A novel pre-defined CDR library for selection of affinity reagents

DESCRIPTION provided by applicant The most commonly used method for generating antibodies is through immunization of animals However this method is generally low throughput expensive time consuming and the antibodies generated are not always renewable Recombinant antibodies rAb like single chain variable fragments scFv have many attractive attributes compared to polyclonal antisera and monoclonal antibodies derived from hybridomas They are renewable through overexpression in the appropriate heterologous host they are easily stored and transferred as DNA and they can be genetically engineered as fusions to various enzymes fluorescent proteins and epitope tags While a number of approaches for generating recombinant affinity reagents exist the cost and throughput of current technologies represent significant roadblocks to the development of a comprehensive and broadly available resource of renewable affinity reagents We believe that improvements to both gene synthesis technologies and the increased affordability of high throughput DNA sequencing can be leveraged to create antibody discovery pipelines based on synthetic biology that rival animal immune systems In this proposal we will build a high throughput pipeline for recombinant antibody development in as few as days The proposed platform takes advantage of pre designed diversity next generation sequence analysis and advanced molecular biology techniques to enable the rapid identification of specific antibodies Although our screening platform is being developed with single chain variable fragment antibodies scFvs the technology is applicable to both Fab and yeast display libraries High throughput conversion of the scFvs to full immunoglobulin G IgG will be integrated within the pipeline so that the antibodies can be directly validated in the desired final format In genomics it was thought that the $ genome would be the inflection point at which whole genome sequencing would become commonplace But even at $ per genome researcher uptake was phenomenal and new ways of using the NextGen sequencers like ChIP Seq RNA seq etc were invented In a similar fashion we believe that at $ and weeks researchers will begin to develop new applications where the cost of producing antibodies is no longer a relevant factor and speed becomes everything The ready availability of low cost high quality affinity reagents will potentially accelerate all aspects of basic science research provide diagnostics for disease biomarkers and serve as a proof of concept for therapy Like the $ genome we think antibody identification and production can eventually go to under $ and less than weeks At that point whole proteome analyses for many different organisms and disease states will be possible And new methods will arise PUBLIC HEALTH RELEVANCE At the moment it costs $ and $ to contract out production of rabbit polyclonal and monoclonal antibodies respectively Unfortunately these reagents take months to deliver may not be renewable cannot be engineered and their sequences are not known Recombinant antibodies rAb have many attractive attributes compared to polyclonal antisera and monoclonal antibodies derived from hybridomas They are renewable through overexpression in the appropriate host they are easily stored and transferred as DNA and they can be genetically engineered as fusions to various enzymes fluorescent proteins and tags While a number of approaches for generating recombinant affinity reagents exist the cost and throughput of current technologies represent significant roadblocks to the development of a comprehensive and broadly available resource of renewable antibodies We believe that improvements to both gene synthesis technologies and the increased affordability of high throughput DNA sequencing can be leveraged to create antibody discovery pipelines based on synthetic biology that rival animal immune systems Here we present a platform for the rapid generation of recombinant monoclonal antibodies in as few as days at a cost comparable to that of polyclonals The platform takes advantage of pre designed library diversity that more closely mimics the natural diversity in human antibodies next generation sequence analysis to decode the enriched sequences and advanced molecular biology techniques to enable the rapid identification and production of recombinant antibodies In genomics it was thought that the $ genome would be the inflection point at which whole genome sequencing would become commonplace But even at $ per genome researcher uptake was phenomenal and new ways of using the NextGen sequencers like ChIP Seq RNA seq etc were invented In a similar fashion we do not know what the uptake and inflection points will be for antibodies At $ and weeks researchers may begin to develop new applications where the cost of producing antibodies is no longer a relevant factor and speed becomes everything The ready availability of low cost high quality affinity reagents will potentially accelerate all aspects of basic science research provide diagnostics for disease biomarkers and serve as a proof of concept for therapy Like the $ genome we think antibody identification and production can eventually go to under $ and less than weeks At that point whole proteome analyses for many different organisms and disease states will be possible And new methods will arise