A high throughput next generation sequencing (NGS) pipeline to generate high quality recombinant therapeutic antibodies

Abstract Therapeutic antibodies are used to treat many different diseasesincluding cancerauto immunityinfection and atherosclerosisThey are the fastest growing class of pharmaceuticalswith annual revenues of$B inOne of the most important methods to select therapeutic antibodies is by selection from vast antibody librariesHowevergood libraries are extremely challenging to produceand commercially available libraries are subject to onerous royalty and milestone paymentsSpecificaandapos s business model is to offer selected antibodies and antibody libraries to customers unencumbered by intellectual property rights or downstream royalty and milestone paymentsSpecifica already has customers for both productsvalidating the business modelWe have previously shown that by combining phage displayyeast display and next generation sequencingNGSwe are able to identify overdifferent antibodies from a selectionreflecting the far deeper sampling that NGS providesHoweverunless antibodies identified by NGS can be easily produced as clones for further analysistheir specificities and properties cannot be assessedrisking the loss of potentially valuable antibodiesthe greater the number of different antibodiesthe greater the number of different epitopesand consequently biological activitiesthat can be targetedHowevergoing easily from sequence to clones presently represents the primary bottleneck in the full exploitation of NGS for in vitro antibody selection that we address in this proposalWe propose to develop three different methods to easily generate antibody clones identified from next generation sequencing based on rolling circle amplificationPCR and gene synthesisIn Specific Aimwe will compare the ease with which clones can be derived using these methods and how deep the different methods are able to reach into the abundance rankcan they isolate a clone sequenced only onceIn Specific Aimwe will examine how deeply into the NGS abundanceantibodies continue to be positive for the targetwill an isolated antibody sequenced only once bind the targetThe approaches described here will allow us to integrate the use of NGS routinely into antibody isolationallowing us to improve both aspects of our servicesto increase the diversity of antibodies selected against particular targets for clients in our therapeutic antibody selection businessandBy providing the most effective NGS interrogation and clone isolation methods to our library clientswe will empower them to maximally exploit the full diversity of our librariesIn phase IIwe will apply the lessons learnt and methods developed in phase I to develop a highly efficient antibody selection pipeline that will integrate NGS into all aspects of selection and down stream antibody production and testingThis pipeline will comprisenovel informatic analysis approaches that will allow us to automatically identify HCDRand VH clonotypesand extract appropriate primers or gene sequencesnovel vectors that will allow the direct mammalian expression of antibodies confirmed as positive by yeast display without additional cloningautomation of the phage and yeast display selection processes Public Health Relevance Therapeutic antibodies are selected from vast antibody librariesIn generalthe greater the number of different antibodies in a librarythe higher the quality of the antibodies that can be selected from itHere we propose to develop novel methods to isolate many more specific antibodies from a selectionusing next generation sequencing