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.