Our overarching goal is to develop conformationally-specific recombinant monoclonal antibodies (R-mAb) including Immunoglobulin G (IgG), single chain variable fragments (scFv) and nanobody (nAb) formats as a novel class of biologics to target voltage-gated sodium (Nav) channels involved in pain signaling. Recent breakthroughs in the structural biology of ion channels and Rosetta computational approaches for enhanced design and refinement of antigens, antibodies (Abs) and stable peptides have set the stage for applying rational design approaches to create conformationally-selective antibodies as superior therapeutic candidates to treat chronic pain. Advances recombinant Ab technology allows for the generation of a broader set of candidate therapeutics in different formats, yet with complementary attributes, that when used in conjunction further increases the likeliehood of success. To pursue the goals of this project we will assemble a diverse and interdisciplinary research team that will include experts in pain biology, development of therapeutics, development of Abs in R-mAb, scFv and nAb formats, computational protein design, neuroscience, electrophysiology, pharmacology, biostatistics, and preclinical models of pain. This project will establish our expert research team and generate preliminary data that would support rationale, feasibility, and validity of our rational design approach for a subsequent Team Research U19 grant application (RFA-NS-21-015). Human genetic studies have identified the Nav1.7, Nav1.8, and Nav1.9 channel subtypes as critical mediators of action potential generation in C-fiber nociceptors, and established these channels as molecular targets for pain therapy. There is a growing trend toward targeting ion channels with biologics, and we will use this approach to identify novel biological therapeutics for the treatment of pain. In particular, mAbs have emerged as prominent therapeutics due to their low immunogenicity, high selectivity, and favorable half-lives, and there are currently >130 different FDA approved mAbs in various formats in clinical use. Following initial studies with polyclonal Abs that demonstrated the technical feasibility, multiple preclinical programs are now using the full spectrum of available technologies to generate diverse forms of Abs against extracellular loops of ion channels. An immunogen design approach, using the Rosetta modelling software, has been recently developed to stabilize protein structural motifs as effective antigens to generate Abs targeting precisely defined epitopes. Our research team will be in a unique position to use our novel structure-based approach and apply our interdisciplinary expertise to develop conformationally-specific mAbs. We propose to design small proteins presenting epitope mimetics from human Nav1.7, Nav1.8, and Nav1.9 channels followed by generation and characterization of mAbs in IgG, scFv and nAb formats against the stabilized epitopes to develop therapeuti...