Our collaborative efforts are focused on validation of promising humanized single chain Fragment variable (hscFv) antibodies targeting a unique amino acid peptide sequence of P2 × 4 receptor (P2 × 4R). Data indicate single dose post-treatment with our newly engineered hP2 × 4 scFv antibody restores mechanical and cold pain-related behaviors to baseline in our trigeminal nerve compression model (FRICT-ION). The reversal to naïve baseline persists until experiment end at 10 weeks. Anxiety-like behaviors that develop in mice with chronic pain do not develop in hscFv antibody treated mice. We are characterizing the efficacy of lead P2 × 4R hscFv antibodies on neuronal cell-type specific activation patterns in medullary spinal V nucleus slices from Fos-TRAP(targeted recombination in active populations) mice and cultured trigeminal ganglia (TG) neurons from FRICT-ION mice. Engineered scFv antibodies feature binding affinity and activity similar to or better than monoclonal antibodies because they are much smaller in size. Thus, they are brain/nervous tissue penetrant due to their small size and have promising biotherapeutic applications for both the nervous and immune systems, now recognized as interactive in chronic pain. The ribosome display method utilized to generate the scFvs is a powerful cell-free technology widely used to select single-chain antibody fragments against the target of choice. This method is inexpensive, rapid, and was used to quickly develop repertoires of high-affinity antibodies targeting P2 × 4. In our hands the scFvs developed have high affinity in the picomolar range, superior stability and solubility. The patent was filed and published. Moving forward we currently continue evaluating these humanized P2 × 4R scFv antibodies that have strong nanomolar binding affinity, reduced self-immunogenicity, and relatively inexpensive large-scale production toward new drug application status based on the successful reversal of pain related measures and reduction of neuronal activation. This project opens new avenues for development of highly effective non-opioid biologic therapeutic interventions for chronic pain. Grant support from DoD grant W81XWH2010930 (KNW, SRAA); VA Merit grant BX002695 (KNW); NIH NIDCR R21 DE028096; NIH UG3 NS123958.