Fig. 2: ART5803 blocks NMDAR internalization and NMDAR hypofunction driven by pathogenic autoantibody (#003-102 Ab).

a HEK293 cells expressing human NMDAR GluN1 and GluN2B subunits, were used in an NMDAR internalization assay to assess antibody effects on GluN1 surface expression by flow cytometry. Representative histograms of data used to generate Fig. 2b, show changes in geometric mean fluorescence intensity (GeoMFI) of allophycocyanin fluorescence (APC-A) normalized for visual comparison. Dotted lines indicate 100% GluN1 surface expression. GluN1/GluN2B expressing cells were treated for 2 h with either #003-102 Ab, ART5803 or control antibody (0-10 µg/mL). b NMDAR internalization by #003-102 Ab, ART5803 and isotype control at 2 h post-treatment. Data are mean of n = 4 independent replicates ± SEM. c NMDAR internalization by ART5803, bivalent (two-armed) ART5803 and Fab ART5803 at 2 h post-treatment. Data are mean of n = 4 independent replicates ± SEM. d ART5803 block (#003-102 Ab + ART5803 co-incubation for 2 h) and rescue (#003-102 Ab pre-treatment for 2 h, ART5803 post treatment for 2 h) of #003-102 Ab induced NMDAR internalization. Data are mean of n = 3 independent replicates ± SEM. e Assessment of ART5803’s agonistic or antagonistic effects on NMDAR was performed using a Fluorometric Imaging Plate Reader (FLIPR) assay to measure intracellular Ca²⁺ levels with a calcium-sensitive fluorescent dye in HEK293 cells expressing human NMDAR GluN1 and GluN2B subunits. In agonistic activity assessments, ART5803, human IgG isotype control antibody, MK-801, or assay buffer only were added, and fluorescence data was acquired over 5 min. Sequentially for antagonistic activity assessments, NMDA or assay buffer only was added, and fluorescence data was acquired over 5 min. Data are mean ± SEM of n = 3 independent replicates and were analyzed by unpaired, two-tailed Student’s t-test. ** p < 0.01, ns = not significant. RFU relative fluorescence units. f Blocking activity of ART5803 on NMDAR hypofunction (Ca²⁺ influx reduction) induced by #003-102 Ab. Cells preincubated with control antibody and subsequently treated with control antibody (conditions on the left side of graph) were either stimulated with assay buffer only or NMDA to demonstrate baseline activity and maximum activation, respectively. Data are mean ± SEM of n = 3 independent replicates. Statistical analysis were performed using unpaired, two-tailed, Student’s t-tests. *p < 0.05, ** p < 0.01. RFU relative fluorescence units. Source data is provided as a Source Data file.