Chronic Lymphocytic Leukemia (CLL) is the most common form of adult leukemia and is incurable with currently available therapies. One promising form of treatment has been antibody therapy, where CLL cells are targeted by anti-CD20 antibodies such as rituximab and ofatumumab. This has led to significant improvements in survival, especially in combination with other therapies, but complete remissions are still relatively rare. Monocytes and macrophages are critical mediators of antibody therapy, and this depends upon Fc? receptor (Fc?R) activity. We have found that activation of these Fc?R leads to production of sFlt-1, a soluble, inhibitory form of the VEGF receptor. sFlt-1 can inhibit VEGF signaling, which has been shown to play a major role in CLL cell survival. These results led us to hypothesize that Fc?R-mediated sFlt-1 production can dampen anti- apoptotic signals in CLL cells, and that this accounts for a significant portion of antibody-mediated antitumor effects. Hence, strengthening monocyte / macrophage sFlt-1 production within the context of antibody therapy may be a powerful means of enhancing its effectiveness. To test the predictions of this hypothesis we propose:
Aim 1 : Analysis of sFlt-1 production and function in response to anti-CD20. Here, we will a) identify the cells primarily responsible for sFlt-1 production upon binding anti-CD20 antibodies, examine the effect of sFlt-1 on CLL cell survival, and study the mechanism(s) by which VEGF-mediated survival in tumor cells is inhibited. We will also b) examine whether sFlt-1 production by monocytes makes CLL cells more susceptible to direct apoptosis in response to agents in use preclinically and clinically for the treatment of CLL.
Aim 2 : Analysis of sFlt-1 production and function in antibody treatment in vivo. We will use established murine models of CLL to a) test whether sFlt-1 is produced during antibody-mediated B cell depletion using a murine CD20 antibody, b) identify relevant Fc?R-bearing effector cell(s) responsible for sFlt-1 production, c) test whether neutralizing sFlt-1 reduces the efficacy of anti-CD20 antibody in a murine CLL model, and d) Whether sFlt-1 levels in CLL patients receiving monotherapy with ofatumumab correlate with response and progression free survival.
Aim 3 : Elucidation of mechanism of sFlt-1 induction by Fc?R clustering. Here, we will determine a) which activating Fc?R is responsible for sFlt-1 induction and whether this induction is negatively regulated by Fc?RIIb and SHIP, b) whether Fc?R-induced sFlt-1 production is a direct or indirect effect, and c) the signaling pathway(s) involved in sFlt-1 induction. Summary: At completion of this study we will have fully explored an entire new mechanism of anti-CD20 mediated killing of tumor cells by monocytes and macrophages. These mechanistic studies will provide information to further enhance the efficacy of both anti-CD20 antibodies such as ofatumumab in CLL but potentially a wide variety of other tumors where similar antibody based treatments are utilized.
We have found that monocytes and macrophages secrete the soluble form of the Vascular Endothelial Growth Factor Receptor (VEGFR1, sFlt-1) upon encountering antibody-coated targets, and this molecule is known to inhibit the pro-survival and angiogenic signaling of VEGF. Here, we propose to examine the mechanism(s) of antibody-mediated sFlt-1 production, to test whether sFlt-1 can enhance antibody-mediated killing, and to elucidate the role of sFlt-1 during antibody therapy in vivo. Because this is a completely unexplored area, results from this study will likely lead to novel ways of enhancing the effectiveness of antibody therapy for CLL, as well as for other forms of cancer.
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