Development of Novel Stem Cell Transplantation and Gene-Immunotherapy Using WT1-Specific T-Cell Receptor Gene.

The Wilms' tumor 1 (WT1) is one of the zinc-finger transcriptional regulators, and its expression level is very low in most tissues of adults. In contrast, various kinds of leukemia and solid tumors express WT1 abundantly, and high expression level of WT1 is correlated with disease aggressiveness and poor prognosis. These findings indicate that WT1 is a promising target antigen for anti-cancer cellular immunotherapy. Following identification of immunogenic epitopes derived from WT1 which are recognized by HLA class I-restricted and HLA class II-restricted T cells, phase I/II WT1 peptide vaccination trials have been conducted. Although the positive correlation between the clinical efficacy and vaccine-induced WT1-specific T-cell response has been reported, the clinical efficacy is not satisfactory. Adoptive transfer of WT1-specific T cells seems to be the promising approach to achieve marked improvement in clinical efficacy of WT1-targeting immunotherapy, however, it still remains difficult to expand WT1-specific T cells sufficiently ex vivo. To overcome these problems, we attempted to establish gene-immunotherapy targeting WT1 using T-cell receptor (TCR) gene isolated from the WT1-specific T-cell clone. We also verified the feasibility of novel stem cell transplantation by transducing WT1-specific TCR gene into hematopoietic stem cells.

We cloned the full length TCR-αa and -β genes from a WT1_235-243-specific and HLA-A*2402-restricted cytotoxic T lymphocyte (CTL) clone. The WT1-specific TCR gene-repressing retroviral and lentiviral vectors were constructed. Retroviral vector was transduced to human peripheral T cells in retronectin-coated plate. WT1-specific functions of TCR gene-transduced CD8⁺ T cells and CD4⁺ T cells were examined by evaluating WT1 peptide-specific cytotoxicity by ⁵¹Cr-release assay and WT1 peptide-specific Th1 cytokine production, respectively. To improve the efficacy of WT1-specific TCR expression, we developed the novel retroviral vector which can inhibit selectively intrinsic TCR expression (si-TCR vector). Finally, we transduced the WT1-specific TCR lentiviral vector into human cord blood CD34⁺ cells, and transplanted them to NOD/SCID/common-γ^null mice. Then, we examined whether WT1-specific human mature T cells can differentiate in mice. The presence of WT1-specific human T cells in mice was determined by tetramer assay and IFN-γ production in response to stimulation with WT1 peptide.

Following transfer of WT1-specific TCR gene into peripheral blood lymphocytes, WT1 peptide-specific CD8⁺ and CD4⁺ T cells could be expanded easily in vitro. TCR gene-transduced CD8⁺ T cells exerted cytotoxicity against WT1 peptide-pulsed target cells and human leukemia cells in an HLA-A*2402-restricted manner. Similarly, TCR gene-transduced CD4⁺ T cells showed WT1-specific Th1 cytokine production in response to stimulation with human leukemia cells in HLA-A*2402-restricted fashion depending on the interaction of CD4 and HLA class II molecules. The newly developed si-TCR vector appeared to inhibit expression of endogenous TCR efficiently and improved the efficacy of WT1-specific TCR expression 3 to 5-fold higher as compared to the conventional vector. Three months after transplantation of WT1-specific TCR gene-transduced human hematopoietic stem cells in NOD/SCID/common-γ^null mice, differentiation of WT1-specific human T cells in murine spleen was evaluated. Tetramer assay revealed that human mature T cells expressing WT1-specific TCR on their cell surface were clearly detected. Furthermore, these WT1-specific CD8⁺ T cells appeared to produce IFN-γ in response to stimulation with WT1 peptide-loaded HLA-A*2402-positive cells.

The adoptive gene-immunotherpay using WT1-specific TCR gene against leukemia seems to be promising. Moreover, the novel stem cell transplantation using WT1-specific TCR gene-transduced hematopoietic stem cells might open the door to induce long-lasting anti-leukemic cellular immunity in patients with leukemia.

No relevant conflicts of interest to declare.