Combining drug resistant bone marrow with immunotherapy for the treatment of cancer

Chemotherapy is effective for eliminating tumors but has toxic secondary effects on the hematopoietic system, impairing host immunity. This dissertation describes studies aimed at investigating the therapeutic benefit of combining chemotherapy, immunotherapy and drug resistance gene therapy for the treatment of cancer. In the first study, experiments were conducted to test the feasibility of combining cell-based immunotherapy with gene therapy. Human gamma/delta T-cells and NK cells, isolated from whole blood, were cultured and infected with retrovirus encoding GFP. Due to ineffective expansion and reduced viability of the cells further human studies were not pursued. In the second study, experiments were performed to assess the benefit of a novel viral concentration method for improving gene transfer into hematopoietic cells. Retrovirus encoding GFP or the drug resistance gene dihyrofolate reductase (DHFR) was concentrated to assess the effect of viral concentration on transduction efficiency. At 40-fold concentration, a 10-15 fold improvement in transduction efficiency was observed relative to controls. In addition, hematopoietic stem cells transduced using these methods were transplanted into mice. Following reconstitution, it was observed that transgene expression in peripheral blood lymphocytes was > 30%. These results suggest that concentrating virus by this method improves transduction efficiency in vitro and in vivo. In the third study, experiments were conducted to determine whether combining chemotherapy, immunotherapy and drug resistance gene therapy has a therapeutic benefit over chemotherapy or immunotherapy alone. Mice were transplanted with bone marrow transduced with DHFR and treated with chemotherapy plus the immunomodulatory antibody anti-CD137. The effect of this treatment on an experimental sarcoma (AG104) was compared with trimetrexate chemotherapy or anti-CD137 treatment alone. After 40 days of monitoring, no mice were cured by chemotherapy alone and only 40% were cured by immunotherapy alone. Treatment with the combined regimen resulted in a 100% cure. In addition, transfer of splenocytes from cured mice into tumor-bearing mice extended survival when compared with transfer of splenocytes from non-cured mice. These results suggest that chemotherapy combined with immunotherapy is an improvement over individual treatments when the hematopoietic system is protected from the cytotoxic effects of chemotherapy by drug resistance gene expression.