A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions.
We describe a novel approach for high-throughput screening of recombinant antibodies, based on their immobilization on solid cellulose-based supports. We constructed a large human synthetic single-chain Fv antibody library where in vivo formed complementarity determining regions were shuffled combinatorially onto germline-derived human variable-region frameworks. The arraying of library-derived scFvs was facilitated by our unique display/expression system, where scFvs are expressed as fusion proteins with a cellulose-binding domain (CBD). Escherichia coli cells expressing library-derived scFv-CBDs are grown on a porous master filter on top of a second cellulose-based filter that captures the antibodies secreted by the bacteria. The cellulose filter is probed with labeled antigen allowing the identification of specific binders and the recovery of the original bacterial clones from the master filter. These filters may be simultaneously probed with a number of antigens allowing the isolation of a number of binding specificities and the validation of specificity of binders. We screened the library against a number of cancer-related peptides, proteins, and peptide-protein complexes and yielded antibody fragments exhibiting dissociation constants in the low nanomolar range. We expect our new antibody phage library to become a valuable source of antibodies to many different targets, and to play a vital role in facilitating high-throughput target discovery and validation in the area of functional cancer genomics.
Azriel-Rosenfeld
Ronit
R
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Green Building, Room 202, Tel-Aviv University, 69978, Ramat Aviv, Israel
Valensi
Moran
M
Benhar
Itai
I
D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology; D002482:Cellulose / Q000737:chemistry; D020650:Combinatorial Chemistry Techniques; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D015151:Immunoblotting; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D000951:Antigens, Neoplasm; D022801:Complementarity Determining Regions; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D002482:Cellulose