Development and analysis of Wilms’ tumor protein (WT1) specific T cells for adoptive immunotherapy of epithelial ovarian cancer

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The Wilms’ tumor protein (WT1), which is overexpressed by a number of human cancers, is an emerging target for cancer immunotherapy. Epithelial Ovarian Cancers (EOC) differentially express certain onco fetal proteins normally present on other tissues only during embryonal development. We sought to evaluate whether the onco fetal protein WT1 was present in EOC and whether it represented a potential immunotherapeutic target for patients with EOC. We evaluated 86 primary ovarian tumor specimens, representing all major histologic subtypes, for WT1 expression by immunohistochemistry (IHC). We found that WT1 overexpression was present in 28/86 (33%) of tumor samples and was associated with serous histology (16/25, 64%) (p=0.001) while only 1/17 (6%) borderline tumors overexpressed the antigen. Thus, WT1 tumor expression was correlated with poor clinical outcome. Furthermore, ELISA evaluation of patient serum revealed 8/32 (25%) of EOC patients had native anti-WT1 antibody responses at a dilution of 1:400. Recently, we and others have identified potentially immunogenic peptides derived from WT1 which bind the common HLA alleles, HLA A0201, A0301, and A2402. These peptide antigens have been used to stimulate and expand WT1-specific T cells. We use autologous EBV transformed B cells (BLCL), isolated from EOC patients, either pulsed with immunogenic WT1 peptides or transfected with vectors encoding the WT1 gene to sensitize and expand WT1-specific T cells form EOC patients, in vitro. This sensitization reliably generated WT1-tetramer positive T cells (range 2-7% tetramer positive) from patients after 4 stimulations over 28 days. Thirty to 210 x 10⁶ cells, with a predominance of CD8+ cells, could be generated over the 28 days. The T cells sensitized with WT1 generate IFNg and other cytokines in response to peptide and specifically lyse primary HLA A2+ WT1+ ovarian or leukemia tumor cells in vitro. Non-WT1 and non-HLA matched tumor cells were not lysed. In a SCID mouse, human tumor xenograft model, we found that adoptively transferred WT1 specific HLA A2+ T cells (two doses of 16 x 10⁶ T cells) home to and specifically induce regressions of established HLA A2+ WT1+ ovarian cancer while the T cells did not home to nor regress non-WT1 or non-HLA matched tumors in the same animals. Due to the frequent expression of WT1 in EOC and the practicable approach of using autologous EBV BLCL to sensitize tumoricidal WT1 T cells from patients, WT1 represents a viable target for adoptive immunotherapy of EOC.