Characterization of granulocyte-macrophage colony-stimulating factor/interleukin-2 fusioncDNA and the use of marrow stromal cells for cancer immunotherapy.

摘要

Immunotherapy of cancer aims at achieving systemic anticancer responses capable of eradicating disseminated malignant cells. The disappointing outcomes associated with several immune-based clinical trials have highlighted the need to improve upon existing therapeutic strategies. The main objective of my thesis was to develop novel means in order to improve current cytokine-based anticancer strategies. The delivery of cytokines, or their encoding cDNA sequences, can induce antitumor immune responses. Interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) are among the most potent cytokines able to induce tumor-specific systemic immunity, both in experimental models and clinical trials. Paradoxically, the combination of GM-CSF and IL-2 has been reported to downregulate certain immune functions, highlighting the unpredictability of dual cytokine use. In the first section of my thesis, I hypothesized that a GM-CSF and IL-2 fusion transgene (GIFT) could circumvent the limitations associated with dual cytokine expression yet preserve synergistic features. B16 mouse melanoma cells were gene modified to express GIFT (B16GIFT) and GIFT gene product was characterized in vitro. When injected into syngeneic mice, B16GIFT cells were unable to form tumors. When used as a whole cell tumor vaccine, irradiated B16GIFT could induce absolute protective immunity against wild type B16 tumors. In mice with established melanoma, B16GIFT therapeutic cellular vaccine significantly improved tumor-free survival when compared to B16 expressing both IL-2 and GM-CSF. Mechanistically, GIFT induced a significantly greater tumor site recruitment of macrophages and NK cells than combined GM-CSF and IL-2. I thus demonstrated that a fusion between GM-CSF and IL-2 can invoke greater antitumor effect than both cytokines in combination and that novel immunobiological properties can arise from such chimeric constructs.Therefore, despite their previously reported immunosuppressive effects on allogeneic immune responses, I provided evidence that primary MSCs can be used as transgenic delivery vehicles to enhance immune responses in syngeneic hosts. In order to further characterize the effect of MSCs on autologous immunity, I investigated the immunomodulatory properties of MSCs during syngeneic antigen-specific immune responses. I provide experimental evidence that syngeneic MSCs behave as conditional antigen-presenting cells. My research demonstrated that IFNgamma can induce mouse MSCs to process and present antigenic peptides derived from a soluble xenoprotein (i.e. ovalbumin) and activate in vitro antigen-specific T cells. When injected in vivo into syngeneic mice, ovalbumin-pulsed IFNgamma-treated MSCs induced potent ovalbumin-specific cellular immune responses and protected mice against ovalbumin-expressing tumors. My studies further showed that human MSCs can also acquire antigen-presenting functions upon IFNgamma stimulation, thereby activating antigen-specific T cell hybridomas. Taken together, my results strongly suggest that in syngeneic conditions, IFNgamma-stimulated MSCs behave as conditional antigen presenting cells able to activate antigen-specific immune responses.Overall, my research opens the door for the development of new immunotherapeutic strategies based on (i) the improvement of cytokine potency by molecular fusion and (ii) the improvement of cytokine delivery by the use of gene modified somatic MSCs, and may reveal MSCs as previously unrecognized cellular regulators of physiological immune responses.Another means to improve current cytokine-based strategies is to limit the severe side-effects associated with their systemic administration. In view of that, I tested the hypothesis that primary marrow stromal cells (MSCs) can be used as a cellular vehicle for the tumor-localized delivery of immunostimulatory cytokines. Specifically, I investigated whether IL-2 gene-modified MSCs can be used to mount an effective immune response against the poorly immunogenic B16 melanoma model. My research demonstrated that primary mouse MSCs can be efficiently gene-modified to secrete IL-2. Remarkably, IL-2 secreting MSCs embedded in a collagen-like matrix and injected in the vicinity of pre-established B16 tumors led to absence of tumor growth in 90% of treated mice. Injection of IL-2 secreting MSCs induced CD8 mediated tumor specific immunity and was dependent upon CD8 and NK cells, but not CD4 cells.