PROJECT SUMMARY AND ABSTRACT 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. The overall goal is to bring this technology through Phase 1 and 2 human trials after meeting the following milestones. In this Diversity Supplement to our funded UG3 grant to support Ms. Aleyah Goins, we propose the following mechanistic studies of our lead humanized CCKBR scFv: Milestone A. Functional studies of humanized CCKBR scFv on neuronal hyperexcitability. Patch-clamp electrophysiological and calcium imaging studies of murine sensory neurons from neuropathic mice and sensitized hiPSC-derived sensory neurons treated with and without humanized CCKBR scFv will be performed. Milestone B. Cytokine and exosomal analysis. Exosomes will be isolated from culture supernatant of sensitized hiPSC-derived sensory neurons treated with humanized CCKBR scFv or vehicle. The exosomes will be lysed, microRNAs isolated and then analyzed using RNA sequencing. Milestone C. Neuropathic mice tissue and blood analysis: Characterization of the immune cell populations will advance understanding of how our humanized CCKBR scFv mediates chronic pain alleviation. Whole blood, brain, spinal cord will be obtained from mice with neuropathic pain treated with humanized CCKBR scFv or vehicle after euthanasia and perfusion. Flow cytometry analysis of blood and tissues, as well as immunohistochemical staining of trigeminal nerve, brain, and spinal cord will be performed. Staining of known injury markers, excitatory and inhibitory neuron markers will be performed. These 3 core goals of Ms. Goins research project will elucidate the mechanism of action of our humanized CCKBR scFv on human neurons and its reversal of chronic pain- and anxiety-like behaviors in mouse models.