Functional Evaluation of T-Cells Generated from WT1-TCR Transduced Human Hematopoietic Stem Cells Using the OP9-DL1 Coculture System

Introduction: Chemotherapy leads to cure of acute myeloid leukemia (AML) in less than half of the patients. Stem cell transplantation can be used as an immunotherapeutic treatment to cure the patient, but carries a high risk of toxicity and mortality. Moreover, not all patients have a suitable donor. We have developed a novel immunotherapeutic treatment, in which we generate in vitro, starting from hematopoietic precursor cells, T-cells that recognize WT1, a tumor antigen that is overexpressed on 70% of the AMLs.

Aims: In this study we have evaluated the functionality and specificity of the generated WT1-directed T-cells both in vitro and in vivo. The ultimate goal is to use these cells in patients, as this form of immunotherapy is promising and could be an option for cure in patients who are not eligible for stem cell transplantation. In contrast to the more widely used immunotherapy using TCR-transduced peripheral T-cells, our therapy is expected to be more effective and carry less risk of autoreactivity.

Methods: CD34⁺ cells isolated from cord blood and mobilized peripheral blood mononuclear cells were cultured on OP9-DL1 in the presence of the cytokines IL-7, Flt3-L and SCF, for 2 weeks, until T-cell commitment. Subsequently, they were transduced with a WT1-TCR (H. Stauss) or a CMV-TCR (M. Heemskerk), and again co-cultured until CD4⁺CD8⁺ double positive cells were abundantly present (generally after another 2-3 weeks). At that point, the agonist peptide WT1 or CMV resp was added to the culture together with IL-7, and 5 days later cells were harvested and expanded (polyclonally or using agonist peptide), in the presence of IL-2, or IL-7+IL-15.

T-cells were evaluated using a ⁵¹Chromium release assay, for cytotoxicity against WT1 and HLA-A2 positive and negative targets. Also, upon activation, production of IFN-g was evaluated using ELISA.

Immunodeficient 6-8 weeks old NSG mice were irradiated (200 cGy), and 24 hours later injected intravenously with either a luciferase-positive, WT1, HLA-A2 transduced K562 cell line (R. Stripecke), or luciferase-transduced, HLA-A2+, WT1+ primary AML cells (expanded on MS-5 in the presence of cytokines) in the current experiments, and 24 hours later, with 5x10⁶ or 10⁷WT1-TCR T-cells or CMV-TCR T-cells (negative control). Mice were evaluated using the IVIS bioluminescence assay.

Results: We observed that a nice mix of WT1-TCR CD8+ and CD4+ T-cells (50%/50%) was generated with cells expanded after harvest from the coculture using the combination of the agonist peptide, IL-7 and IL-15. Using ⁵¹Cr release assay and ELISA, we could show that upon activation, the T-cells showed specific cytokine production and efficient killing of tumor cells. We observed that the luciferase+, WT1, HLA-A2 transduced K562 cell line homed to ovaria and brain (female mice) or liver, testes and brain (male mice) when injected intravenously, and these are largely sanctuary sites, not reached by the T-cells, therefore resulting in low efficiency. When this cell line was injected subcutaneously in the hind flank, mice showed significant swelling of the resp limb and needed to be euthanized for ethical reasons before full evaluation was possible.

Currently, experiments are ongoing evaluating the efficacy of the WT1 T-cells against luciferase transduced primary AML cells (after long term expansion culture on MS-5), as these cells are expected to home to the bone marrow and blood of the mice, and therefore reflect more the physiological situation, and can be more easily reached by the T-cells. Results of these experiments will be presented at ASH.

Summary/Conclusion: We have shown that, using the OP9-DL1 model, we were able to generate large numbers of high-avidity tumor-specific naïve and resting T-cells, after a process similar to thymic positive selection. After expansion (polyclonal or antigen specific -in the presence of the agonist peptide-) and activation, these cells show specificity and functionality in vitro and are currently evaluated in an in vivo immunodeficient mouse model.