Current methods for converting an antibody fragment (for example, scFv, Fab, Fab', etc.) involves subcloning from an M13 phagemid or yeast display vector by either restriction enzyme digestion or PCR, followed by ligation into 1 or more vectors to produce the heavy and light chains, transformation into E. coli and subsequent DNA sequencing validation. The proposed scFv --> IgG method using recombineering changes this paradigm for subcloning antibody fragments to produce IgG molecules into a simple transduction of a properly modified phagemid or yeast-display vector into a genetically-modified strain of E. coli harboring a specially-modified IgG expression shuttle plasmid. The costs and labor are reduced from approximately $100-$200 for traditional subcloning and DNA sequencing analysis to under $20 per clone conversion. Additionally, automation equipment is unnecessary and the low error-rate of recombineering is expected to obviate the need for DNA sequence validation. AxioMx is developing a pipeline for the rapid discovery (less than two weeks) of recombinant Abs. Completion of the objectives of this proposal will allow researchers to develop and quickly assemble IgG molecules which could be useful for high throughput proteome analysis, diagnostics, and immunotherapeutics. The ability to clone immuno-pools from phage, yeast and other display technologies while keeping the heavy and light chains linked is a significant advantage to the proposed method. We expect this method to increase the efficient production of better antibodies with implications for both diagnostics and therapeutics.
Public Health Relevance Statement: Public Health Relevance: The market for antibodies in research is approximately $2B, with the market for custom antibodies >$100M and expected to grow at least until 2016 [C. Bird, lead analyst, Frost and Sullivan report, Strategic Analysis of the Research Antibody Market, March 2012]. Most recombinant antibodies are screened in display as antibody fragments. Current methods for converting an antibody fragment (for example, scFv, Fab, Fab', etc.) involves subcloning from an M13 phagemid or yeast display vector by either restriction enzyme digestion or PCR, followed by ligation into 1 or more vectors to produce the heavy and light chains, transformation into E. coli and subsequent DNA sequencing validation. The proposed method will reduce the costs and labor from $100-$200 for traditional subcloning (including DNA sequencing analysis) to under $20 per clone conversion. Additionally, the need for automation equipment will be limited, even in high-throughput mode. And the low error-rate of recombineering is expected to obviate the need for DNA sequence validation. Finally, unlike almost all other methods, the recombineering method links the heavy and light chains together. So that transformation of the novel strain of E. coli with a pool of display clones will keep together within the bacterium the heavy and light chains that bind to the target. Clones will be able to be tested in mammalian cells faster and cheaper.
Project Terms: Antibodies; Attachment Sites (Microbiology); Automation; Bacteria; Bacteriophages; Binding (Molecular Function); Birds; cost; Custom; design; Diagnostic; Digestion; DNA Sequence; DNA Sequence Analysis; Elements; Equipment; Escherichia coli; Genes; Genetic Recombination; Immunoglobulin Constant Region; Immunoglobulin Fragments; Immunoglobulin G; Immunoglobulin Variable Region; Immunotherapeutic agent; in vivo; Integrase; Lead; Libraries; Ligation; Light; Link; Mammalian Cell; Marketing; Methods; mutant; novel; Peptides; Phage Display; Plasmids; Production; Proteome; public health relevance; Recombinant Antibody; Recombinants; Reporting; Research; Research Personnel; Resistance; restriction enzyme; System; Systems Integration; Technology; Testing; Therapeutic; Validation; vector; Yeasts
