IL-2/B7.1 Fusagene Transduction of AML Blasts by a Self-Inactivating Lentiviral Vector Stimulates T Cell Responses in Vitro: A Strategy To Generate Whole Cell Vaccines for AML.

Despite recent treatment advances prognosis for the majority of AML patients remains bleak. AML patients over 60 years of age treated with intensive chemotherapy have an estimated 2-year survival of 20% and only 10% at 4–5 years. This is attributed to unfavourable cytogenetics, preceding myelodysplastic syndrome and limited abilities to tolerate intensive chemotherapy. We and others have previously demonstrated that tumour cells engineered to express B7.1 and IL-2 can stimulate T cell and NK cell responses against the unmodified tumour cells and prolong the survival of vaccinated animals in mouse models of leukaemia. However, development of whole cell vaccines for AML have been severely hampered by the fact that primary AML cells are poorly infectable with RV, AdV, AAV and HSV. Therefore we have constructed self-inactivating lentiviral vectors incorporating enhancer elements and a myeloid efficient promoter to express B7.1 and IL-2 in a monocistronic “fusagene” configuration as a single fusion protein. This polyprotein is post-synthetically cleaved to generate biologically active membrane anchored B7.1 and secreted IL-2. This provides transduction efficiencies of between 40%–99% for B7.1 and 2-15ng/10⁶/24hrs of IL-2 secretion in both established and primary AML cells, following a single round of infection at an MOI of 10. The combined expression of IL-2 and B7.1 in AML blasts enables increased in vitro stimulation of allogeneic T cells and autologous T cells obtained from both non-remission and remission patients (post-BMT or non-BMT recipients). In addition, IL-2/B7.1 AML cells stimulated an increase in lymphocyte numbers, expansion of CD8+ cells and increased expression of T cell activation markers. Furthermore, the stimulated lymphocytes secrete greater levels of Th1 cytokines and show evidence of specificity, as indicated by their increased proliferation in the presence of unmodified AML compared to remission bone marrow cells. This data supports the notion that IL-2/B7.1 whole cell vaccination could stimulate therapeutically useful immune responses in AML patients.