title: Mouse monoclonal antibodies to transient receptor potential ankyrin 1 act as antagonists of multiple modes of channel activation.
abstract: The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in different pathophysiologies that include asthma, cough, itch, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and pain behaviors in rodents. Hence, TRPA1 antagonists are being pursued as potential therapeutics. With the goal of generating monoclonal antibodies (mAbs) to human TRPA1 that could act as selective antagonists, we immunized mice with a variety of antigens expressing TRPA1 channels. After generation of hybridomas, the hybridoma conditioned media were screened to identify the mAbs that bind TRPA1 channels by a flow cytometry assay utilizing U2OS or Chinese hamster ovary (CHO) cells stably expressing TRPA1. The purified IgGs from the hybridomas that showed selective binding to TRPA1 were evaluated for antagonism in agonist-induced (45)Ca(2+) uptake assays using CHO-TRPA1 cells. Several of the mAbs showed concentration-dependent inhibition of AITC and cold (4°C) activation of TRPA1. The most potent mAb, 2B10, had IC50 values of approximately 260 and 90 nM in the two assays, respectively. These antagonist mAbs also blocked osmotically activated TRPA1 as well as activation by an endogenous agonist (4-oxo-2-nonenal). In summary, we generated mouse mAbs against TRPA1 that act as antagonists of multiple modes of TRPA1 activation.
authors.affiliation: Departments of Therapeutic Discovery (K.J.L., R.P., V.B., C.A.P.) and Neuroscience (W.W., N.R.G.), Amgen Inc., Thousand Oaks, California.
authors.forename: Ki Jeong
authors.initials: KJ
authors.lastname: Lee
authors.affiliation: Departments of Therapeutic Discovery (K.J.L., R.P., V.B., C.A.P.) and Neuroscience (W.W., N.R.G.), Amgen Inc., Thousand Oaks, California.
authors.forename: Weiya
authors.initials: W
authors.lastname: Wang
authors.affiliation: Departments of Therapeutic Discovery (K.J.L., R.P., V.B., C.A.P.) and Neuroscience (W.W., N.R.G.), Amgen Inc., Thousand Oaks, California.
authors.forename: Rupa
authors.initials: R
authors.lastname: Padaki
authors.affiliation: Departments of Therapeutic Discovery (K.J.L., R.P., V.B., C.A.P.) and Neuroscience (W.W., N.R.G.), Amgen Inc., Thousand Oaks, California.
authors.forename: Vivian
authors.initials: V
authors.lastname: Bi
authors.affiliation: Departments of Therapeutic Discovery (K.J.L., R.P., V.B., C.A.P.) and Neuroscience (W.W., N.R.G.), Amgen Inc., Thousand Oaks, California.
authors.forename: Cherylene A
authors.initials: CA
authors.lastname: Plewa
authors.affiliation: Departments of Therapeutic Discovery (K.J.L., R.P., V.B., C.A.P.) and Neuroscience (W.W., N.R.G.), Amgen Inc., Thousand Oaks, California ngavva@amgen.com.
authors.forename: Narender R
authors.initials: NR
authors.lastname: Gavva
mesh_terms: D000447:Aldehydes / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels; D003412:Cricetulus; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors*; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000037:antagonists & inhibitors*
chemical_list: C403894:4-oxo-2-nonenal; D000447:Aldehydes; D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels; D002118:Calcium