In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.

Directed evolution of novel binding proteins

http://biscweb.uark.edu/ipinto/biol5334/5.pdf

Protein-protein interactions: methods for detection and analysis.

Publisher Summary Combinatorial antibodies will play a significant role in the design of vaccines and new antiviral agents. Antibodies may also provide a way of determining receptor function in vivo and serve as templates for the design of small molecules. Combinatorial antibodies have been shown to provide an accurate functional reflection of the natural response as demonstrated by the ability of cloned antibodies to compete with serum antibodies for binding antigens. Combinatorial antibodies also provide a useful (if somewhat incomplete) guide to the molecular biology of the response. With its ability to provide large numbers of human antibodies directed against a single antigen, the combinatorial approach allows for the rapid assessment of immune dominant as well as neutralizing epitopes in the context of the human response. This information should be utilized in the future to guide the design of more effective vaccines. The new-found ability to generate human antibodies and to evolve their specificities and affinities, ex vivo promises increased the use of this class of molecules in the service of human health.

Human antibodies from combinatorial libraries.

We have developed a strategy for guiding the selection of human antibody fragments from phage display repertoires to a single epitope of an antigen, using rodent monoclonal antibodies as a template. Thus the heavy chain of a rodent antibody (MAb32) directed against human tumor necrosis factor alpha (TNF alpha) was cloned and paired as a template chain with a repertoire of human light chains for display as Fab fragments on filamentous phage. The phage were selected by binding to the antigen. The selected human light chains were in turn paired with a repertoire of human heavy chains displayed on phage, and the phage selected again. The isolated phage displaying human antibody fragments binding to TNF alpha also bound to a peptide comprising the N-terminal region of TNF alpha as with MAb32. One of the human Fab fragments was recloned for expression as a glycosylated human antibody in mammalian cells: Binding to TNF alpha could be competed with MAb32 or with anti-serum to the peptide, indicating the same epitope. The human antibody was found to have a binding affinity (Kd = 15 nM) similar to MAb32 (Kd = 26 nM). The process contrasts with existing means of "humanizing" rodent monoclonal antibodies in that the antibodies derived are completely human.

Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen.

Antibodies from phage antibody libraries.

Human antibody Fab fragments that bind to hepatitis B surface antigen (HBsAg) were generated by using a recombinant phage surface-display expression system. Characterization of HBsAg-specific Fab fragments isolated from two vaccinated individuals reveals diversity in specificity of antigen binding and in the sequences of the complementarity-determining region. The sequence results show examples of human light-chain promiscuity that result in fine specificity changes and a strong relationship to a human germ-line gene. This application illustrates further that this technique is a powerful tool to isolate distinct human antibodies against immunogenic viral targets.

Human combinatorial antibody libraries to hepatitis B surface antigen.

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Human combinatorial antibody libraries to hepatitis B surface antigen.