A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

Abstract

Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila 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 protein. Using this approach, we targeted human Kv1.3, a voltage-gated potassium channel widely recognized as a therapeutic target for the treatment of a variety of T-cell mediated autoimmune diseases. Recombinant Kv1.3 was used to generate and recover 69 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<10 nM) and specific for Kv1.3 over related Kv1 family members, hERG and hNav1.5.

Ion channels as therapeutic antibody targets

“…46 The DNA immunization approach has been used to generate K v 1.3 nanobodies using the Ablynx platform, 93 as well as in combination with purified antigen to generate conventional K v 1.3 antibodies. 77 These are described in a later section.…”

“…Efficient recovery of purified channel proteins (in many cases, in the order of > 1mg/L culture) has enabled the development of antigen preparations and screening tools that have recently been used to generate a panel of blocking anti-K v 1.3 antibodies. 77 Besides the propensity for this organism to encode hundreds of native ion channels, it remains unclear why it bypasses the limiting factors associated with low expression yields in mammalian cells where there appears to be an upper limit on how many functional recombinant channels can reach the plasma membrane before creating a toxic metabolic environment. No such toxicity is observed in Tetrahymena, allowing many more recombinant channels to reach the cell surface.…”

Screening Strategies for the Discovery of Ion Channel Monoclonal Antibodies

“…Due to the structural and functional diversity of ion channels, different strategies need to be considered for generation of antigen formats of the ion channel or antigens derived from the channel (Wilkinson, Gardener, & Williams, ). While peptides derived from the ion channel can be used as antigens (Cox et al., ), it is usual to use a conformationally relevant form of the channel as the antigen (Bednenko et al., ). Ion channels are normally expressed in low abundance in vivo and the heterologous expression of the proteins needed to achieve high expression levels requires considerable experimentation to optimize the host expression system, particularly as heterologous overexpression can be toxic (Vu, Bautos, Hong, & Gelli, ).…”

“…conformationally relevant form of the channel as the antigen (Bednenko et al, 2018). Ion channels are normally expressed in low abundance in vivo and the heterologous expression of the proteins needed to achieve high expression levels requires considerable experimentation to optimize the host expression system, particularly as heterologous overexpression can be toxic (Vu, Bautos, Hong, & Gelli, 2009).…”

“…channel activity may be used as controls to demonstrate that the ion channel is expressed and that binding can be detected on the cell surface. For example, Bednenko et al (2018) demonstrated that the recombinant K v 1.3 channel correctly assembled into a tetramer by staining channel expressing cells with fluorescently labelled Stichdactyla toxin. The use of functional positive controls, if available, combined with the EC 50 values for channel activation can facilitate the understanding of assay sensitivity and the likelihood of detecting antibody ion channel modulators under a given set of assay conditions.…”