Skip Navigation

Find Funding

Contract Topics

330 Generation of Site-Specific Phospho-Threonine Protein Standards for use in Cancer Assays

Fast-Track proposals will be accepted.

Number of Anticipated Awards: 3-5

Budget (total costs, per award): Phase I: $150,000 for 9 months; Phase II: $1,000,000 for 2 years

It is strongly suggested that proposals adhere to the above budget amounts and project periods. Proposals with budgets exceeding the above amounts and project periods may not be funded.

The deadline for receipt of all contract proposals submitted in response to this solicitation is: November 25, 2013 by 4:30 p.m. ET.

Summary:
As phosphorylated proteins play a significant role in normal and abnormal cellular function, there is a critical need for the production of pure, analytically characterized phospho-proteins/polypeptides for use in assays designed to capture the phosphorylation signatures of different cancers. Phospho-protein/polypeptide standards can serve as reference controls for quantitative immunoassays, immunogens for generating phospho-specific antibodies, and can be utilized in kinase inhibitor screens as well as protein-protein interaction studies. Technologies for generating phospho-serine and –tyrosine proteins are currently progressing, whereas the production of phospho-threonine (pThr) proteins has been unsuccessful due to imprecise chemical, enzymatic, and recombinant techniques. For example, biological systems that generate pThr-reagents are lacking, and those that exist provide poor control over site-directed phosphorylation and the phosphorylation percentage at each site. Additionally, commercially available reagents often do not meet purity guidelines for specific analytic applications. Therefore, the development of an appropriate methodology for the production of high quality and analytically verified pThr-protein/polypeptide standards will be a valuable step toward the commercialization of assays that detect target proteins associated with disease and drug action. The identification of such phosphorylated biomarkers has significant potential for the development of safer and more effective targeted cancer therapies.

Project Goals:
The primary goal of this contract topic is to develop innovative site-directed phospho-threonine protein synthesis technologies for the production of marketable pThr protein/polypeptide standards that enhance the analytic capacity of cancer assays. Technologies must demonstrate the reproducible generation of high quality, analytically verified phosphorylated threonine protein standards with 50 – 80% modification of the specified site. Protein standards listed below are of particular interest to the NCI; however, any phospho-protein target of clinical relevance to cancer may be developed. Peptide domains should contain 100 – 1000 amino acid residues that span the phospho-site of interest. A minimum of 30 amino acids in length may be acceptable, specifically if both a capture and detection antibody can bind to the peptide fragment.

Ultimately, the development of robust, quantitative, phospho-threonine specific assays will require:
  1. production of phosphorylated polypeptide standards;
  2. verification of the specific phospho-sites;
  3. quantification of site-specific phospho-residues.
Priority Name Phospho-Site(s)
1 ATR (ataxia telangiectasia and Rad3-related protein) pT1989
2 mTOR (mechanistic target of rapamycin) pT2446
3 Akt/PKB (v-akt murine thymoma viral oncogene homolog) pT308
4 Chk2 (CHK2 checkpoint homolog) pT68
5 53BP1 (tumor suppressor p53-binding protein) pT543
6 XRCC1 (X-ray repair complementing defective repair in Chinese hamster cells 1) pT519/T523/T488

Phase I Activities and Expected Deliverables:
  • Develop proof-of-concept methodology to reliably produce threonine site-directed, high content (>50%), pThr-protein/polypeptide standards for at least two NCI-approved targets. Full length protein is desired, but if too difficult to produce, polypeptides of 100-1000 amino acid domains will be acceptable. Under certain circumstances, shorter peptides with a minimum 30 amino acids in length may also be acceptable, pending NCI approval.
  • Provide data that demonstrates reproducibility and accuracy of the methodology that includes analytic (quantitative) measurements of the produced pThr-calibrators, including but not limited to site of phosphorylation and level of phosphorylation.
  • Demonstrate preliminary product stability (<10% degradation of the phospho-content) for at least 1 month when stored at company-determined optimized conditions (e.g., -80°C).
  • Deliver to NCI a minimum of 0.5 mg each of two NCI-approved pThr-protein/polypeptide standards selected for production. A corresponding Certificate of Performance that contains the analytic characterization strategy and measurements for each protein must also be submitted.
  • Provide the written methodology for the generation of the pThr-calibrators using a Standard Operating Procedure (SOP) template to be provided by NCI.
  • If requested, provide NCI with sufficient reagents to perform ten test runs for independent validation of the methods used for phospho-protein production and characterization.
  • Provide technical support, and if requested, one on-site training session for the NCI.
Phase II Activities and Expected Deliverables:
  • Processes should be optimized to reproducibly generate quantitative, threonine site-specific phosphorylation with the highest level of modification feasible (>80% preferred) for six NCI-approved targets.
  • Demonstrate preliminary product stability (<10% degradation of the phospho-content) for at least 1 month when stored at company-determined optimized conditions (e.g., -80°C).
  • Perform full validation of the method with three runs for all designated pThr-protein targets, and provide data that characterizes reproducibility, variability, and accuracy of the optimized methods with Quality Control measures implemented.
  • Deliver to NCI:
    • A minimum of 1 mg each of all of the specified pThr-recombinant proteins/peptides
    • All data
    • Final versions of the Certificate of Performance for each polypeptide
    • An ISO- and CLIA- quality SOP of method
  • If requested, provide NCI with sufficient reagents to perform ten test runs for independent validation of the methods used for phospho-protein production and characterization.
  • Provide technical support, and if requested, one on-site training session for the NCI.
  • Provide the program and contract officers with a letter of commercial interest.



[Back to Contract Topics]