Rescue of in vitro models of CSF1R-related adult-onset leukodystrophy by iluzanebart: mechanisms and therapeutic implications of TREM2 agonism

Abstract

Microglia dysfunction is implicated in several neurodegenerative disorders, including a rare microgliopathy; CSF1R-related adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (CSF1R-ALSP). CSF1R-ALSP is caused by heterozygous loss-of-function mutations in the colony stimulating factor 1 receptor (CSF1R) gene, which encodes a receptor required for the differentiation of myeloid cells, as well as for microglial survival and proliferation. Similar functions have also been ascribed to triggering receptor expressed on myeloid cells 2 (TREM2), which shares an analogous microglia enrichment profile and converging intracellular signaling pathway mediated by spleen associated tyrosine kinase (SYK) and phosphoinositide-3-kinase (PI3K). Iluzanebart is a human monoclonal IgG1, human TREM2 (hTREM2) agonist antibody under development for the treatment of CSF1R-ALSP. To explore the therapeutic hypothesis that loss of CSF1R signaling and related microglial hypofunction can be circumvented via activation of TREM2, we evaluated the potential of iluzanebart to compensate for CSF1R loss-of-function. Herein, we demonstrate that iluzanebart is a potent, dose-dependent, and specific activator of TREM2 signaling in human primary cells. Iluzanebart treatment rescued viability of human monocyte-derived macrophages (hMDM) and induced pluripotent stem cell-derived human microglia (iMGL) in multiple in vitro models of CSF1R-ALSP, including in induced pluripotent stem cell (iPSC) differentiated microglia carrying the heterozygous I794T mutation found in CSF1R-ALSP patients. Additionally, iluzanebart treatment in microglia modulated surface levels of CSF1R, resulting in increased receptor activation as measured by phosphorylation of CSF1R. Differentially expressed genes identified in the hippocampus of mice treated with iluzanebart were exemplary of TREM2 activation and were related to cell proliferation, regulation of inflammatory processes, and innate immune response pathways. Proliferation of microglia, changes in protein levels of specific chemokines identified by gene expression analysis, and increased CSF1R levels were also confirmed in vivo. These findings demonstrate that iluzanebart is a potent and selective TREM2 agonistic antibody, with pharmacology that supports the hypothesis that TREM2 activation can compensate for CSF1R dysfunction and its continued clinical development for individuals with CSF1R-ALSP.

Fig. 1

Potency of iluzanebart across in vitro human model systems. Activation of TREM2 via protein phosphorylation was assessed using AlphaLISA assays. A HEK-hTREM2 Cells. In HEK293T cells expressing human TREM2 and DAP12, pDAP12 in response to iluzanebart for 45 min was analyzed, resulting in an EC50 potency of 1.09 nM (n = 2 independent experiments). Results are shown as normalized to IgG control, which did not stimulate SYK phosphorylation, and reported as % of maximal activation. B HEK-hTREM2 Cells. Similarly, in the same HEK293T cell line, iluzanebart had a potency of 0.19 nM (n = 3 independent experiments) when measuring phosphorylation of SYK. Results are shown as normalized to IgG control, which did not stimulate SYK phosphorylation, and reported as % of maximal activation. C Human Monocyte Derived Macrophages (hMDM). In human MDM cells, treatment with iluzanebart for 45 min stimulated phosphorylation of SYK with an average EC50 potency of 4.77 nM (n = 4 independent experiments). Results are shown as normalized to IgG control, which did not stimulate SYK phosphorylation, and reported as % of maximal activation. D iPSC Derived Human Microglia (iMGL). In human iPSC-derived microglia cells, iluzanebart treatment for 5 min stimulated phosphorylation of SYK with an average EC50 potency of 0.63 nM (n = 6 independent experiments), and is shown as normalized to IgG control, which did not stimulate SYK phosphorylation, and reported as % of maximal activation

Fig. 2

Iluzanebart is a specific agonist of human TREM2. A hTREM1-HEK. In a HEK293T cell line expressing hTREM1 and hDAP12, an anti-TREM1 antibody (green) stimulated phosphorylation of SYK after 45-min treatment, whereas iluzanebart (blue) did not (n = 3 independent experiments), confirming that iluzanebart is a TREM2-specific agonist with no cross-reactivity to the related TREM1 receptor. B TREM2 -/- iMGL. Iluzanebart (blue) does not stimulate pSYK in TREM2^−/− human iPSC-derived microglia. Concanavalin A (green) was used as a positive control demonstrating that TREM2^−/− cells can activate pSYK in response to TREM2-independent activators. Data shown is normalized to IgG control after 5 min exposure (n = 2 independent experiments) and reported as % of maximal activation. C iPSC Derived Human iMGL. Treatment of human iPSC microglia with iluzanebart reduced levels of soluble TREM2 levels after 24 h, compared to IgG control which had no effect (n = 2 independent experiments)

Fig. 3

Iluzanebart increases surface levels of CSF1R. A iPSC Derived Human Microglia (iMGL). Treatment of iMGLs with iluzanebart increased levels of soluble CSF1R levels after 24 h, compared to IgG control which had no effect (n = 3 independent experiments). B Representative image of population analysis of iMGL post treatment with iluzanebart show increases the population of CSF1R + CD45 + iMGL compared to IgG or untreated control after 24 h. C Representative histogram of iMGL shows an increase of surface CSF1R iMGL with iluzanebart. D Quantification of mean fluorescence intensity (MFI) demonstrates a TREM2-dependent increase in surface levels of CSF1R by iluzanebart. N = 7 independent experiments for wildtype, n = 5 independent experiments for TREM2 knockout iMGL. Significance was determined by 2-way ANOVA with Sidak’s multiple comparison’s test. * < 0.05

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 survival and restores morphology of human MDMs and iPSC microglia following CSF1 withdrawal in vitro. A-B Human Monocyte Derived Macrophages (hMDM). Iluzanebart counteracts the effects of CSF1 withdrawal-induced apoptosis and morphology in hMDM cultures from 2 independent monocyte donors (A) hMDM. Withdrawal of CSF1 from the culture media increased caspase 3/7 + cells in human MDM cultures (grey bar). Treatment of cells with iluzanebart (10 µg/ml, blue bar) led to a significant reduction in caspase 3/7 + cells, whereas treatment with IgG (green bar) did not. B hMDM. Withdrawal of CSF1 reduced confluence of cells in human MDM cultures (grey bar). Treatment of cells with iluzanebart (10 µg/ml, blue bar) led to a significant increase in confluence, whereas treatment with IgG (green bar) did not. C-E iPSC Derived Human Microglia (iMGL). C Withdrawal of CSF1 decreased iMGL cell viability (n = 3 independent experiments), as measured by CellTiter Glo (grey bar). Treatment with iluzanebart (75 µg/ml, blue bar) led to a significant restoration of ATP levels, whereas treatment with IgG control (green bars) did not. D iMGL. Withdrawal of CSF1 from the culture media decreased morphological eccentricity (n = 5 independent experiments) of human iPSC-derived microglia in vitro (grey bar). Treatment with iluzanebart (75 µg/ml, blue bar) restored cellular morphology to a more ramified state, whereas treatment with IgG control (green bar) did not. E iMGL. Withdrawal of CSF1 increased caspase 3/7 + cells (n = 5 independent experiments) in iPSC microglia cultures (grey bar). Treatment with iluzanebart (75 µg/ml, blue bar) led to a significant reduction in caspase 3/7 + cells, whereas treatment with IgG control (green bar) did not. F hMDM. Representative images of cells in complete media (CM), cells in withdrawal media treated with IgG control (WD + IgG), and cells in withdrawal media exposed to iluzanebart (WD + ILU). G iMGL. Representative images of cells in complete media (CM), cells in withdrawal media treated with IgG control (WD + IgG), and cells in withdrawal media exposed to iluzanebart (WD + ILU). Significance was determined by one-way ANOVA with Tukey’s multiple comparison’s test, * < 0.05, ** < 0.05, *** < 0.0005, **** < 0.0001

Fig. 6

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

Fig. 7

Pharmacokinetic analysis of iluzanebart in hTREM2-CV mice. Terminal iluzanebart brain concentrations and corresponding fold exposures over iluzanebart in vitro sCSF1R EC₅₀ and sTREM2 IC₅₀ pharmacologically align with on-target TREM2 agonism. Values are reported as mean ± standard deviation, n = 4–6 mice per group. A hTREM2-CV mice were dosed intraperitoneally with a single dose of iluzanebart, and brain and plasma concentrations were measured 24 h later. A minimal dose of 10 mg/kg was shown to achieve brain levels equal to or higher than the pSYK EC50 in iPSC derived microglia. B Terminal iluzanebart brain concentrations and corresponding fold exposures over iluzanebart in vitro sCSF1R EC₅₀ and sTREM2 IC₅₀. Iluzanebart Plasma concentrations of iluzanebart in the 100 and 200 mg/kg dose groups was above the limit of quantitation and was therefore excluded

Fig. 8

Bulk RNAseq analysis of iluzanebart-treated (ILU) mouse hippocampal tissue reveals differentially expressed genes. A Volcano plots of differentially expressed genes in hippocampus harvested from iluzanebart-treated (10, 30, 100, and 200 mg/kg) hTREM2 knock-in mice vs. IgG control-treated, n = 4–6 mice per group. B STRING analysis of genes of interest revealed gene clusters related to proliferation/ DNA replication, innate immune response, endoplasmic reticulum and protein folding, and AP-1/ transcription activation. C Gene ontology terms found to be associated with the differentially expressed genes (p < 0.05) are listed, ranked by adjusted p-value

Fig. 9

MSD analysis of iluzanebart-treated mouse cortical tissue reveals dose-dependent, TREM2-dependent changes in cytokine levels. A-C hTREM2-CV mice (n = 4–6 mice per group) were treated with iluzanebart (IP; 1, 3, 10, 30, 100, or 200 mg/kg), IgG (100 mg/kg), or saline. Cortical tissue was harvested 24 h after treatment. Levels of the cytokine IP10 (A), MIP1α (B), and MCP1 (C) showed a dose–response effect in response to iluzanebart treatment, whereas IgG-treated had no change in levels in any of these factors compared to saline-treated controls. D Cytokine levels measured by MSD in cortical tissue from hTREM2^+/+ mice or hTREM2.^−/− mice, perfused and collected 24 h after intraperitoneal dose of 200 mg/kg iluzanebart. Three chemokines expected to respond to TREM2 agonism demonstrated a significant increase relative to vehicle after 24 h, while no significant increase in these chemokines was observed in mice lacking TREM2. IL15, a cytokine not expected to respond to TREM2 agonism, was included as a negative control. Data shown is reported as % of vehicle control. Significance was determined by one-way ANOVA with Dunnett’s multiple comparison’s test, * < 0.05, ** < 0.05, *** < 0.0005, **** < 0.0001

Fig. 10

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. 11

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