Prostate Specific Membrane Antigen (PSMA) is a marker in prostate tumors and solid tumor neovasculature that has recently become a focus for anti-cancer therapies. Prostate cancer is the second leading cause of cancer deaths in American males. An estimated 232,000 new cases of prostate cancer will be diagnosed and approximately 30,000 deaths will occur in 2005. Although there have been marked improvements in the early detection, diagnosis and treatment of cancer in general, there is still a great need for the development of new technologies that will result in even better management of these diseases. It has been shown that tumors often express specific antigens, such as PSMA, that are either absent or are expressed in low amounts in normal tissue. In addition to prostate cancer, examples of tumors that express specific antigens include breast, lung, and colon cancers. In this proposal, we will focus on prostate cancer as a model disease to demonstrate technology for the rapid selection of high affinity ligands to a specific tumor marker, in this case, PSMA. Dr. Gregory Weiss at the University of California, Irvine has created phage-displayed libraries that are based on randomization of several different repeat protein scaffolds. The """"""""Randomized Repeat Proteins"""""""" (RRPs) that can be obtained from these libraries contain loops that are reminiscent of complementarity-determining regions (CDRs) on antibodies. RRPs offer many advantages over monoclonal antibodies, including rapid turnaround and economical expression. The goal of our proposal is to demonstrate that the phage-displayed RRP libraries can be used to rapidly select a novel ligand specific for a tumor antigen, PSMA. In general, a system that reliably generates ligands against tumor antigens would have broad diagnostic and therapeutic applications including targeted delivery of imaging agents or antineoplastic drugs. However, cancer treatment is merely 1 potential use of RRPs, and phage-displayed RRP libraries have the potential to contribute significantly to a broad range of research and biomedical applications. In this SBIR Phase I proposal, we plan to utilize phage-displayed RRP libraries to identify ligands that specifically recognize PSMA. We have chosen to demonstrate this concept using prostate cancer as a model disease because it is a leading cause of mortality in the male population. PSMA is highly expressed on the surface of prostate and other cancer cells, making it an ideal target for anti-tumor or imaging agents. PSMA is also a possible serum marker for early detection and diagnosis of prostate and other cancers. Finally, PSMA is expressed in solid tumor neovasculature, but not in normal vasculature and thus is a potential target for anti- angiogenic therapies that can be used for the treatment of cancer in general. Our primary objective is to use the phage display system to identify anti-PSMA RRPs and explore their application in the early detection, diagnosis and treatment of prostate cancer and anti-angiogenic therapies. In the future, the phage display system can be used to identify RRPs against any protein of interest, broadening its application to other cancers and/or other diseases.
The Specific Aims of this SBIR Phase I proposal are thus focused on first demonstrating the ability of the phage-displayed libraries to identify RRPs that are specific for PSMA expressed at the surfaces of prostate cancer cells in tissue culture. The PSMA RRPs identified from these studies will undergo further development in the Phase II studies with an eye towards potential commercial applications in the area of improving the delivery of imaging and therapeutic agents to tumors expressing PSMA. In order to demonstrate this key Phase I Milestone, the Specific Aims are summarized as follows:
Specific Aim 1. Identify RRPs specific for the PSMA ectodomain. The Weiss laboratory has already demonstrated the synthesis of RRP libraries and identified binding partners to PSMA. Additional libraries will be constructed and used to identify high affinity RRPs against purified soluble PSMA ectodomain.
Specific Aim 2. Express and purify anti-PSMA RRPs. Anti-PSMA RRP genes identified from the RRP libraries will be inserted into plasmids for expression with a terminal thioester or free cysteine for site-specific attachment of fluorescent probes. Each anti-PSMA RRP fusion protein will be expressed in a bacterial system and purified by affinity chromatography. Anti-PSMA RRPs will then be tested in vitro for binding to purified PSMA ectodomain using qualitative pulldown experiments and quantitative biosensor binding experiments.
Specific Aim 3. Screen anti-PSMA RRPs against panels of cancer and normal cell lines. Once binding to purified PSMA ectodomain has been verified, anti-PSMA RRPs will be labeled with fluorescent dyes and PSMA specific binding to prostate and other cancer cells will be assessed by fluorescence microscopy, quantitative binding analysis and FACS. To further confirm the selection of PSMA specific RRPs, a panel of non-PSMA expressing cell lines representing normal tissues will also be screened. The most promising anti- PSMA RRPs will then be coupled to the surface of fluorescently labeled liposomes and PSMA specific binding will be confirmed in the cell assays. Successful execution of the proposed Specific Aims would establish the initial proof of concept of the utility of phage-displayed RRP libraries to select high affinity ligands for a molecular target, in this case, PSMA. In the Phase II studies, potential commercial applications of the anti-PSMA RRPs for improving the diagnosis and management of prostate and other cancers, and in anti-angiogenic therapies will be developed. The major thrust of the PSMA commercial development program will be to conduct studies aimed at establishing the RRPs as targeting ligands to improve the delivery of imaging or pharmacologically active agents to cancer cells that express PSMA. Further, it is anticipated that the phage-displayed RRP technology developed here will be generally applicable for rapid discovery of ligands that specifically recognize other disease specific markers. ? ? ?