Development of monoclonal antibody reagents and peptide tools for the identification and characterization of human embryonic stem cells.
Tools and Technologies I
Antibodies and peptides reactive with human embryonic stem cells, either differentiated or undifferentiated, are important tools that enable the identification, isolation and characterization of specific cell types. Antibodies and peptides that detect specific proteins on the surface of human embryonic stem cells allow the characterization and comparison of different human embryonic stem cell lines and enable the isolation of defined populations of live cells. Furthermore, understanding the differences between normal cells and embryonic stem cells in their responses to oncogene activation will also help discern whether stem cell therapies would have a cancer-inducing side effect. Previous studies have disappointingly only utilized monoclonal antibodies (mAbs) targeting antigens raised against human EC cells and mouse embryos. In this application, we propose the development of antibody reagents and peptide tools specific for human embryonic stem cells which will greatly increase the safety of future stem cell based therapies. To this end, our overall objective in this research proposal is the identification of a panel of human and mouse antibody reagents and peptide tools that specifically bind and internalize to/into live hESCs, by targeting different molecular features differentially expressed on undifferentiated hESCs. Key features of this proposal are 1) our expertise in antibody and peptide phage display methodology, 2) the capability for the generation of innovative antibody conjugates as required for successful applications, 3) a highly collaborative scientific network that has been assembled, and 4) an ability to timely evaluate the efficacy of the prepared antibodies and peptides.
Statement of Benefit to California:
Health care needs are unlimited, whereas resources are finite. Public expenditures on health care have to compete with other societal priorities such as education, the environment, defense and infrastructure. Even in relatively wealthy, developed countries, scarcity is the defining characteristic of resource allocation problems. Therefore, new therapies are desperately needed to enhance medical treatment options, to improve our quality of life and to lower healthcare costs. California has a large and diverse population that poses challenges for the future of medical care. A major goal for California’s supporters of stem cell research is the development of stem cell-directed tools/reagents that have broad medical applications. Stem cells have the potential to improve the health of all Californians, but they are especially important for research and the development of treatments for disease states that currently have no cure. For example, neurological diseases are especially difficult to treat with classical medicines, and these diseases are becoming increasingly prevalent, hence an ideal target for stem cell research. This proposed project is designed to generate materials of natural origin as a source of new tools and reagents for the analysis and discovery of human embryonic stem cells. The development of new therapeutics/drugs will depend on further characterization of these cells and the development of assays that allow for the evaluation of well defined molecules that can identify, and potentially monitor human embryonic stem cells.
This application focuses on generation of antibody and peptide tools that recognize hESC surface markers. Using phage display technology and immunization of mice, the applicants will select antibodies that bind with high affinity and specificity to the hESC surface, with particular emphasis on those that become internalized. The applicants also propose to identify cyclic peptides with similar activities. In addition to these major aims, the applicants discussed additional goals including identification of corresponding antigen targets and adaptation of internalized peptide/antibodies for potential delivery of cellular therapies. The reviewers were enthusiastic about the proposed tools but expressed significant concerns about the feasibility and scope of the effort. The proposal was well written and structured, and reviewers were confident that the applicant will eventually find antibodies that could be potentially valuable, although it is unclear that their specificity and affinity would be sufficient for all of the proposed uses. The perceived strengths in the approach were diminished by the omission of pertinent details, the overreliance on a single methodology, and the inclusion of tangential lines of investigation that complicated rather than refined the focus. Finally, the reviewers questioned the extent to which the eminently qualified investigators could balance their contribution to this project with significant, ongoing commitments to other scientific endeavors. The impact of the proposed tools could be substantial in both basic and clinical research. In terms of utility, this technology offers the potential to eliminate undifferentiated stem cells from differentiated populations that are destined for human use, thereby minimizing the risk of tumor formation. In addition to this clinical value, this technology could provide purer populations of differentiated cells for use in a wide variety of research and discovery efforts. While this is an active area of pursuit, the reviewers appreciated the applicants’ focus on identifying markers for undifferentiated cells rather than differentiated ones, the latter of which is more typical of the endeavors in this field. Finally, insights gained from use of this technology, or even the derived antibodies alone, could drive better understanding of the mechanisms of pluripotency and self-renewal. The most serious concern expressed by reviewers was that the research plan was too ambitious, and that the goals of this proposal would not be possible to achieve in two years with the proposed budget. There are several experiments, such as an attempt to identify antigen targets, which are sufficiently complex and ambitious to warrant separate aims but were described superficially as part of a larger aim. Furthermore, the applicant proposes a number of projects that are technically difficult, time- and resource-consuming, and are unlikely to be achieved within this application timeframe. These include conversion of peptides into peptidomimetics and therapy / siRNA delivery with peptides. Reviewers also commented that Specific Aim 3, based on cyclic peptide phage display, is a risky screen that may not result in applicable molecules. Reviewers were also disappointed that certain details were not provided – for instance, the applicant does not identify the particular hESC lines that will be used to create the library, and it was unclear whether the cell-surface membrane fractions to be used as an immunogen would be isolated from the same or different hESC line as that used to immunize mice. In addition, the applicant did not rationalize the decision to use both mouse and human antibodies, and thus the reviewers were uncertain as to the advantage of this approach. Some reviewers felt that the applicant relied too heavily on phage display and did not propose alternative or parallel methodologies, such as hybridoma generation, to improve the chances that useful antibodies would be obtained. As a final criticism, the reviewers noted that the approaches geared towards potential therapies were not convincingly integrated with the overall theme of this effort. It is unclear why the PI goes in such detail as far as intracellular antibody localization tracking. If therapeutic studies are planned relying on specific sub-cellular antibody localization, and if that is important for payload delivery efficacy, a better background/explanation would have been useful. The reviewers agreed that the applicants are eminently qualified to conduct the research described in this proposal. The principal investigator is an expert with phage display technology, and the co-investigator leads cutting edge research in hESC biology. One reviewer noted that the PI is involved in a large number of unrelated projects and wondered if his/her responsibilities might be spread too thin. The reviewers felt that the budget seemed appropriate or perhaps slightly excessive for the phage display experiments but is likely to be insufficient if the more ambitious projects, such as the target antigen identification, are pursued. In summary, while this proposal describes tools that could be of great value, the lack of focus and overly ambitious scope cast major doubt on its overall feasibility.