Phase I Amount
$1,080,240
We present a complete platform for the rapid generation and validation of recombinant mAbs. The innovations of this proposal include: (1) the pATHENA vector system for overnight conversion of phage display scFv or Fab clones into IgG molecules, (2) the Epivolve method for the isolation of site-specific Abs, (3) the incorporation of yeast display into pATHENA to allow biophysical measurement of binding affinities without the need for protein isolation, and (4) our MILKSHAKE technology for the validation of Abs for IHC, Western, and ELISA applications. This proposal is a culmination of- and largely a focused integration of several independent technologies that have been developed (and de-risked) by the principal investigator (PI). We now want to incorporate these technologies and add them into a single Abbratech platform. In essence, the proposal is a very robust set of molecular biology modules for: (i) library construction, (ii) library screening using both phage- and yeast display, (iii) biophysical and kinetic analysis, (iv) directed evolution, (v) affinity maturation ("AffMat"), (vi) protein engineering, (vii) production of site- directed Abs, (viii) protein expression integrated into a single platform, and (viii) Ab validation for flow cytometry, ICC, ELISA and Western applications. All parts of the proposed workflow have either can be- or have already been automated for high-throughput (HT) production with off-the-shelf robotic solutions. The proposed platform represents an innovative way to deliver recombinant mAbs, at ideally a retail total cost of less than several thousand dollars (costs are discussed in the Commercialization plan). This accuracy, specificity, projected timeline, and cost cannot be currently achieved by any other commercially available mAb discovery platform. This proposal meets a need that has not been markedly improved upon since the advent of monoclonal Abs forty years ago and phage display, around the same time. And it can finally place phage display as a primary engine of diversity, replacing the inefficient and time-consuming immunization schedules that even phage display advocates fall back on. The significance is high, the team is well-qualified, the innovation, tactical and strategic, is imaginative, and even though the scope of this Phase II is daunting, the team is well-equipped to do it.
Public Health Relevance Statement: NARRATIVE Improved methods for deriving high quality affinity reagents will be needed to keep up with the pending impact when single molecule protein sequencing instrumentation comes "online' in the next few years. When that time comes, it is not a realistic expectation to be able to go to an existing Ab catalog for mAbs that are well-validated, cloned, and sequenced, and that work in specific applications against newly discovered biomarkers. The high costs and low throughput of current antibody-generating technologies represent significant roadblocks to the development of a comprehensive and broadly available resource of renewable affinity reagents. Thus, there is an urgent need for all Abs to be defined by their sequences and made recombinant.
Project Terms: Antibodies; Clinical Treatment Moab; mAbs; monoclonal Abs; Monoclonal Antibodies; Antigenic Determinants; Binding Determinants; Epitopes; Back; Dorsum; Bacteriophages; Phages; bacterial virus; Biophysics; biophysical foundation; biophysical principles; biophysical sciences; Biotechnology; Biotech; Capital; Capital Financing; Capital Funding; Cells; Cell Body; Employment; Engineering; Enzyme-Linked Immunosorbent Assay; ELISA; enzyme linked immunoassay; Escherichia coli; E coli; E. coli; Flow Cytometry; Flow Cytofluorometries; Flow Cytofluorometry; Flow Microfluorimetry; Flow Microfluorometry; flow cytophotometry; Future; Genes; Immunoglobulin G; 7S Gamma Globulin; IgG; Immunization Schedule; instrumentation; Kinetics; Libraries; Methods; Molecular Biology; DNA Molecular Biology; Pain; Painful; Plasmids; pressure; Production; Protein Engineering; genetic protein engineering; protein design; Proteins; Publications; Scientific Publication; Publishing; Reagent; T-Cell Receptor; MHC Receptor; Major Histocompatibility Complex Receptor; T-Cell Antigen Receptors; Genetic Recombination; DNA Recombination; Recombination; Resources; Research Resources; Risk; Robotics; Specificity; Technology; Testing; Time; Work; Yeasts; Integrase; Generations; Measures; Cost Savings; falls; promotor; promoter; timeline; improved; Site; Area; Surface; Phase; Failure; Measurement; integrated system; system integration; Systems Integration; directed evolution; Directed Molecular Evolution; tool; Catalogs; catalog; System; experience; expectation; Peptide Sequence Determination; Amino Acid Sequence Determinations; Protein Sequence Determinations; Protein Sequencing; Protein Sequencing Molecular Biology; single molecule; Molecular Interaction; Binding; protein expression; Advocate; Affinity; Data; Qualifying; Recombinants; Phage Display; Validation; validations; technology validation; technology implementation; Principal Investigator; Development; developmental; vector; cost; scale up; Consumption; innovate; innovative; innovation; commercialization; bio-markers; biologic marker; biomarker; Biological Markers; screenings; screening; biophysical characteristics; biophysical characterization; biophysical measurement; biophysical parameters; biophysical properties; biophysical studies; biophysical analysis
