TCR-gene therapy, TCRs were sequenced, and transferred into peripheral blood derived CD8+ T cells.ResultsIn total we identified, 7 novel CT-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6 and MAGE-A9 expressing tumors cells in the context of HLA-A1, -A2, -A3, -B7, -C7 and -B35.ConclusionsWith this set of 7 novel CT-specific TCRs we expand the arsenal of tumor specific TCRs. With this expanding library of TCRs it would be possible to select in future for each cancer patient, based on HLA typing and gene expression, a useful TCR to generate a personalized TCR-gene therapy products. In addition, patients could be treated with multiple TCRs to enhance the efficacy and increase the durability of clinical responses by reducing the likelihood of tumor escape.Disclosure InformationM.A.J. de Rooij: None. D.M. Steen: None. D. Remst: None. A. Wouters: None. M.G.D. Kester: None. R.S. Hagedoorn: None. P.A. van Veelen: None. E.M.E. Verdegaal: None. J.H.F. Falkenburg: None. M.H.M. Heemskerk: None.

TCR Gene Therapy: Challenges, Opportunities, and Future Directions

Adoptive immunotherapy with gene-engineered T cells has provided new treatment options for cancer patients [...]

The First Functional HLA-a*01:01-Restricted EBV-LMP2-Specific T-Cell Receptors for TCR Gene Therapy of Patients with EBV-Associated Type II/III Malignancies

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.

Isolation and characterization of NY-ESO-1–specific T cell receptors restricted on various MHC molecules

Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2–restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2.