This is a revision of a training proposal by Stuart C. Helfand, DVM, designed to provide him with the basic knowledge and research skills necessary to become an independent investigator in cancer immunotherapy. The program will provide him with laboratory training in cellular and molecular immunology techniques and encourage development of conceptual and practical approaches for hypothesis testing. This training will build on his unique strengths as a veterinary clinical oncologist, enabling him to bridge in vitro and animal models to clinical problems of cellular immunity and cancer immunotherapy in humans and animals. The objectives for Phase I are to provide basic training through graduate course work and participation in programs within the University of Wisconsin Comprehensive Cancer Center. Phase II will develop an intensive project to improve cancer immunotherapy by activating tumoricidal monocytes/macrophages targeted by antitumor monoclonal antibodies.
Aim 1 is to examine the effects of the immune cell activators liposome-encapsulated muramyl tripeptide (L-MTP-PE) and granulocyte/monocyte-colony stimulating factor (GM-CSF) on tumor killing mediated by human monocytes/macrophages in antibody-dependent cellular cytotoxicity (ADCC). Enhancement of the tumoricidal effects of activated monocytes/macrophages, specifically targeted toward cancer cells labeled with tumor-reactive monoclonal antibody, will be examined using radioisotope-release assays. Healthy human donors and cancer patients will be sources of monocytes for study.
Aim 2 is to elucidate the effector mechanisms utilized by monocytes/macrophages activated by L-MTP- PE and GM-CSF to kill tumor targets in ADCC. Membrane changes in activated monocytes/macrophages that contribute to tumor cell recognition will be analyzed by flow cytometry binding assays and calcium flux quantitated using augmented cytotoxicity will be examined by Northern analysis of mRNA specific for these molecules and their surface expression analyzed by flow cytometry. The contribution of nitric oxide, tumor necrosis factor-alpha, and interleukin-1 to cytotoxicity will be ascertained by blocking assays, ELISA, and Northern analysis.
Aim 3 is to examine tumoricidal function and effector mechanisms of monocytes/macrophages activated with L-MTP-PE in vivo in a canine tumor model. The relevance of in vitro results from Aims 1 and 2 will be verified in vivo in dogs treated with L-MTP-PE for metastatic melanoma. This project has significance to treatment of human cancer using immunologic approaches and builds on a promising animal model.
