Westlund, Karin N

Chronic pain affects nearly 100 million adults in the U.S., yet few novel therapeutics have emerged in recent decades. P2X4 receptor (P2X4R), implicated in pain signaling, represents a promising target. We evaluated a humanized single-chain variable fragment (hscFv) targeting P2X4R for its ability to reduce ATP-induced currents and modulate excitability in human dorsal root ganglion (hDRG) neurons. Voltage-clamp recordings confirmed that human P2X4R (hP2X4R) hscFv significantly reduced ATP-evoked currents in HEK-293T cells expressing human P2X4, likely by relocalization of the receptor to the perinuclear region after hscFv treatment. Immunohistochemistry and transcriptomic analyses demonstrated widespread P2X4R ( P2RX4 ) expression across hDRG neuronal subtypes in both male and female donors. Current-clamp recordings revealed that hP2X4R hscFv selectively increased action potential (AP) threshold in multi-firing hDRG neurons, without affecting single-firing neurons. Spontaneous activity at rest and depolarizing spontaneous fluctuation (DSF) amplitude were also reduced. Analysis confirmed consistent effects of hP2X4R hscFv on excitability parameters. These findings suggest that hP2X4 hscFv exerts modest but targeted effects on human sensory neurons, supporting its potential as a novel therapeutic for chronic pain.

Chronic pain affects a significant portion of the population, with fewer than 30% achieving adequate relief from existing treatments. This study describes the humanization methodology and characterization of an effective non-opioid single-chain fragment variable (scFv) biologic that reverses pain-related behaviors, in this case by targeting P2X4. After nerve injury, ATP release activates/upregulates P2X4 receptors (P2X4R) sequestered in late endosomes, triggering a cascade of chronic pain-related events. Nine humanized scFv (hscFv) variants targeting a specific extracellular 13-amino-acid peptide fragment of human P2X4R were generated via CDR grafting. ELISA analysis revealed nanomolar binding affinities, with most humanized molecules exhibiting comparable or superior affinity compared to the original murine antibody. Octet measurements confirmed that the lead, HC3-LC3, exhibited nanomolar binding kinetics (KD = 2.5 × 10−9 M). In vivo functional validation with P2X4R hscFv reversed nerve injury-induced chronic pain-related behaviors with a single dose (0.4 mg/kg, intraperitoneal) within two weeks. The return to naïve baseline remained durably reduced > 100 days. In independent confirmation, the spared nerve injury (SNI) model was similarly reduced. This constitutes an original method whereby durable reversals of chronic nerve injury pain, anxiety and depression measures are accomplished.

A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of KD = 1.794 × 10–8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.

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.