Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice.

Karin N Westlund,Marena A Montera,Aleyah E Goins,Adinarayana Kunamneni,Nikita Suri,Sascha R A Alles,Ravi V Durvasula,Robyn Bartel,Sabrina L Mcilwrath

doi:10.3390/ijms222413612

Abstract

Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.

Highlights

Targeting P2X4R with an scFv for Chronic Pain ReversalP2X4R is an ATP-gated purinergic receptor ion channel found sparingly in the cell membrane under normal conditions but not typically involved in nociception itself [1]
Extracted RNA from full-length antibody was isolated from five mouse spleens afthree immunizations with a unique custom 13 a.a. extracellular sequence of P2X4R
These results show that P2X4R scFv reduces excitability of large-diameter trigeminal ganglia (TG) neurons by neurons by increasing their rheobase, which may underlie the anti-allodynic action of the increasing their rheobase, which may underlie the anti-allodynic action of the scFv

Summary

Introduction

Targeting P2X4R with an scFv for Chronic Pain ReversalP2X4R is an ATP-gated purinergic receptor ion channel found sparingly in the cell membrane under normal conditions but not typically involved in nociception itself [1]. Spinal microglia produce and release brainderived neurotrophic factor (BDNF), initiating persisting activity in second-order neuronal cells and the disruption of neuronal Cl- homeostasis causal in persistence of pain and opioid tolerance [3,17,18,19,20,21]. Both phosphorylated p38 and P2X4R are initially exclusively

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