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Spranger, Stefani (2011): In vitro and in vivo analysis of human cell-based immunotherapies for acute myeloid leukemia. Dissertation, LMU München: Faculty of Biology
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Abstract

Conventional cancer therapies like surgery, radiation and chemotherapy help to eliminate primary tumor masses but often fail to eradicate disseminated tumor cells. However, it is such residual tumor cells that frequently underlie metastasis and relapse. Major obstacles for targeting such cells are wide spread dissemination and long-term persistence in niches that are difficult to reach. For example, many patients with acute myeloid leukemia (AML) show persistence of leukemia after chemotherapy – so-called minimal residual disease (MRD) – which confers a life-threatening risk for relapse in over 70% of patients. Arming the immune system to attack residual tumor cells has high therapeutic potential since immune cells can patrol the body to find and destroy residual tumor cells. Therapeutic approaches using the immune system - so-called immunotherapies - can take several forms. My project concentrated on preclinical studies of two strategies: 1) use of dendritic cells (DC) for therapeutic vaccination and 2) adoptive T cell therapy with lymphocytes expressing transgenic T cell receptors (TCR) specific for tumor-associated antigens (TAA). In therapeutic vaccination a highly potent vaccine is needed to induce a valid immune response in patients with cancer. Effective antitumor immunity requires mobilization of IFN-γ-producing CD4+ T cells (Th1 cells) and lymphocytes with cytotoxic function, including cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In my studies, high potency vaccines were developed using mature DC generated in 3 days (3d-mDC) that were stimulated with synthetic Toll-like receptor TLR3 and/or TLR7/8 agonists. This TLR stimulation mimics DC interaction with viruses and causes mDC to secrete the bioactive form of IL-12, supporting induction of effector cells. Characterization in vitro showed that TLR-activated 3d-mDC were superior to conventional 7d-mDC in capacity to induce Th1 cells as well as CTL. A humanized mouse model was established to verify these observations in vivo. NOD/scid IL2Rgnull mice, lacking murine T, B and NK cells, were reconstituted with human peripheral mononuclear cells and vaccinated with 3d-mDC, stimulated or not with TLR agonists, and conventional 7d-mDC. Induction of CTL was quantified ex vivo using splenocyte populations containing human lymphocytes. The in vivo results were concordant with in vitro observations, demonstrating the superior capacity of 3d-mDC that were stimulated with TLR agonists to induce CTL. Adoptive T cell therapy using TCR-modified lymphocytes represents a second powerful way to provide patients with specific antitumor immunity. Here previously isolated TCR gene sequences are introduced into activated patient-derived lymphocytes, assigning them new antigen specificities. First, T cells must be isolated with TAA specificities that express high-affinity TCR which effectively recognize tumor cells. It was contended that T cell stimulation using peptide-epitopes from TAA presented on foreign MHC would allow isolation of high-affinity TCR, since these T cells had not yet undergone negative selection in the thymus. This contention was proved in individual experiments, as described in this thesis, for the antigens tyrosinase, survivin and HMMR (hyaluronan-mediated motility receptor). Since survivin and HMMR are broadly expressed in AML, TCR specific for these TAA were isolated and subsequently transferred into recipient lymphocytes. Expression of survivin-specific TCR resulted in MHC-restricted death of transduced lymphocytes due to their elevated survivin expression after activation. This precludes use of survivin-specific TCR for therapy of AML. In contrast, transfer of an HMMR-specific TCR yielded effector lymphocytes that effectively killed AML cells in vitro. The behavior in vivo of TCR transduced lymphocytes is crucial for therapeutic outcome. To explore this capacity a xenograft mouse model was established using solid and disseminated human tumor cells injected into NOD/scid IL2Rgnull mice. Adoptive transfer of lymphocytes expressing an HMMR-specific TCR into tumor-bearing mice resulted in significant retardation of tumor outgrowth. Adoptive transfer of memory-like lymphocytes with higher proliferative potential and prolonged in vivo survival may also affect tumor growth. Analyses in vivo and in vitro showed that IL-15-induced effector memory T cells conferred the most potent antitumor immunity. In summary, this work provides evidence for potent in vivo antitumor effects by either using DC-based vaccines or adoptive transfer of TCR transduced lymphocytes, opening application of both strategies for immunotherapy of cancer.