Development and characterization of a monoclonal antibody blocking human TRPM4 channel

Abstract

TRPM4 is a calcium-activated non-selective monovalent cation channel implicated in diseases such as stroke. Lack of potent and selective inhibitors remains a major challenge for studying TRPM4. Using a polypeptide from rat TRPM4, we have generated a polyclonal antibody M4P which could alleviate reperfusion injury in a rat model of stroke. Here, we aim to develop a monoclonal antibody that could block human TRPM4 channel. Two mouse monoclonal antibodies M4M and M4M1 were developed to target an extracellular epitope of human TRPM4. Immunohistochemistry and western blot were used to characterize the binding of these antibodies to human TRPM4. Potency of inhibition was compared using electrophysiological methods. We further evaluated the therapeutic potential on a rat model of middle cerebral artery occlusion. Both M4M and M4M1 could bind to human TRPM4 channel on the surface of live cells. Prolonged incubation with TRPM4 blocking antibody internalized surface TRPM4. Comparing to M4M1, M4M is more effective in blocking human TRPM4 channel. In human brain microvascular endothelial cells, M4M successfully inhibited TRPM4 current and ameliorated hypoxia-induced cell swelling. Using wild type rats, neither antibody demonstrated therapeutic potential on stroke. Human TRPM4 channel can be blocked by a monoclonal antibody M4M targeting a key antigenic sequence. For future clinical translation, the antibody needs to be humanized and a transgenic animal carrying human TRPM4 sequence is required for in vivo characterizing its therapeutic potential.

Investigation of Novel Small Molecular TRPM4 Inhibitors in Colorectal Cancer Cells

“…Recently, a new monoclonal antibody, M4P, against TRPM4 was developed and was shown to specifically inhibit TRPM4 currents. M4P could contribute to research on TRPM4 in disease models [ 41 ]. However, small TRPM4 inhibitors will possibly have an advantage because of their lower price and smaller size resulting in the ability to diffuse through the membrane.…”

Transcriptional Profiling of Tumorspheres Reveals TRPM4 as a Novel Stemness Regulator in Breast Cancer

“…Our results also suggest that TRPM4 can be used as a druggable target in breast cancer. Indeed, the cell membrane localization of the protein renders it an ideal candidate for novel anti-cancer drugs that could either fall into the small-molecule inhibitors category [71] or they could be specific antibodies that block the protein [72].…”

Pharmacological Modulation and (Patho)Physiological Roles of TRPM4 Channel—Part 1: Modulation of TRPM4

“…The selectivity of these compounds is, in many cases, rather weak, necessitating the search for and testing of newer and newer com- [78,79]. Moreover, especially in in vivo studies, either specific antibodies M4P, M4M, and M4M1 were used to block TRPM4 [80,81], or small-interfering RNAs (siRNA) were applied to silence TRPM4 [82]. This latter approach was used in vitro as well [35].…”

“…M4P was selective for TRPM4, as it did not influence either TRPM5 [80] or TRPM2 and TRPM7 channels [136]. Recently, Low et al developed two mouse monoclonal antibodies (M4M and M4M1) targeting an extracellular epitope of human TRPM4 [81]. Interestingly, these antibodies reduced ion currents mediated by expressed human TRPM4 channels but failed to be effective in vivo in rats, highlighting the specificity of TRPM4 inhibition by these truly selective antibodies.…”