The objective of this application is to establish a pilot, high-throughput, three-tiered antibody selection pipeline. Tier one antibodies will be renewable recombinant polyclonal antibodies that can be amplified from 1 ml to >/=100,000 liters with no loss of diversity. Eventually they will be selected against all human proteins. Structurally, the provided antibodies will be similar to traditional antibodies, allowing scientists to use them without modifying protocols. However, unlike traditional polyclonals, they will resemble affinity-purified polyclonals, with most antibodies directed towards the target of interest. These tier one polyclonals should satisfy most research needs. Where required, second tier monoclonal antibodies will be isolated from first tier polyclonal antibody pools. Third tier monoclonal antibodies will be custom projects requiring the creation of derivative libraries from second tier antibody leads, and the selection of monoclonals with improved affinities or specificities. The broad availability of antibodies against all human proteins will facilitate the understanding of human disease, and provide likely targets for therapeutic intervention, with the antibodies themselves having the potential to become therapeutic leads. The first two specific aims involve the creation of quality control pipelines for target antigen and antibody library production;the third combines these in the antibody selection pipeline, while in the fourth, selected antibodies are characterized. This project will combine phage and yeast antibody display, with initial selections carried out from phage antibody libraries, and subsequent selections from yeast libraries created by cloning phage outputs into yeast display vectors. Polyclonal pools will be expressed as single gene scFv-Fc fusions, using murine or rabbit Fc domains evolved for high expression levels. After internal antibody validation, characterization will be carried out at the Human Protein Atlas, which has an extensive pipeline characterizing antibodies for specificity, tissue and cellular distribution. This three-tiered approach will increase throughput by ~100 fold compared to the selection and characterization of monoclonals against each target, resulting in a dramatic reduction in costs. The renewable polyclonals will be highly functional, and provide a source for monoclonals when needed.

Public Health Relevance

The availability of antibodies against all human proteins will allow the assessment of the role of different proteins in human disease, such as cancer, diabetes, dementia and heart disease. Not only will this allow the identification of proteins that can be targeted in novel treatments, but the antibodies, or derivatives of them, may be usable as potential therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54DK093500-02
Application #
8335448
Study Section
Special Emphasis Panel (ZRG1-BST-M (51))
Program Officer
Sechi, Salvatore
Project Start
2011-09-25
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
2
Fiscal Year
2012
Total Cost
$1,411,039
Indirect Cost
$532,632
Name
Los Alamos National Lab
Department
Type
DUNS #
175252894
City
Los Alamos
State
NM
Country
United States
Zip Code
87545
D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato et al. (2014) The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires. MAbs 6:160-72
D'Angelo, Sara; Kumar, Sandeep; Naranjo, Leslie et al. (2014) From deep sequencing to actual clones. Protein Eng Des Sel 27:301-7
Jha, Ramesh K; Gaiotto, Tiziano; Bradbury, Andrew R M et al. (2014) An improved Protein G with higher affinity for human/rabbit IgG Fc domains exploiting a computationally designed polar network. Protein Eng Des Sel 27:127-34
Deantonio, Cecilia; Cotella, Diego; Macor, Paolo et al. (2014) Phage display technology for human monoclonal antibodies. Methods Mol Biol 1060:277-95
Ferrara, Fortunato; Naranjo, Leslie A; D'Angelo, Sara et al. (2013) Specific binder for Lightning-LinkĀ® biotinylated proteins from an antibody phage library. J Immunol Methods 395:83-7