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Therapeutic anti‐P2X4 receptor scFv antibody for chronic neuropathic pain
Author(s) -
Kunamneni Adinarayana,
Montera Marena,
Suri Nikita,
Westlund Karin N,
Alles Sascha R,
Pappu Suguna,
Durvasula Ravi
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.05215
Subject(s) - panning (audio) , antibody , neuropathic pain , recombinant dna , receptor , microbiology and biotechnology , in vivo , pharmacology , phage display , medicine , chemistry , immunology , biology , biochemistry , paleontology , zoom , gene , lens (geology)
The P2X purinoceptor 4 (P2X4) plays a crucial role in signaling leading to chronic inflammatory and neuropathic pains, thus, the P2X4 receptor provides a new potential therapeutic target for their treatment. We have generated a panel of mouse single‐chain Fv‐antibodies (scFvs) recognizing extracellular peptide of P2X4 using cell‐free ribosome display, for use to pain‐signalling neurons. A mouse scFv library was generated, and ribosome display was applied for anti‐P2X4 scFv recombinant antibody selection. After three rounds of bio‐panning, a panel of recombinant antibodies was isolated followed by ELISA, cross reactivity analysis, western blotting and immunofluorescence staining. Anti‐P2X4 scFv clones with high specificity and affinity were successfully selected. Indirect ELISA assay revealed differential P2X4 peptide binding capability and specificity for three scFvs, with no reaction to the negative control anti‐Zika scFv 7‐2. The panning process was efficient in selecting clones of high affinity, as was evident from affinity differences of the scFvs: scFv95 and scFv12 had the highest and second highest affinities, respectively, while scFv103 had lower affinity. The highest affinity P2X4 receptor scFvs (12 and 95) were selected for in vivo behavior testing in the F oramen R otundum I nflammatory C onstriction T rigeminal I nfraorbital N erve injury ( FRICT‐ION ) Model. Single dose intraperitoneal injection (4 mg/kg, 100 μl) reversed mechanical allodynia and anxiety in mice with long standing neuropathic pain (8–10 weeks). Reversal to naïve baseline within one week was maintained for more than 10 weeks post‐treatment at experiment end. Continued reversal was maintained at baseline for >20 weeks in a second cohort. The Western blot of the medullary brainstem provides evidence of brain penetration of both these scFv antibodies that reduce hypersensitivity and anxiety behaviors. We also performed whole‐cell patch clamp recordings from dissociated trigeminal ganglia neurons of nerve‐injured vs naïve mice and determined the effect of P2X4 receptor scFvs on neuronal excitability. These data provide support for pursuit of P2X4 receptor scFvs as translational therapy for pain relief. Support or Funding Information 1. GRANT FUNDING NIH NIDCR R21 DE028096 2. Start‐up Funds to Dr. Durvasula 3. Startup Funds to Dr. Westlund