Therapeutic antibodies are used to treat many different diseases, including cancer, auto-immunity, infection and atherosclerosis. They are the fastest growing class of pharmaceuticals, with annual revenues of ~$75B in 2013. One of the most important methods to select therapeutic antibodies is by selection from vast antibody libraries. However, good libraries are extremely challenging to produce, and commercially available libraries are subject to onerous royalty and milestone payments. Specifica's business model is to offer selected antibod- ies and antibody libraries to customers unencumbered by intellectual property rights or downstream royalty and milestone payments. Specifica already has customers for both products, validating the business model. We have previously shown that by combining phage display, yeast display and next generation sequencing (NGS), we are able to identify over 1000 different antibodies from a selection, reflecting the far deeper sam- pling that NGS provides. However, unless antibodies identified by NGS can be easily produced as clones for further analysis, their specificities and properties cannot be assessed, risking the loss of potentially valuable antibodies: the greater the number of different antibodies, the greater the number of different epitopes, and consequently biological activities, that can be targeted. However, going easily from sequence to clones pres- ently represents the primary bottleneck in the full exploitation of NGS for in vitro antibody selection that we ad- dress in this proposal. We propose to develop three different methods to easily generate antibody clones iden- tified from next generation sequencing based on rolling circle amplification, PCR and gene synthesis. In Specif- ic Aim 1 we 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 rank (can they isolate a clone sequenced only once?). In Specific Aim 2, we will examine how deeply into the NGS abundance, antibodies continue to be positive for the target (will an isolated antibody sequenced only once bind the target?). The approaches described here will allow us to integrate the use of NGS routinely into antibody isolation, allowing us to improve both aspects of our services: 1) to increase the diversity of antibodies selected against particular targets for clients in our ther- apeutic antibody selection business; and 2) By providing the most effective NGS interrogation and clone isola- tion methods to our library clients, we will empower them to maximally exploit the full diversity of our libraries. In phase II, we will apply the lessons learnt and methods developed in phase I to develop a highly efficient an- tibody selection pipeline that will integrate NGS into all aspects of selection and down stream antibody produc- tion and testing. This pipeline will comprise: 1) novel informatic analysis approaches that will allow us to auto- matically identify HCDR3 and VH clonotypes, and extract appropriate primers or gene sequences; 2) novel vectors that will allow the direct mammalian expression of antibodies confirmed as positive by yeast display without additional cloning; 3) automation of the phage and yeast display selection processes.
Public Health Relevance Statement: Public Health Relevance Therapeutic antibodies are selected from vast antibody libraries. In general, the greater the number of different antibodies in a library, the higher the quality of the antibodies that can be selected from it. Here we propose to develop novel methods to isolate many more specific antibodies from a selection, using next generation sequencing.
Project Terms: Affinity; Anchored Polymerase Chain Reaction; Antibodies; Antibody Diversity; Antibody Formation; antibody libraries; Atherosclerosis; Autoimmunity; Automation; B-Lymphocytes; Bacteriophages; base; Binding; Biological; Biotechnology; Businesses; Client; Cloning; combinatorial; cost; Development; Disease; drug discovery; Epitopes; gene cloning; gene synthesis; Generations; Genes; Genetic Recombination; Goals; Human; improved; In Vitro; Infection; Informatics; innovation; Intellectual Property; interest; Libraries; Malignant Neoplasms; method development; Methods; Modeling; Molecular; Molecular Target; next generation sequencing; novel; payment; Phage Display; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Process; Property; Property Rights; public health relevance; Recombinant Antibody; Recombinants; Risk; Role; Sampling; Services; Small Business Innovation Research Grant; Specificity; Stream; Testing; Therapeutic antibodies; vector; Yeasts
