Kurzfassung

Minimal residual disease (MRD) remains as the main obstacle to improve the long-term survival in leukemia. Induction of anti-leukemia immune responses during remission will be likely to promote rejection of residual leukemia cells and therefore cure. This proposal aims to stimulate autologous immune responses against MRD by genetically modifying antigen-presenting cells with an oncogene such that they will present a profile of putative antigenic proteins similar to the original leukemia to...
Minimal residual disease (MRD) remains as the main obstacle to improve the long-term survival in leukemia. Induction of anti-leukemia immune responses during remission will be likely to promote rejection of residual leukemia cells and therefore cure. This proposal aims to stimulate autologous immune responses against MRD by genetically modifying antigen-presenting cells with an oncogene such that they will present a profile of putative antigenic proteins similar to the original leukemia to the immune system. Dendritic cells (DCs) are the most potent antigen-presenting cells in the body, and it has been recently shown that their frequency decreases significantly with aging (Della Bella, Bierti et al. 2007). Therefore, in order to improve the survival of high-risk elderly leukemia patients who are not eligible for high co-morbidity treatment modalities, a potential approach would be the adoptive transfer of autologous, ex vivo expanded DCs. Our group has recently developed a method of genetically programming monocytes with designed lentiviral vectors co-expressing tumor antigens, GM-CSF and IL-4, which can autonomously differentiate into long-lived and immunologically potent dendritic cells in vitro and in vivo (Koya, Weber et al. 2004) (Koya, Kimura et al. 2007). Since this DC genetic programming is readily achieved in one day and DCs self-differentiate subcutaneously, this method requires significant lower amounts of monocytes for DC production and bypasses the cell culture requirement, facilitating implementation in larger cohorts of patients. We propose to advance towards pre-clinical development of these programmed DCs for prophylactic vaccination of elderly patients with Acute Myeloid Leukemia (AML). Our focus will be on patients carrying the FLT3-ITD oncogene, which occurs in approximately 20-30% of AML cases and is one of the pivotal high-risk prognostic markers for AML at presentation and relapse (Schlenk, Dohner et al. 2008). Our hypothesis is that FLT3-ITD co-expression in engineered DCs will lead to of a similar protein expression signature as present in the original FLT3-ITD leukemia cells (e.g.. (Fig. 1). Our plan of attack is: Aim 1: To engineer safety-enhanced tetracistronic non-integrating lentiviral vectors for simultaneous co-expression of GM-CSF, IL-4 the suicide gene HSV-TK and prototypic FLT3-ITD oncogenes. Mouse bone marrow (BM) and human monocytes obtained from AML patients in remission will be transduced with the lentiviral vectors to self-differentiate into DC/LV-FLT3-ITD. We will evaluate if FLT3-ITD expression and/or up-regulated protein products influence DC/LV self-differentiation. Aim 2: To validate in a mouse transplantable FLT3-ITD leukemia model (32D) that mice immunized with DC/LV-FLT3-ITD will mount effective immunity against the oncogene and/or up-regulated protein products. Aim 3: To validate in human in vitro systems that DC/LV-FLT3-ITD can prime and expand naïve and memory autologous T cell responses and to characterize the mechanism(s) of immune response. Aim 4: To advance towards development of standard operating procedures for DC/LV-FLT3-ITD production and pre-clinical testing for future clinical trials. Significance: The lentiviral vector system here described for use in DC engineering is novel and likely to be efficient for gene delivery of other types of onco-antigens. The technical and conceptual knowledge that will arise from these studies will be certainly applicable to the development of immunotherapy to treat various types of hematological malignancies.


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