Embryonic stem cells (ES) are a major focus of research, because of their experimental utility and great promise as therapeutic tissue regeneration and gene replacement agents. ES cells are rapidly growing pluripotent cells with the capacity to differentiate into multiple cell types. Despite the important role that human ES (hES) cells could have in biomedical applications, research faces some obstacles. For example, only a limited number of hES cell lines have been approved for study. Additionally, few hES cell lines are completely stable and subpopulations often develop that are partially differentiated toward different cell lineages. The stage of differentiation of hES cells is often accompanied by the expression of one or a few plasma membrane antigens (PMAs) such as the cell surface marker SSEA-4. Monoclonal antibodies (mAbs) to novel PMAs on hES cells are of great biomedical interest as research reagents to monitor and standardize the developmental stage of hES cells and also have potential as diagnostic reagents and in the development of therapeutic stem cells. Abeome Inc. has developed a high-throughput hybridoma based platform, DISH (Direct Selection of Hybridomas), for producing mAbs with unprecedented speed and at substantially lower costs than by any other method. DISH works because a proprietary, genetically modified myeloma fusion partner allows the generation of hybridomas that robustly secrete and surface present Ig. This innovation eliminates the labor intensive and problem-plagued steps of limiting dilution cloning because these hybridomas can be selected by Fluorescent Activated Cell Sorting (FACS) and plating can be automated. The goal of this Phase I proposal is to isolate of a battery of mAbs to novel PMAs from pluripotent hES cells derived from the hESBGN-01 or hESBGN-02 (NIH registry #BG-1 and BG-2) cell lines as well as to less pluripotent cells that have differentiated from these lines. In brief, formalin fixed low passage SSEA-4+ and SSEA-4- populations derived from hES cell lines will be used to immunize two sets of mice. DISH will be used to isolate individual hybridomas prepared from these mice. PMA-specific mAbs will be sub-screened to identify those reactive for the surface of SSEA-4+ cells, but not with the SSEA-4- stem cells and vice-versa. This Phase I project will take the Applicant through the development of this novel variation on its DISH technology and could lead to the rapid commercial production of mAbs to thousands of stem cell PMAs. ? ?
