DESCRIPTION (provided by applicant): Although the human immune system has evolved to repel many forms of biological invasion, its ability to identify which cells are detrimental to the body is not perfect. The research proposed herein has two main objectives: to explore a new fundamental strategy for directing the human immune system to kill target cells and to apply that strategy to treating several forms of metastasis-prone cancer beginning with HT-29 human colon cancer cells. The strategy is partially inspired by the concept of prodrugs. A molecule will be made such that when it interacts with urokinase-type plasminogen activator (uPA, an enzyme overexpressed on the surface of many types of cancer cells, especially those that are prone to metastasize), it will be chemically transformed into an electrophile that will covalently attach to the cancer cell's surface. The molecule will be bifunctional, and the other end will bind naturally occurring human antibodies. Once antibodies have bound the cancer cell, the human immune system will be activated to kill the target. This general strategy should also be applicable to treating a variety of other medical conditions. The research is divided into the following specific aims. (1) To design and synthesize a substrate analogue that functions as a latent electrophile to be activated by uPA- mediated hydrolysis. (2) To use this enzyme-activated electrophile to covalently attach antigens to the surface of cancer cells. And, (3) to study the immunological response against human cancer cells labeled by this target-activated antigen. This research will employ various methods. Bifunctional molecules will be chemically synthesized using standard organic chemistry reactions. Analysis of uPA-mediated activation of these molecules will be assayed by high-performance liquid chromatography (HPLC). Analysis of covalent attachment of the activated electrophiles to the activating protein will be assayed by mass spectrometry. Noncovalent binding between the antigen-labeled protein and human antibodies will be assayed by enzyme- linked immunosorbent assay (ELISA). Binding of antibodies to cells will be assayed by immunofluorescence microscopy. Antibody-mediated cytotoxicity will be assayed in various cancer cell lines beginning with commercially available HT-29 colon cancer cells.
PUBLIC HEALTH RELEVANCE: The research proposed herein offers a strategy to use small organic molecules to direct the natural human immune system to fight cancer. Specifically, this approach will use organic chemistry to make molecules that can selectively attach to cancer cells and direct the immune system to destroy those cells. Although the first application will be against the HT-29 human colon cancer cell line, the method proposed is broad enough in scope to target several types of cancer, especially those that are most likely to metastasize throughout the body. |