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Background The 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 Methods In 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. Results In 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. Conclusions With 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 Information M.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.

Background Cancer Testis Antigens (CTAs) are highly expressed in multiple different tumor types, but silent in normal tissue, except the testis. This tumor-restricted expression pattern makes them an ideal target for adoptive T-cell therapy. However, the responsiveness in clinical setting may be hampered because high-affinity T cells against self-antigens presented in the context of self-HLA are deleted in the thymus by negative selection. In this study, we aim to identify high-affinity T cell receptors (TCRs) specific for CTAs from the allogeneic-HLA repertoire. Materials and Methods In this study, HLA class I binding peptides derived from different CTA genes were identified by HLA-peptide elution experiments and subsequent mass spectrometric analysis. From the identified peptides HLA tetramers were generated to isolate peptide specific CD8+ T cells from healthy allogeneic donors. Efficacy and safety of the TCRs was determined by various different stimulation assays. The most potent TCRs were sequenced, analyzed and transduced into peripheral CD8+ and CD4+ T cells to confirm CTA specific cytokine production and cytotoxicity. Results MAGE and CTAG peptides were eluted from multiple myelomas, EBV-transformed lymphoblastic cells, acute myeloid leukemia and ovarium carcinomas. We selected TCRs recognizing 3 different MAGE-A1 peptides in the context of HLA-A*02:01, HLA-A*03:01 and HLA-B*07:02. Furthermore, we selected TCRs specific for MAGE-A3 in the context of HLA-B*35:01 and HLA-A*01:01; TCRs specific for MAGE-A9 in the context of HLA-A*01:01 and TCRs specific for CTAG1 in the context of HLA-A*02:01. The selected T-cell clones demonstrated efficient recognition of MAGE-A1, MAGE-A3 or CTAG1 positive multiple myeloma and solid tumor cell lines without detectable cross-reactivity. Conclusions We identified multiple different TCRs from the allogeneic-HLA repertoire specific for CTA genes. These TCRs demonstrate efficient recognition and killing of CTA positive multiple myeloma and solid tumor cell lines and did not show any cross-reactivity. The peptides recognized by the TCRs are presented in different HLA alleles. Since, 71% of the world population contains one of these HLA-alleles, a large percentage suffering from a MAGE or CTAG positive tumor could potentially be treated with the identified TCRs by TCR-gene therapy. Disclosure Information M.A.J. de Rooij: None. D.M. van der Steen: None. D. Remst: None. A. Wouters: None. M. van der Meent: None. R.S. Hagedoorn: None. M.G.D. Kester: None. P.A. van Veelen: None. F.J.H. Falkenburg: None. M.H.M. Heemskerk: None.

Meeting abstracts Studies using T-cell receptor (TCR) or chimeric antigen receptor (CAR) transduced T-cells have shown the effectiveness of adoptive immunotherapy to treat different malignancies. The efficacy and safety of such interventions greatly depends on good target selection to prevent on

TCR-transfer to engineer tumor-specific T cells may be a strategy for adoptive immunotherapy. For complete eradication of leukemic cells and to achieve long-term protection, potent effector T cell function and long-term T cell persistence are necessary. Therefore, we propose to use virus specific T cells for TCR transfer since such engineered dual specific T cells can be triggered via their endogenous TCR by latent presence of viral antigens, improving their long-term persistence. We have previously shown that virus specific T cells can be redirected towards anti-leukemic reactivity by transfer of the hematopoietic minor histocompatibility antigen HA-2 specific TCR (HA-2-TCR). The TCR-transferred virus specific T cells showed differences in TCR cell surface make up, which was stable for months after repetitive non-specific TCR triggering. The T cells expressed either both TCRs intermediately at the cell surface, or the endogenous TCR was highly expressed with a low expression of the introduced TCR, or the introduced TCR was highly expressed with a low expression of the endogenous TCR. It may be anticipated that frequent encounter with viral antigens in vivo leads to selective outgrowth of TCR-transferred dual specific T cells with high expression of the endogenous viral specific TCR but low expression of the introduced tumor specific TCR, resulting in reduced anti-leukemic reactivity. To address this issue, we generated CMVA2-specific T cells transduced with the HA-2-TCR. This resulted in dual specific cells with different TCR cell surface make up. The dual specific T cells were repetitively stimulated specifically either via their endogenous virus specific TCR or via the introduced HA-2 specific TCR. In time, the cell surface expression of the endogenous and introduced TCRs as measured with CMVA2 and HA-2A2 tetramers diverged. Repetitive stimulation of the endogenous TCR skewed the dual specific T cells towards a cell population that predominantly expressed the endogenous TCR. In contrast, repetitive stimulation of the introduced TCR skewed the cells towards T cells that predominantly expressed the introduced TCR. However, this divergence in tetramer stainings was shown to quickly revert after a single stimulation via the other TCR. To study whether this divergence was the result of a difference in TCR cell surface distribution or of selective outgrowth of different T cells, T cells were sorted that predominantly expressed either the endogenous or the introduced TCR. These cells were subsequently stimulated on the endogenous or introduced TCR, and compared regarding TCR cell surface expression and functional activity. Directly after sorting dual specific T cells preferentially expressing the endogenous TCR were still reactive against HA-2+ target cells, although the reactivity was reduced compared to cells preferentially expressing the introduced TCR. However, when restimulated on the introduced HA-2-TCR, the dual specific T cells expanded antigen specifically, and reverted within several days into cells with high expression of the introduced TCR that exerted potent HA-2 specific anti-leukemic effector functions. In conclusion, we demonstrate that these dual specific T cells are likely to persist in vivo due to repetitive encounter with viral antigens with preservation of anti-leukemic effector function. Moreover, in vivo exposure to the tumor associated antigen will further enhance the relevant specificity.