Control of Cell Death Levels Using Th9402-Based PDT Treatment on Fresh PBMC, and Potential Application to Patients with cGvHD.

Recently, extracorporeal photopheresis (ECP) has shown interesting clinical activity for the treatment of drug-refractory chronic graft-vs-host disease (cGvHD), inducing Th1/Th2 immunomodulation to restore immune tolerance. Several studies indicate that target cell apoptosis may have a role in the control of cGVHD, and increasing apoptotic levels may favor immune modulation. We have developed an approach to eliminate immunoreactive cells using the Theralux™ photodynamic cell therapy (PDT) system based on the use of the rhodamine-derivative TH9402 illuminated ex vivo with a visible light source (l=514nm). The capacity of TH9402 PDT to induce increasing levels of T cell apoptosis has not been investigated. The induction of apoptotic cell death was studied using peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers (HV) and cGvHD patients. We found a good correlation between PDT conditions and levels of cell death induced. In comparison to controls showing 8±4 percent (%) of apoptosis, as measured by TUNEL assay on cells harvested 3 days post-treatment, PBMC from HV subjected to PDT using 0.33, 0.66, or 1.32 micromolar of TH9402 showed 28±16%, 49±17%, and 78±11% of apoptosis, respectively. These studies have also shown that the intra- and inter-donor variability in TH9402 incorporation are very low (~5% and 10%, respectively). To ensure that these findings could also be applied to a clinical setting, PBMC from cGvHD patients were treated with 0.33 and 1.32 micromolar of TH9402 to trigger either low or high levels of apoptosis. PBMC from cGvHD patients showed a sensitivity similar to that of PBMC from HV, with 38±9% and 73±13% of apoptosis when treated with 0.33 and 1.32 micromolar TH9402, respectively. Data obtained from the analysis of various cell death parameters such as the loss of DΨm, AnV/PI staining, TUNEL assay and agarose DNA ladder on PBMC and human Jurkat T cells treated with escalating doses of TH9402 suggested strongly the higher sensitivity of proliferating cells to the treatment. Moreover, together with the use of the caspase-inhibitor ZVAD-fmk, these data suggested that upon irradiation, the photoactivation of TH9402 will trigger the formation of reactive oxygen species (ROS) and the release of proapoptotic factors from mitochondria triggering various cell death mechanisms, such as caspase-dependent apoptosis, caspase-independent apoptosis, or a mixture of apoptosis and necrosis. Finally, preliminary data showed that PDT-treated cells were able to induce in vitro the maturation of monocyte-derived dendritic cells. Based on these data, we are beginning a pilot clinical study evaluating two controlled PDT conditions inducing low and high levels of apoptosis in order to assess the efficacy and biological effect of TH9402-based ECP to treat cGVHD in humans.