High Avidity PRAME Specific T Cells Derived From In Vivo HLA Mismatched Transplantation Setting Potentially Useful for Immunotherapeutic Strategies.

Abstract 4087

Poster Board III-1022

Adoptive T cell therapy is an attractive strategy to provide cancer patients with antigen specific T cells. For this approach T cells with specificity for non-polymorphic tumor-associated self-antigens that are shared between various tumors are promising candidates. However, the isolation of high avidity T cells specific for nonpolymorphic tumor associated self-antigens is difficult, because of self-tolerance. During thymic selection T cells that exhibit high avidity for self-antigens presented by self-HLA are deleted by positive and negative selection. This explains why most T cells directed against non-polymorphic tumor associated self-antigens characterized to date exhibit low to intermediate avidity. After HLA mismatched stem cell transplantation (SCT) however, T cells educated in the donor have not encountered the allogeneic HLA molecules from the patient during thymic selection. Consequently, these T cells can exhibit high avidity for tumor associated antigens presented by allogeneic patient HLA molecules. In this study we aimed to identify T cells directed against non-polymorphic tumor associated antigens using an in vivo HLA mismatched transplantation setting. Alloreactive T cell clones were isolated and expanded from a patient that experienced graft versus leukemia as well as acute graft versus host disease following HLA-A2 mismatched SCT and donor lymphocyte infusion for the treatment of AML. All isolated T-cell clones were allo-HLA-A2 reactive, and by loading of T2 cells with HPLC fractionations of peptides eluted from HLA-A2 we were able to demonstrate that all alloreactive clones recognized one single fraction, indicative for peptide specific recognition. By two additional peptide HPLC fractionation rounds and mass spectrometry we were able to characterize the peptides of 8 different allo-HLA-A2 reactive T cell clones. One of the T cell clones, was of particular interest since this T cell clone recognized the peptide SLLQHLIGL derived from the preferentially expressed antigen on melanomas (PRAME). Recognition by the clone of HLA-A2 positive COS cells transfected with PRAME, and tetramer staining of the clone, confirmed the specificity against the PRAME derived peptide. Peptide titration demonstrated that the PRAME specific T cell clone exhibited high affinity for the SLL peptide. Since PRAME is overexpressed in a large fraction of tumors, we analyzed whether the PRAME specific clone could recognize HLA-A2 expressing tumor cell lines. The results demonstrated that all 8 tested melanoma cell lines, 2 of 3 RCC cell lines, 1 of 2 mamma carcinoma cell lines and 1 of 2 lung carcinoma cell lines were recognized by the clone. In addition, 5 out of 10 primary acute myeloid leukemia cells, and 2 out of 3 acute lymphoblastic leukemia cell lines were recognized. Since it has been described that also certain normal tissues express low levels of PRAME, the clone was tested against numerous HLA-A2 positive non-malignant cells. No reactivity against fibroblasts, keratinocytes, bronchus epithelial cells, hepatocytes, billiair duct epithelial cells, colon epithelial cells, mesenchymal stem cells, (activated) B-cells, (activated) T cells, monocytes and CD34+ cells was observed. However, the clone demonstrated high reactivity against monocyte derived DC's and a low but significant reactivity against primary tubular epithelial cells. By quantitative PCR we demonstrated that the level of recognition of the different cell types is correlated with the expression levels of PRAME. The results demonstrate that the high avidity PRAME specific T cells clone, derived from an in vivo allo-HLA-A2 immune response, is solely PRAME specific and exerts high reactivity against numerous tumors and limited of target toxicity. Based on these results we conclude that the high affinity TCR from this high avidity PRAME specific T cell may be an effective tool for adoptive T cell therapy using TCR gene modified T cells for the treatment of cancer patients.