In contrast to gene therapy of solid tumors, only a few preclinical studies exist about gene therapy of acute lymphoblastic leukemia. Previously, we showed that vaccination of mice with syngeneic BCR-ABLp185 expressing leukemia cell lines modified to express costimulatory molecules and cytokines induce a systemic immunity against wild type leukemia. However, the difficulties to culture and transfect human leukemia cells limit the clinical application of leukemia cell based vaccines. Thus, we evaluated the pre-immunization of mice with DNA based vaccines subsequently challenged by the cell line BM185. In order to avoid the limitations and serious side effects associated with viral vectors we used nonviral gene delivery methods. Minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide or GM-CSF were used as DNA vaccine and double stem-loop immunomodulators (dSLIM), containing three CpG-motifs were used as immune adjuvant. We provide biometrical and CTL data that shows specific immunization and protection of mice which received the complete vaccine BCR-ABL/GM-CSF/dSLIM by GeneGun delivery of naked DNA. Mean tumor-free survival (p=0.019) and overall survival (p=0.008) were significantly longer compared to non-vaccinated mice. Furthermore we show that BCR-ABL specific sequences are required to prevent Ph+ acute lymphoblastic leukemia by DNA vaccination. However, survival rate was moderate and only 26.7% of vaccinated mice survived and remained tumor-free. While current nonviral genetic tumor vaccine systems are less effective than viral vaccines, in particular when tumor associated antigens are weakly immunogenic, microencapsulation of tumor-specific DNA with poly (β-amino esters) might be a promising, safe and efficient nonviral delivery method for genetic vaccines. These biodegradable cationic polymers can bind and condense DNA into nanoparticles, deliver large DNA payloads and show low toxicity. Therefore, we prepared DNA-polymer complexes of MIDGE-vectors and end-modified poly (butane diol diacrylate co amino pentanol) C32-117 or C32-118 which self-assemble into particles with effective diameters of 200 nm and zeta potentials of 9.8 mV in PBS. We compared the immunization of mice receiving the vaccine BCR-ABL/GM-CSF/dSLIM by intradermal injection as naked DNA or alternatively, complexed with C32-117 or C32-118. We present data about tumor growth, survival rate, tumor free and overall survival of vaccinated and control mice.
Chemotherapy and tyrosine kinase inhibitors provide high remission rates. However, prognosis of adult patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia (Ph(+) ALL) still remains poor. Because most adults eventually relapse without allogeneic stem cell transplantation, which is not available for all patients, novel strategies are required for relapse prevention. As the integrity of the immune system is essential for the control of remaining leukaemia cells, we compared the efficacy of anthocyanins, imatinib and a DNA-based vaccine as non-immunosuppressant components with 6-mercaptopurine (6-MP) to control minimal residual disease in vitro and in vivo using different leukaemia cell lines and syngeneic mice. Proliferation of Ph(+) ALL was significantly better inhibited by anthocyanin-rich berry extract or imatinib compared with 6-MP. Although anthocyanins induced apoptosis in some leukaemia cell lines, the level of caspase-3, caspase-8 and caspase-9 was significantly lower compared with imatinib and 6-MP. When used as single components, anthocyanins and imatinib mesylate failed to eradicate pre-existing Ph(+) ALL in syngeneic mice, while 6-MP led to 10% and DNA vaccination to 56% survival. Intriguingly, only the combination of DNA vaccination with berry extract but not with the isolated anthocyanin, cyanidin-3-rutinoside or imatinib further increased leukaemia-free and overall survival, and 90% of lethally challenged mice survived. We suggest that induction and enhancement of a leukaemia-specific immunity by DNA vaccination and anthocyanin-rich berry extract can also decrease the relapse rate in patients with Ph(+) ALL. Furthermore, this approach may serve as strategy for maintenance therapy of other malignancies.
Although 40–50% of adults and 70–80 % of children with acute lymphoblastic leukemia (ALL) can be cured by poly chemo therapy, the prognosis of patients with Philadelphia chromosome positive (Ph+) ALL remains poor. Therefore, new relapse prevention strategies are needed for patients with Ph+ ALL during remission. We have shown previously, that vaccination of mice with leukemia cell lines modified to express costimulatory molecules and cytokines induce a systemic immunity against the syngeneic BCR-ABLp185 expressing leukemia cell line BM185. However, the difficulties to culture and transfect human leukemia cells limit the clinical application of leukemia cell based vaccines. Thus, we evaluated the immunization of mice with DNA-based vaccines subsequently challenged by the cell line BM185. Combinations of minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide, GM-CSF, IL12, IL27 or CD40L were used for in vivo transfection of murine skin. In addition, we used natural DNA-based double stem-loop immunomodulators (dSLIM), containing three CpG-motifs as non-specific immune adjuvant. In order to increase transfection efficacy, MIDGE-vectors were microencapsulated into poly(β-aminoester) nanoparticles with diameters of 200 nm. Mice immunized with the BCR-ABL/GM-CSF/dSLIM vaccine showed a significant longer mean tumor-free (p=0.019) and overall survival (p=0.008) compared to nonvaccinated mice. BCR-ABL specific sequences were required to prevent Ph+ acute lymphoblastic leukemia. Furthermore, CTL assays showed that specific lysis was significantly higher after vaccination with BCR-ABL/GM-CSF/dSLIM compared to GMCSF/dSLIM (p<0.05) and to naïve mice (p<0.005). The vaccine efficacy was clearly dosedependent. Microencapsulation of MIDGE vectors increased the efficacy of the vaccine compared to the naked DNA-vaccine. Mice immunized with the microencapsulated vaccine BCR-ABL/GM-CSF/dSLIM showed a significant longer mean tumor-free (p<0.0001) and overall survival (p<0.0001) compared to non-vaccinated mice and 70% survived and never developed leukemia. Cotransfection with IL27 or IL12 lead to significant longer tumor free (IL27: p=0.02; IL12: p<000.1) and overall survival (IL-27: p=0.03; IL12: p<000.1) compared to the vaccine BCR-ABL/GM-CSF/dSLIM. The best protection with a survival rate of 91% was observed in mice which received the vaccine BCR-ABL/GMCSF/IL12/dSLIM. We have shown previously in T-cell depletion studies that CD8+ T cells were the effector cells in the BM185 cell-based vaccine model and currently we evaluate whether CD8+ T cells also play a major role in the BM185 DNA-based vaccine model. In conclusion, we provide survival and functional data that show immunization and protection of mice with optimized leukemia specific DNA-vaccines.
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