Humanization of therapeutic antibodies is a common step in adapting murine-derived antibodies for human use to reduce their immunogenicity. While single chain Fragment variable antibodies (scFvs) lack constant domains, the proposed humanization project will replace framework amino acids in the murine cholecystokinin B rector (CCKBR) scFvs with well-characterized human framework sequences. Affinity maturation of the target-binding complementarity-determining regions (CDRs) will ensure that the humanized CCKBR scFv acquires high affinity to both the rodent and human CCKBR peptide targets, which exhibit 85% identity. CHO cell expression and purification of top 10 candidate clones will provide protein to support efficacy and a thorough examination of binding kinetics by surface plasmon resonance. Comparison of humanized/affinity matured CCKBR scFv candidates to the parent murine scFv will enable selection of a lead scFv candidate(s) with binding kinetics (Kon and Koff) and target affinity (KD) anticipated to provide efficacy in animal models of neuropathic pain and be suitable for commercial development and clinical evaluation. The overall goal is to bring this technology through Phase 1 and 2 human trials after meeting the following milestones. Milestone 1 Year 1 Identify 3-5 humanized scFv clones that exhibit a KD of <130 nM affinity for the human peptide that defines the binding site for the parent scFv and its efficacy. Expression and purification will be provided in sufficient quantity (>90% purity, low endotoxin) to conduct validation assays. Milestone 2 Year 1 Select 1-2 ranked hits based on demonstrated in vitro efficacy using hiPSC-derived sensory neurons as the screening platform. Year 2 Select 1-2 ranked hits based on in vitro characterization in murine trigeminal (TG) and dorsal root ganglia (DRG) primary sensory neurons derived from experimental pain models. Milestone 3 Year 1-3 Determine dose response and validate efficacy of 1-2 hCCKBR scFv therapeutic candidates in murine models of neuropathic and back pain based on in vitro efficacy in Aim 2. Comparisons will include naïve, sham, and neuropathic pain model mice, both untreated and treated with humanized scFv (n =10 mice/group) administered once intranasally. Mechanical threshold increases of >15-fold and full recovery from anxiety and depression-like behaviors denote success. Comparisons with standard analgesics morphine and duloxetine will serve as proof of concept. Milestone 4 Year 3-5 Characterize and validate hCCKBR scFv lead in conjunction with NIH contractors. We will focus on IND enabling studies including CMC activities needed to support cGMP production and GLP pre-clinical Tox/PK/PD. Milestone 5 Years 4-5 Solidify interactions with NIH contractors and other pharmaceutical contractors for final formulation, upscaling, manufacturing, approvals for clinical trials, and commercialization.