Development of KV1.3-Blocking Monoclonal Antibodies using Tetrahymena thermophila
2018; Elsevier BV; Volume: 114; Issue: 3 Linguagem: Inglês
10.1016/j.bpj.2017.11.1749
ISSN1542-0086
AutoresJanna Bednenko, Rian Harriman, Lore Mariën, Hai M. Nguyen, Alka Agrawal, Ashot Papoyan, Yelena Bisharyan, Joanna Cardarelli, Ted Clark, Donna Cassidy-Hanley, Bas van der Woning, Hans de Haard, Ellen J. Collarini, Heike Wulff, Paul A. Colussi,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoExpression of the voltage-gated Kv1.3 potassium ion channel, whose role involves regulation of membrane potential and calcium signalling in both excitable and non-excitable cell types, is upregulated in activated immune cells associated with inflammatory and autoimmune diseases such as ischemic stroke, Alzheimer's disease, type 1 diabetes, multiple sclerosis, and psoriasis. Many studies have shown that specific Kv1.3 blockers are effective in a number of animal models of these inflammatory conditions demonstrating that Kv1.3 is an attractive target for therapeutic intervention. Despite its clinical relevance, however, current small molecule and peptide toxin drugs targeting Kv1.3 suffer a lack of selectivity, less than optimal bioavailability and/or poor pharmacokinetics. Monoclonal antibodies (mAbs) are widely considered superior to classical small molecule and peptide drugs in these aspects but technical difficulties in producing sufficient recombinant immunizing channel proteins remain a serious challenge in their development. We discuss here a general strategy to overcome this critical hurdle by combining overexpression of recombinant Kv1.3 in Tetrahymenathermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the channel protein. We show that the resulting recombinant Kv1.3 traffics to the cell surface, is correctly folded, adopts a native tetrameric conformation and is readily purified from membranes. Recombinant Kv1.3 was then used to generate and recover 70 full-length anti-Kv1.3 mAbs from immunized chickens and llamas, of which, 10 were able to inhibit Kv1.3 current. Select antibodies were shown to be potent (IC50<10nM) and specific for Kv1.3 over related Kv1 family members, hERG and hNav1.5.
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