title: Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies.
abstract: Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080, anatoly.shcherbatko@pfizer.com.
authors.forename: Anatoly
authors.initials: A
authors.lastname: Shcherbatko
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Davide
authors.initials: D
authors.lastname: Foletti
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Kris
authors.initials: K
authors.lastname: Poulsen
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Pavel
authors.initials: P
authors.lastname: Strop
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Guoyun
authors.initials: G
authors.lastname: Zhu
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Adela
authors.initials: A
authors.lastname: Hasa-Moreno
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Jody
authors.initials: J
authors.lastname: Melton Witt
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Carole
authors.initials: C
authors.lastname: Loo
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Stellanie
authors.initials: S
authors.lastname: Krimm
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Ariel
authors.initials: A
authors.lastname: Pios
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Jessica
authors.initials: J
authors.lastname: Yu
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Colleen
authors.initials: C
authors.lastname: Brown
authors.affiliation: the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, and.
authors.forename: John K
authors.initials: JK
authors.lastname: Lee
authors.affiliation: the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158.
authors.forename: Robert
authors.initials: R
authors.lastname: Stroud
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: Arvind
authors.initials: A
authors.lastname: Rajpal
authors.affiliation: From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.
authors.forename: David
authors.initials: D
authors.lastname: Shelton
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D000918:Antibody Specificity / Q000276:immunology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005260:Female; D005620:Freund's Adjuvant; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D010146:Pain / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control; D055503:Protein Multimerization / Q000276:immunology; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology*; D051381:Rats; D058476:Receptors, Purinergic P2X2 / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D058477:Receptors, Purinergic P2X3 / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D014302:Trinitrobenzenesulfonic Acid; D059265:Visceral Pain / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control
chemical_list: D000911:Antibodies, Monoclonal; D007473:Ion Channels; D058920:Purinergic P2X Receptor Antagonists; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D014302:Trinitrobenzenesulfonic Acid; D005620:Freund's Adjuvant
keywords: P2X3; cell surface receptor; monoclonal antibody; pain; patch clamp; purinergic receptor; receptor internalization