) The long-term goal of this project is to develop immunotherapy with anti-neuroblastoma antibodies that will be effective against primary and metastatic tumor cells, even if they are resistant to other agents. The hypotheses are (1) Neutrophil mediated ADCC is primarily due to loss of plasma membrane integrity (necrosis) and so will not be affected by resistance to apoptosis. (2) Neutrophil ADCC will be increased (a) by promoting effector-target cell adhesion that activates degranulation and production of reactive oxygen species; and (b) by sensitizing tumor cells to cytotoxic effects of neutrophils. (3) Neutrophil migration into tumor sites will occur with antibody targeted delivery of GM-CSF. Our preliminary studies indicate (1) that and retinoid resistant neuroblastoma cell lines are susceptible to neutrophil ADCC mediated by anti-ganglioside GD2 antibody, especially an anti-GD2/GM-CSF fusion protein (immunocytokine); (2) that neutrophil FcyRII and Mac-1 (CD11b/CD18) are required for ADCC; and (3) that treatment of neuroblastoma cells with 13-cis-retinoic acid, interferon-y, fenretinide, or buthionine sulfoximine increases ADCC.
The specific aims are (1) to determine if there is cross resistance between neutrophil ADCC mediated by anti-GD2/GM-CSF (hu14.18/GM-CSF) and other agents and to understand why tumor cell lines vary in their sensitivity to ADCC; (2) to determine if maximizing neutrophil - neuroblastoma cell conjugates increases ADCC; (3) to determine if subjecting neuroblastoma cells to oxidative and ceramide related stress increases ADCC and (4) to evaluate localization of hu14.18/GM-CSF and neutrophils to neuroblastoma cells in murine models. Research Design. Neutrophil ADCC mediated by hu14.18/GM-CSF against 29 MYCN amplified and non-amplified neuroblastoma cell lines with a range of sensitivities to chemotherapeutic agents, retinoic acid, and fenretinide will be evaluated with digital image microscopy to quantify loss of calcein fluorescence from pre-loaded target cells. A new three-color flow cytometry ADCC assay will be used to evaluate expression of cell surface molecules by neuroblastoma cells (e.g., GD2) and neutrophils (e.g., Mac-l, FcyR, CD63) and the relationship to (a) the frequency of neutrophil-conjugated vs. non-conjugated tumor cells; (b) neutrophil: tumor cell ratio in conjugates and (c) kinetic aspects of ADCC (neutrophil-tumor cell conjugates; degranulation; production of ROS; and tumor cell membrane disruption). These assays will be used to maximize neutrophil ADCC with the agents that increase neutrophil - neuroblastoma conjugates and that subject neuroblastoma cells to oxidative and ceramide related stress. Immunodeficient SCID mice with local and metastatic human neuroblastomas will be used to test localization of hu14.18/GM-CSF to primary and metastatic sites, including bone marrow. Agents that enhance ADCC in vitro, as well as anti-angiogenic agents, will be tested for their effect upon immunocytokine and neutrophil localization. This research should establish immunocytokine mediated neutrophil ADCC as a potentially effective therapy for high-risk neuroblastoma.
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