Development and characterization of functional antibodies targeting NMDA receptors

Abstract

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.

Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation

“…For developing better and specific antibody-selective immunotherapies, the identification of patient-derived monoclonal antibodies and the characterization of their binding properties to further investigate the disease pathologies is an essential step ( Kreye et al, 2021 ). The binding pattern of patient-derived monoclonal antibodies can be studied using purified full-length receptors or domains in distinct conformations bound to ELISA plates followed by structural analysis, e.g., single particle cryo-EM of combined receptor and autoantibody proteins and functional analyses ( Chou et al, 2022 ; Noviello et al, 2022 ; Tajima et al, 2022 ). Such combined approaches will provide detailed binding pattern pictures of patient-derived autoantibodies opening novel windows for clinical treatment using antibody depletion strategies.…”

“…In case of conformational epitopes, however, there are also limitations with such approaches. Recently, binding patterns of patient-derived monoclonal antibodies have been studied using purified full-length receptors or domains in distinct conformations bound to ELISA plates followed by structural analysis, e.g., single particle cryo-EM of combined receptor and autoantibody proteins and functional analyses ( Chou et al, 2022 ; Tajima et al, 2022 ).…”

Protein quality control of N-methyl-D-aspartate receptors

“…Novel approaches to target specific subunits of NMDARs can be achieved through structural-based drug design. Alternative structural-based approaches are demonstrated in the novel study in which, Tajima et al generated an antibody specific for GluN2B-containing receptors that acts to inhibit the function of the channel by stabilizing the closed state of the channel (Tajima et al, 2022). The introduction of structural-based stabilizing antibodies into the CNS to promote the stability of variant receptors is an appealing future application.…”

Excitatory and inhibitory D-serine binding to the NMDA receptor

“…The conformational transitions of the LBDs from open to closed clamshells result in the generation of tension in the LBD-TMD linkers, which in turn facilitates gating of the TMD channel ( Tajima et al, 2016 ; Chou et al, 2020 ). The ATDs allosterically regulate channel activities in a subtype-dependent manner via distinct interactions with the LBDs ( Yuan et al, 2009 ; Gielen et al, 2009 ; Karakas et al, 2011 ; Tajima et al, 2022 ). Therefore, the LBDs can be considered the fundamental vehicles for driving ligand gating.…”