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 21 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 IC 50 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.

Antibodies to the Extracellular Pore Loop of TRPM8 Act as Antagonists of Channel Activation

“…Second, in single concentration experiment, we identified three monoclonal antibodies that achieved 60% to 100% blockade of icilin-induced activation of the human TRPM8 channel. Only two monoclonal antagonist antibodies, one to hERG potassium channel [37], and another to TRPA1 channel [38], have been published to date, whereas two other monoclonal antibodies against the pore loop of TRPV1 did not block channel activation [20]. Our data support the view that it is feasible to generate monoclonal antagonist antibodies that show full block of channel activation by different mechanisms.…”

“…Kv1.2 and Kv3.1 [34], TRP channels, e.g. TRPC1, TRPC5, TRPV1 and TRPM3 [20], [22], [35] and CaV1.2 channels [36], as well as monoclonal antibodies to hERG [37] and TRPA1 channels [38].…”

Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization

“…Many published studies describing antibody production for membrane proteins that used genetic immunization have included boosts with some form of the protein, such as purified protein or cells expressing the protein (Sanders et al, 2002; Koch-Nolte et al, 2005; Lee et al, 2014). However, there are several reports for individual proteins that evidently used only DNA as immunogen.…”

Discovery of Functional Antibodies Targeting Ion Channels

“…This particular approach has shown some success using adenovirus, although it is not entirely clear in the published work which of several strategies led to the final functional monoclonal antibody. 76 Examples of strategies that have been used to isolate functional monoclonal antibodies described in the public domain are described in the case studies in this review.…”