Phase II year
2015
(last award dollars: 2016)
Phase II Amount
$1,277,966
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 21 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 $1000 genome would be the inflection point at which whole genome sequencing would become commonplace. But even at $10,000 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 $5000 and 4 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 $1000 genome, we think antibody identification and production can eventually go to under $100 and less than 2 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 Statement: Public Health Relevance: At the moment it costs ~$1000-3000 and $10,000-20,000 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 21 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 $1000 genome would be the inflection point at which whole genome sequencing would become commonplace. But even at $10,000 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 $5000 and 4 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 $1000 genome, we think antibody identification and production can eventually go to under $100 and less than 2 weeks. At that point, whole proteome analyses for many different organisms and disease states will be possible. And new methods will arise.
NIH Spending Category: Bioengineering; Biotechnology; Genetics; Human Genome
Project Terms: Affinity; Algorithm Design; Animals; Antibodies; Antibody Affinity; base; Basic Science; Biological Assay; Biological Markers; Bromodomain; chromatin immunoprecipitation; Cloning; combinatorial; commercialization; Contracts; cost; Data; design; design and construction; Development; Diagnostic; Digit structure; Disease; Diversity Library; DNA; DNA Modification Methylases; Engineering; Enzyme-Linked Immunosorbent Assay; Enzymes; Epitopes; gene synthesis; Generations; Genetic Engineering; Genome; genome sequencing; Genomics; Goals; High-Throughput DNA Sequencing; High-Throughput Nucleotide Sequencing; Human; Hybridomas; Immune Sera; Immune system; Immunization; Immunofluorescence Immunologic; Immunoglobulin Fragments; Immunoglobulin G; Immunoglobulins; Immunohistochemistry; Length; Libraries; Mammalian Cell; Methods; Molecular Biology Techniques; Monoclonal Antibodies; Mutagenesis; next generation sequencing; novel; Oligonucleotides; Organism; Oryctolagus cuniculus; overexpression; Partner in relationship; Phase; Phosphoric Monoester Hydrolases; Phosphotransferases; polyclonal antibody; Process; Production; Proteins; Proteome; public health relevance; Reading; Reagent; Recombinant Antibody; Recombinants; Research; Research Personnel; Resources; scale up; screening; Sequence Analysis; Speed (motion); synthetic biology; synthetic construct; Technology; Time; transcriptome sequencing; uptake; Validation; Variant; Western Blotting; Yeasts
