Fig 2: Iluzanebart Proposed Mechanism of Action. A Healthy CSF1R signaling. Under normal conditions, ligand binding to CSF1R induces homodimerization and triggers transphosphorylation and activation of the CSF1R intracellular kinase domains, recruiting Src kinase and triggers downstream mediators such as SYK and transmembrane adapter protein DAP12, promoting microglia survival, proliferation, phagocytosis, and motility. Ligand binding to and activation of TREM2 also triggers the same intracellular signaling pathways through pSYK and through the DAP12 adapter protein. B Dysfunctional CSF1R in CSF1R-ALSP. When CSF1R receptors are dysfunctional due to genetic mutations, signaling through microglia health-promoting pathways is reduced (C) Treatment of CSF1R-ALSP with iluzanebart. Iluzanebart binds with high affinity to the extracellular domain of two TREM2 molecules, sequestering them in an active, dimerized state and activating microglia health-promoting downstream signaling to compensate for lost CSF1R signaling as well as increasing signaling through an increase in the amount of CSF1 receptors at the cell surface

Fig 3: Iluzanebart induces proliferation and increases CSF1R levels in microglia in vivo. hTREM2-CV mice were dosed intraperitoneally with a single, 200 mg/kg dose of either iluzanebart or IgG control, or an equal volume of saline. Brain tissue was harvested 48 h after dosing, enzymatically digested, and cells stained with Ki67, CSF1R, CD45, CD68, and human IgG. Only mice with detectable human IgG present in the sample were analyzed. A Analysis of microglia post iluzanebart dosing show a significant increase in the number of Ki67 positive microglia compared to IgG or saline control. B Representative histogram shows an increase in population of cells with high CSF1R expression (blue circle) in microglia with Iluzanebart treatment. C Quantification of mean fluorescence intensity (MFI) demonstrates an increase in cells with high CSF1R by Iluzanebart treatment. N = 5–6 mice per group. Data shown is reported as % of saline control. Significance was determined by one-way ANOVA with Tukey’s multiple comparison’s test, * < 0.05, ** < 0.005, *** < 0.0005

Fig 4: Iluzanebart (ILU) rescues the effects of CSF1R inhibition in human MDM and iPSC microglia. A-B Human Monocyte Derived Macrophages (hMDM). Iluzanebart counteracts the effects of pharmacological CSF1R inhibition on confluence (A) and morphology (B) of MDMs in vitro (n = 3 independent experiments). Treatment of hMDM cells with PLX5622 (iCSF1R) led to a reduction in cell confluence and morphology (striped bars). Treatment with iluzanebart (10 µg/ml; blue bar) restored cellular confluence and ramified morphology, whereas treatment with IgG at the same concentration (green bars) had no effect. C-E iPSC Derived Human Microglia (iMGL). C Treatment with PLX5622 (iCSF1R) decreased iPSC microglia cell viability, as measured by CellTiterGlo (grey bar). Treatment with iluzanebart (75 µg/ml; blue bar) led to a significant restoration of viability, whereas treatment with IgG control at the same concentration (green bar) had no effect (n = 4 independent experiments). D iMGL. Treatment with PLX5622 (iCSF1R) altered iMGL morphology (grey bar). Treatment with iluzanebart (75 µg/ml) restored cellular confluence and ramified morphology (blue bar), whereas treatment with IgG control had no effect (n = 4 independent experiments). E iMGL. Treatment with PLX5622 (iCSF1R) (n = 4 independent experiments) increased numbers of caspase 3/7 positive cells in iPSC microglia cultures (grey bar). Treatment with iluzanebart (75 µg/ml) led to a significant reduction in caspase 3/7 + cells (blue bar), whereas treatment with IgG control had no effect (green bar). Significance was determined by one-way ANOVA with Tukey’s multiple comparison’s test. * < 0.05, ** < 0.05, *** < 0.0005, **** < 0.0001

Fig 5: Iluzanebart (ILU) increases viability and restores CSF1R activity in CRISPR-generated I794T+/- mutant iPSC microglia. iPSC microglia generated to heterozygously express the I794T CSF1R mutation were harvested, plated onto IgG- or iluzanebart-coated surfaces (ILU), and incubated for 7 days before analysis. Cultures were assessed for viability by CellTiterGlo, soluble CSF1R in the media by ELISA, or for phospho-CSF1R levels in the cells by ELISA. A In I794T+/- microglia, viability was significantly increased in iluzanebart-treated cultures compared to IgG-treated controls (n = 9 independent experiments). B While iluzanebart treatment did not significantly affect levels of total CSF1R in either WT or I794T+/- cells, total CSF1R levels were 23% lower in I794T+/- cells compared to WT cells (n = 9 independent experiments). C While soluble CSF1R levels were 38% lower in I794T+/- cell culture media compared to WT, iluzanebart treatment significantly increased levels of soluble CSF1Rin both cultures (n = 9 independent experiments). D While iluzanebart treatment had no impact on phosphorylation of CSF1R in WT cells, phospho-CSF1R levels were 34% lower in I794T+/- cells compared to WT, and iluzanebart treatment restored pCSF1R levels to normal WT levels. (n = 9 independent experiments). E While iluzanebart treatment slightly increased the ratio of activated CSF1R in WT cells, activated CSF1R levels were increased 2.26 × in I794T+/- cell cultures (n = 9 independent experiments). All data were normalized to cell number as determined using Incucyte cell number determination. Significance was determined by two-way ANOVA with Tukey’s multiple comparison’s test, * < 0.05, ** < 0.005, **** < 0.0001