In vivo evaluation of monoclonal antibody M4M using a humanised rat model of stroke demonstrates attenuation of reperfusion injury via blocking human TRPM4 channel

Abstract

Background

Blocking Transient Receptor Potential Melastatin 4 (TRPM4) in rodents by our antibody M4P has shown to attenuate cerebral ischaemia-reperfusion injury. Since M4P does not interact with human TRPM4, the therapeutic potential of blocking human TRPM4 remains unclear. We developed a monoclonal antibody M4M that inhibited human TRPM4 in cultured cells. However, M4M has no effect on stroke outcome in wild-type rats. Therefore, M4M needs to be evaluated on animal models expressing human TRPM4.

Methods

We generated a humanised rat model using the CRISPR/Cas technique to knock-in (KI) the human TRPM4 antigen sequence.

Results

In primary neurons from human TRPM4 KI rats, M4M binds to hypoxic neurons, but not normoxic nor wild-type neurons. Electrophysiological studies showed that M4M blocked ATP depletion-induced activation of TRPM4 and inhibited hypoxia-associated cell volume increase. In a stroke model, administration of M4M reduced infarct volume in KI rats. Rotarod test and Neurological deficit score revealed improvement following M4M treatment.

Conclusion

M4M selectively binds and inhibits hypoxia-induced human TRPM4 channel activation in neurons from the humanised rat model, with no effect on healthy neurons. Use of M4M in stroke rats showed functional improvements, suggesting the potential for anti-human TRPM4 antibodies in treating acute ischaemic stroke patients.

Keywords:

• TRPM4 channel
• monoclonal antibody
• reperfusion injury
• acute ischaemic stroke
• hypoxia
• humanised rat model

Ethics approval and consent to participate

All animal procedures were approved by the Institutional Animal Care and Use Committee (protocol no. A19099) of Lee Kong Chian School of Medicine and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines 2.0.

Authors’ contributions

P.L. conceived and directed the project. C.P.P., S.W., B.C., S.W.L., J.T.S.Q., A.T-H.L, B.N. and P.L. conceived, analysed data, and wrote the paper. C.P.P., S.W., B.C. and S.W.L. performed experiments and analysed data. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

P.L. has a patent WO-2014209239-A1 (Trpm4 Channel Inhibitors for Stroke) for the TRPM4 blocking antibody. The authors declare that they have no other competing interests.

Availability of data and material

All data generated or analysed during this study are included in this published article and its supplementary.

Additional information

Funding

This work was supported by grants MOH-001384-00, MOH-001361-00 and MOH-000522-00 from the Singapore Ministry of Health’s National Medical Research Council.