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.
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
J Pharmacol Exp Ther. 2014 Aug;350(2):223-31. doi: 10.1124/jpet.114.215574. Epub 2014 Jun 3.
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.
瞬时受体电位锚蛋白 1（TRPA1）通道与多种病理生理过程有关，包括哮喘、咳嗽、瘙痒和炎性疼痛。TRPA1 的激动剂，如芥末油及其关键成分丙烯基异硫氰酸酯（AITC），在人类中引起疼痛和神经源性炎症，以及啮齿动物的疼痛行为。因此，TRPA1 拮抗剂被作为潜在的治疗方法进行研究。为了产生能够作为选择性拮抗剂的针对人 TRPA1 的单克隆抗体（mAbs），我们用表达 TRPA1 通道的多种抗原对小鼠进行免疫。生成杂交瘤后，通过利用稳定表达 TRPA1 的 U2OS 或中国仓鼠卵巢（CHO）细胞的流式细胞术测定筛选杂交瘤条件培养基，以鉴定与 TRPA1 通道结合的 mAbs。从显示与 TRPA1 选择性结合的杂交瘤中纯化的 IgG，在使用 CHO-TRPA1 细胞的激动剂诱导（45）Ca（2+）摄取测定中评估其作为拮抗剂的作用。几种 mAbs 显示出对 AITC 和冷（4°C）激活 TRPA1 的浓度依赖性抑制。最有效的 mAb 2B10 在这两种测定中的 IC50 值分别约为 260 和 90 nM。这些拮抗剂 mAb 还阻断渗透压激活的 TRPA1 以及内源性激动剂（4-氧代-2-壬烯醛）的激活。总之，我们生成了针对 TRPA1 的小鼠 mAb，它们作为多种 TRPA1 激活方式的拮抗剂。
