In order to understand the pathogenesis of cancer, it will be critical to elucidate the cellular signaling pathways that contribute to oncogenic transformation. Complex networks of protein kinases and adaptor proteins play a central role. These molecules undergo a series of conformational changes, as they interact with each other and become activated. Therefore, there is a growing demand for monoclonal antibodies (mAbs), which recognize conformational epitopes that are dependent on the activation state of the signaling protein. Since conformation changes are often correlated with phosphorylation events, the most widely used approach has been to raise phosphopeptide-specific antibodies. Such antibodies can be powerful tools to assess the phosphorylation status of the target molecule, but they have certain limitations: 1. Since peptide immunogens may not reflect the native conformation of the corresponding sequences in the intact protein, resulting mAbs often react with target proteins under denatured condition exclusively, and thus many mAbs are only applicable to the Western blotting system, precluding studies on intact cells or tissues. 2. Even if the peptide immunogens do mimic the conformation of the corresponding proteins, the phosphorylation sites are often occupied by binding proteins (e.g., Src-homology domain 2) upon phosphorylation, and are unavailable for antibody binding under native conditions. To bridge this technical gap between targeted immunization (synthetic peptide) and conformational alignment (peptide vs. corresponding protein), we have developed a series of algorithms and databases to optimize peptide design for effective immunization. In addition, we have established a highly efficient rabbit hybridoma system, and have shown that rabbit mAbs raised to synthetic peptide immunogens are superior to both mouse mAbs and rabbit polyclonals. We propose to combine this rabbit mAb system with our peptide-design method, and apply these methods to the development of conformation-specific antibodies to protein kinases involved in signaling pathways that are related to oncogenic transformation. Our approach will focus on predicted immunogenic peptide sequences that do not include phosphorylation sites. Phase I studies are designed to demonstrate the validity of the approach, and will focus on Src family kinases. We have previously developed conformation-specific mouse mAbs to c-Src, which will be compared with rabbit mAbs raised in the current project. If the approach proves to be successful, Phase II studies will be designed to develop optimized antibody reagents for monitoring the activation state of Src family kinases, as well as other protein kinases involved in oncogenic transformation.
Initially, antibodies developed in this project will be commercialized as research reagents. In addition, we will seek alliances with pharmaceutical companies to apply these mAbs as diagnostic reagents for diseases such as cancer, inflammatory diseases, and autoimmune diseases. ? ? ?
