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239 Development of Alternative Affinity Capture Reagents for Cancer Proteomics Research

Number of anticipated awards: 3
(Fast-Track proposals will be accepted.)
Budget (total costs): Phase I: $150,000;
Phase II: $1,000,000

The deadline for receipt of all contract proposals submitted in response to this solicitation was:
5:00 p.m. Eastern Standard Time
Monday, November 6, 2006

Today, existing biotechnology reagent companies produce thousands of antibodies per year. Many of these are commercially available. However, many of these antibodies are known to be poorly characterized and suboptimal across multiple applications. Polyclonal antibodies lack the reproducibility of monoclonal antibodies. Likewise, the production of monoclonals is expensive and may take 6-8 months to produce. Even after production, there is no guarantee that a monoclonal antibody will be specific for the target of interest, will work in the needed assay, or can be used in combination with other antibodies due to an antibody's large size and subsequent competition for overlapping binding domains. As such, the high costs associated with producing even small quantities of monoclonal antibodies represent a large barrier towards cost-effective reagents and resources for proteomic technology research and clinical adaptation. The goal of this project is to develop reproducible, highly qualified/characterized alternative protein capture reagents for the cancer research community. The development of these affinity capture reagents will be done in coordination with NCI's Clinical Proteomic Technologies for Cancer initiative and be targeted to a list of over 100 purified recombinant proteins being constructed and characterized through this initiative.

The National Cancer Institute has developed intensive programs in the field of clinical proteomics to understand and quantify the alterations that occur in proteins that will play a significant role in the early diagnosis and effective treatment of cancer in the 21st century. In an effort to accelerate the development of clinical protein detection systems, the NCI held a series of scientific and technical meetings with leading experts (scientists, physicians, clinicians, and engineers) in this field by addressing technology development, standardization, implementation and integration of the most robust proteomic technologies that could be routinely used in a clinical setting. The first of these meetings was held in April 2002 with the most recent in February 2006. These meetings identified and defined effective strategies that the NCI should take to address the many challenges of clinical cancer proteomics. At the Proteomic Technologies Reagents Resource Workshop held on December 2005, there was strong support for the development of renewable, alternative capture reagents that could be produced more efficiently and cost-effectively than monoclonal antibodies and hence, provide an inexpensive, well-characterized resource for the scientific community. Several technologies such as yeast single chain antibodies, "nanobodies," or synthetically produced capture reagents such as aptamers and nanotechnology reagents demonstrate a potential for alternative, reproducible, cost-effective reagents in proteomics research.

Therefore, the NCI is interested in proposals that focus on developing alternative affinity capture reagents that can effectively compete against ELISA-based antibody technologies in terms of protein recognition, binding affinity, and detection and can be reproducibly produced in a cost-effective and efficient manner. These affinity reagents will ultimately be designed to target proteins developed in coordination with the Clinical Proteomic Technologies Initiative for Cancer that is producing purified cancer-related proteins (approximately 100) for research programs. Furthermore, these capture reagents must pass performance characterization criteria and be made available as a resource to the scientific community. The minimal performance platforms the affinity reagent must be required to surpass are ELISA-based technologies. Other suggested choices of performance applications that could be validated included Western blot, immunohistochemistry, and immunoprecipitation. In addition, other considerations include quantitative information for affinity reagents (Kd, on-rate, off-rate), the actual binding epitope in order to interpret the quantitative characterization, and application in multiplex platforms such as microarrays. While it is expected that initial development and quality assurance/quality control costs may be comparable to that of monoclonal antibodies, it is intended that production costs of these renewable reagents will be significantly lower.

Phase I Activities and expected deliverables:

• Work with the Clinical Proteomic Technologies for Cancer community, private and public sectors to identify appropriate minimum characterization criteria/validation assays (Commercially available ELISA kits can serve as appropriate tests for performance criteria).
• Generate affinity reagents to at least ten protein targets and demonstrate equivalent or improved performance to ELISA and other affinity-based platforms.
• Present findings to an NCI Evaluation Panel and demonstrate how the capture reagents compare in binding affinity to antibody-based platforms such as ELISA and have improved cost effectiveness and throughput capabilities in production compared to antibodies.
• Research should be proposed with quantitative feasibility milestones.

Phase II Activities and expected deliverables:

• Implement strategy and project plan for a fully functional affinity reagent development platform for at least 100 protein targets developed through the Clinical Proteomics Technology Initiative for Cancer. The reagents should be able to capture the target of interest from complex biological mixtures such as blood, plasma, or tissue. The plan should include comparison with ELISA-based technologies for evaluation of performance criteria.
• Test performance criteria against ELISA and other affinity platforms.
• Work with the Clinical Proteomic Technology Initiative for Cancer programs to integrate capture reagents into the proteomic research platforms.

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