Iluzanebart is a potent activator of TREM2 across multiple cell-based model systems

Binding of iluzanebart to the human TREM2 receptor has been epitope mapped to the extracellular Ig-like domain of TREM2, with an average Kd of 1.25 nM (Supplemental Figs. 1 and 2). The cell-based potency of iluzanebart as an agonist of TREM2 was evaluated in HEK293T cells engineered to express both human TREM2 (hTREM2) and human DAP12 (hDAP12). Phosphorylation of both DAP12 and SYK serve as indicators of TREM2 receptor activation, and SYK activation is used as a surrogate measurement for downstream pathway signaling [16]. Levels of both pSYK and pDAP12 were measured following treatment with iluzanebart at a range of concentrations (Fig. 1A and B). Compared to its IgG control, which showed no ability to stimulate SYK phosphorylation, iluzanebart was found to have an average EC50 of 0.27 nM. The high potency of iluzanebart to activate TREM2 signaling was confirmed when activation was measured by pDAP12, which resulted in an average EC50 of 1.14 nM.

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)

To determine if iluzanebart’s ability to activate TREM2 would be maintained in relevant human primary cells, it was next assessed in a human monocyte-derived macrophage (hMDM) cell culture system. Compared to its IgG control, which showed no ability to stimulate phosphorylation of SYK, iluzanebart was found to have an average EC50 of 4.77 nM in hMDM cells (Fig. 1C). Finally, iluzanebart potency was assessed in human induced pluripotent stem cell (iPSC)-derived microglia cells (iMGL) and was found to have an average EC50 of 0.63 nM, while treatment with its IgG control induced no phosphorylation of SYK (Fig. 1D). While potencies may be affected by differences in the levels of TREM2 receptor at the cell surface among the various cell types tested, overall, these data show that iluzanebart is a potent, sub-nanomolar agonist of human TREM2.

Iluzanebart is a specific agonist of human TREM2

Phosphorylation of SYK is not TREM2 specific; phosphorylation of SYK can also be induced through activation of TREM1, another member of the TREM family of receptors, but one that is genetically distinct from TREM2. To determine whether iluzanebart exhibits off-target effects through TREM1, or is specific to TREM2, HEK293T cells engineered to express human TREM1 (but not hTREM2) and human DAP12 were treated with iluzanebart. While a positive control human anti-TREM1 antibody stimulated SYK phosphorylation as expected, iluzanebart was unable to stimulate SYK phosphorylation in cells expressing TREM1 (Fig. 2A). Therefore, activation of SYK phosphorylation by iluzanebart is TREM2 specific.

Iluzanebart specificity for TREM2 was further confirmed in human microglia using TREM2 knockout (TREM2 -/-) iMGL. As shown in Fig. 2C, iluzanebart was unable to activate SYK in TREM2−/− cells (Fig. 2B). Concanavalin A, which activates SYK through a TREM-independent mechanism [27], was used as a positive control. Activation of SYK by Concanavalin A confirmed that SYK was capable of being phosphorylated in this system, even in the absence of TREM2. Together, these data suggest that iluzanebart is a specific, potent agonist of TREM2.

TREM2 undergoes regular proteolytic processing by cleavage and subsequent shedding of its extracellular ectodomain, producing soluble TREM2 (sTREM2) [28,29,30]. Recent studies have correlated neurodegenerative disease stage with levels of sTREM2; increased levels of CSF sTREM2 have been reported to be a biomarker of Alzheimer’s disease severity [31, 32]. Separately, reductions in sTREM2 in the CSF have been used as a biomarker of binding and activation by TREM2 agonist antibodies [33]. Therefore, TREM2 receptor engagement was confirmed in our microglial assay via measurement of reduced levels of soluble TREM2 released into the media. Consistent with TREM2 target engagement, treatment of microglia with iluzanebart for 24 h led to a dose-dependent decrease in sTREM2 levels detectable in the cell media (Fig. 2C) with an average IC50 of 1.14 nM, similar to its EC50 for phosphorylation of SYK and DAP12.

Iluzanebart increases surface CSF1R levels in healthy microglia

Like TREM2, CSF1R undergoes proteolytic cleavage to generate a soluble fragment. However, unlike TREM2, decreased shedding of soluble CSF1R (sCSF1R) is associated with both disease pathology and homeostasis [34]. Both TREM2 and CSF1R receptors are regulated at the cell surface by an ADAM protease, ADAM17, resulting in cleavage of each receptor and the release of its ectodomain into the surrounding environment to generate a soluble fragment. Additionally, binding of ligands to their respective receptor can result in receptor internalization and an equivalent reduction in soluble receptor levels, as was observed with soluble TREM2. As such, increases in levels of soluble CSF1R in the media can function as a surrogate measurement for the abundance of the receptor at the cell surface.

iMGL treated for 24 h with iluzanebart resulted in a dose-dependent increase in soluble CSF1R levels (Fig. 3A), with an average EC50 of 0.52 nM, when compared to treatment with IgG control, providing a functional readout of iluzanebart in vitro efficacy. We further hypothesized that increases in sCSF1R in the media could be explained by increases in the number of CSF1 receptors at the cell surface. Indeed, this was found to be the case as iluzanebart treatment resulted in increased levels of CSF1R at the cell surface of microglia, as measured by flow cytometry (Fig. 3B and C). When this increase in CSF1R positive microglia was quantified (Fig. 3D), iMGL treated with iluzanebart had a significant, 44.5% increase in surface CSF1R as compared to control IgG. Conversely, microglia that did not express TREM2, (TREM2 -/-) showed no increase in CSF1R levels upon iluzanebart stimulation, demonstrating that the observed increase in CSF1R levels is entirely dependent on TREM2 activation. Together, these data confirm that iluzanebart directly increases levels of surface CSF1R, as confirmed by multiple assay methodologies.

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

Iluzanebart rescues the effects of inhibition of CSF1R signaling in human myeloid cells

To investigate the potential of iluzanebart to compensate for the attenuation of CSFR1 signaling that occurs in CSF1R-ALSP, hMDM cells were treated with an IC50 concentration of PLX5622, a highly specific, small molecule inhibitor of CSF1R kinase activity that functionally results in the loss of receptor signaling through inhibition of autophosphorylation [33]. The effect of iluzanebart treatment on cell viability and morphology, which are observed to be altered in CSF1R-ALSP individuals, was assessed following treatment with PLX5622.

Treatment of hMDMs with 1 µM PLX5622 (the experimentally determined IC50 concentration) in the presence of 10 µg/ml IgG resulted in a significant decrease in the percentage of viable cells in the culture (Fig. 4A, green bars). However, in the presence of 10 µg/ml iluzanebart, no significant decrease in viability was detected following PLX5622 treatment (4A, blue bars). Similarly, iluzanebart protected microglia from changes in confluence and cellular morphology in cultures treated with PLX5622, whereas the IgG control did not (Fig. 4B). This effect was likewise seen in iMGL cultures that were treated with PLX5622. Cell viability (Fig. 4C) and morphology (Fig. 4D) were both reduced by PLX5622 treatment and rescued by treatment with iluzanebart, but not by the matched IgG control. This improvement in cell viability was demonstrated to be acting via inhibition of apoptosis, as a statistically significant decrease in PLX5622-induced caspase 3/7 staining in iMGL cultures was observed following treatment with iluzanebart (Fig. 4E). Thus, iluzanebart was able to reverse the effects of CSF1R functional inhibition on all CSF1R-ALSP-relevant phenotypes measured in disease relevant primary cells.

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

Iluzanebart rescues the effects of complete loss of CSF1R signaling in human myeloid cells

While the CSF1 receptor contains multiple intracellular domains that are responsible for transmission of the downstream signaling cascade, the extracellular domain consists of five IgG like domains, and is responsible for binding of its ligands, CSF1 and IL34 [34]. In the absence of a ligand, CSF1R can still dimerize, but remains in an auto-inhibited state, preventing autophosphorylation and subsequent downstream signaling. Due to this autoinhibition, withdrawal of CSF1 from culture media has been demonstrated to induce loss of CSF1R signaling, resulting in loss of viability in human myeloid cells. Conversely, agonism of TREM2 under low CSF1 conditions has been shown to rescue hMDM from apoptosis and promote survival [11, 24, 25].

To investigate whether the protective effect of iluzanebart is seen in the context of complete ligand withdrawal to reduce CSF1R signaling, hMDM and iMGL cultures were each placed in withdrawal media lacking CSF1. In hMDM cultures, CSF1 withdrawal resulted in a significant increase in apoptosis, as measured by caspase 3/7 staining (Fig. 5A, grey bar), and resulted in a significant change in morphology as measured by a decrease in cell confluence (Fig. 5B, grey bar, 5F). While treatment with its IgG control yielded no rescue of these effects (green bars), treatment of hMDM with iluzanebart resulted in a statistically significant reversal of both cell viability and of morphological changes (blue bars). The same effect was observed when cultures of iMGL were placed in CSF1 withdrawal media and subsequently treated with either IgG or iluzanebart; cell viability (Fig. 5C), morphology (Fig. 5D and 5G), and levels of caspase activation (Fig. 5E) were all restored by iluzanebart treatment. Thus, iluzanebart was able to rescue the effects of CSF1R inhibition in two independent models of CSF1R hypofunction, in two different human myeloid cell types.

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

Iluzanebart rescues the effects of a CSF1R-ALSP relevant CSF1R mutation in CRISPR-edited microglia

The I794T mutation in the CSF1 receptor is one of many observed mutations in the kinase domain that renders CSF1R functionally inactive. It is also the most frequently observed mutation reported in an ongoing natural history study of CSF1R-ALSP [35]. This mutation was introduced into an apparently healthy normal iPSC line, ASE-9211 using CRISPR-Cas9. The resulting progenitor cells (I794T+/-) were differentiated to monocytes, harvested, plated onto IgG- or iluzanebart-coated cell culture plates, and incubated for seven days to complete the differentiation process to microglia, utilizing a CSF2/IL34 driven differentiation protocol [36]. At this point, viability, soluble, total, and phospho-CSF1R (pCSF1R) levels were each assessed. In I794T+/- iMGL, viability was significantly increased in iluzanebart-treated cultures compared to IgG-treated controls (Fig. 6A). Additionally, as observed in CSF1R-ALSP patients, levels of both soluble CSF1R, total CSF1R, and phospho-CSF1R were significantly lower in I794T+/- microglia as compared to wildtype microglia; 38%, 23%, and 34% respectively. While iluzanebart treatment did not affect levels of total CSF1R levels in either cell line (Fig. 6B), ILU treatment increased levels of soluble CSF1R in cultures of both I794T+/- and wildtype cells (Fig. 6C). Additionally, while iluzanebart treatment resulted in a modest increase in “activated” CSF1R as measured by phospho-CSF1R levels in wildtype microglia, phospho-CSF1R levels were significantly increased in I794T+/- cells, with levels of phospho-CSF1R restored to that of healthy wildtype cells. This increase translates to a significant increase of 2.26 × in the ratio of phosphorylated to total CSF1R levels in I794T+/- cells (Fig. 6D, E). Interestingly, no effect on total CSF1R levels was observed, suggesting that this effect is limited to alterations in levels of CSF1R at the cell surface, and is not due to changes in transcription of CSF1R or in cell number. Together, these data suggest that iluzanebart has the potential to restore levels of CSF1R signaling in CSF1R-ALSP patient microglia to levels observed in healthy individuals.

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

Iluzanebart concentrations in mouse plasma and brain

Next, as iluzanebart is specific for human TREM2, the effects of iluzanebart treatment were assessed in mice that are engineered to express human TREM2 but lack expression of murine TREM2 (hTREM2-CV). These mice have been extensively profiled by others, and microglia from hTREM2 expressing mice have been shown to fully recapitulate the function and behavior of endogenous microglia [20, 25, 37, 38]. First, to validate that iluzanebart is human specific, we have demonstrated that iluzanebart shows no ability to bind and activate rodent TREM2, as no induction of SYK phosphorylation was observed in HEK293T cells engineered to express rodent TREM2 and DAP12 receptors (Supplementary Fig. 3A and B). Treatment with iluzanebart failed to induce SYK phosphorylation below concentrations of 1000 nM, while a positive control antibody that cross reacts with both mouse and human TREM2 did result in SYK phosphorylation. Therefore, the observed activity in vivo is limited exclusively to agonism of human TREM2.

Iluzanebart concentrations were determined in plasma samples from equal numbers of adult male and female mice at 1, 2, 4, 8 and 24 h post dose, and in brain tissues 24 h post dose, following single intraperitoneal (IP) administration of iluzanebart at 1, 3, 10, 30, 100, and 200 mg/kg in healthy hTREM2-CV mice. All mice survived treatment, and no treatment related signs of toxicity were observed. Plasma concentration–time profiles are shown in Supplemental Fig. 4, with maximal concentrations were observed between 2 and 8 h post dose, and plasma exposure of iluzanebart increased in an apparently proportional manner from 1 to 30 mg/kg (Fig. 7). At 24 h post dose, the measured total brain concentrations were approximately 0.1% of plasma concentrations and increased dose proportionally from 1 to 200 mg/kg. At the 10 mg/kg dose, brain concentrations were within range of the in vitro iMGL pSYK EC50 value, as well as the sCSF1R and sTREM2 EC50 values.

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

Transcriptomic profiling and pathway analysis in iluzanebart-treated mice

Hippocampal tissues were collected from the same hTREM2-CV transgenic mice utilized in the same pharmacokinetic study following single IP administration of iluzanebart at 10, 30, 100, or 200 mg/kg or control IgG at 100 mg/kg. RNA isolated from the hippocampus was analyzed using RNAseq, and differentially expressed genes in the tissues from iluzanebart-treated mice relative to control IgG-treated mice are shown in Fig. 8A and Supplementary File 1. After 200 mpk iluzanebart treatment, 128 genes with adjusted p values of less than 0.05 were determined to be significantly differentially expressed. The 100 mpk dose yielded 97 significant DEGs, but of those genes, a significant amount, 36%, were similarly differentially expressed in the 200 mpk group after iluzanebart treatment. In the 30 mpk and 10 mpk groups, fewer genes were determined to be significantly differentially expressed; with 27 and 16 genes differentially expressed, respectively.

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

Genes that were significantly upregulated by iluzanebart at 200 mg/kg compared to IgG control included genes involved in chemotaxis and immune activation and regulation (Ccl12, Cxcl10, and Gpr84), proliferation (Fam11a, Mki67, Top2a, Uhrf1), and homeostasis (Dusp1, Fcrls, Itgb2, and Olfml3). While these mice do not exhibit any neurodegenerative disease pathology, some neuroprotective, disease associated microglia (DAM) genes were still detected as upregulated. DAM genes whose expression increased greater than 0.25 log2 fold included Cst7, Ccl3, Ccl6, Cd52, Cd68, Clec7a, Cxcl16, Ctsz, Fgl2, Itgax, SlamF9, and Tyrobp [39]. Other DAM genes that were significantly upregulated, but less than 0.25 log2 fold included Axl, B2m, Cd9, Gusb, H2-d1, Hif1a, and Lpl. The most significantly affected pathway was determined to be proliferation related pathways, with 14% of total DEGs at 200 mpk relating to proliferation and having a log2 fold change of > 0.3.

Confirming previous data, no change in expression of CSF1R was observed at 24 h, suggesting that the increase in surface levels of CSF1R on microglia is not due to increased gene expression. All differentially expressed genes (adjusted p value ≤ 0.05) as compared to the IgG control for the 200 mg/kg group were analyzed using STRING (Fig. 8B). STRING analysis showed that differentially expressed genes self-assembled into “nodes” around different TREM2 related processes. These processes were representative of both TREM2 and CSF1R related pathways, including immune system process, regulation of proliferation, regulation of transcription, AP-1 transcription factor and early growth response, among others.

Finally, the gene ontology (GO) terms describing the top differentially expressed pathways affected by changes in gene expression are listed in Fig. 8C. Pathways that were significantly affected by iluzanebart treatment also included TREM2 and CSF1R relevant pathways; response to stress, inflammatory response, chemotaxis, transcription, and cell cycle, among others. Overall, iluzanebart treatment of healthy hTREM2-CV mice resulted in changes in gene expression indicative of TREM2 activation, but also changes in gene expression that overlap with CSF1R-related pathways.

Iluzanebart induces TREM2 specific chemokine increases in vivo

Levels of specific proteins, guided by the genes that were highlighted in the RNAseq analysis, were determined in cortical tissue lysates isolated from the same iluzanebart or IgG-treated hTREM2-CV mice using MesoScale Discovery assays. Of note, IP10 (Cxcl10) was increased 2.59 log2 fold (adjusted p value = 0.0018) in the 200 mpk group compared to IgG control, while Ccl3 (Mip1α) was increased 2.11 log2 fold (adjusted p value = 0.029). Iluzanebart treatment significantly increased protein expression of IP10 and MCP1 at 100 and 200 mg/kg, and MIP1α at doses of ≥ 10 mg/kg in hTREM2-CV mice (Fig. 9A-C). In contrast, iluzanebart treatment (200 mg/kg) had no effect on the protein levels of IP10, MCP1, and MIP1α in the cortical tissues collected from a separate cohort of mice lacking human TREM2 (Fig. 9D), demonstrating that this effect is entirely dependent on TREM2. Additionally, levels of IL15, a non-TREM2 dependent cytokine, were not affected by iluzanebart treatment, and were similar between cortices from hTREM2-CV and hTREM2 -/- mice, demonstrating again that cytokine modulation is specific and TREM2 dependent. Therefore, iluzanebart treatment resulted in gene expression changes in mice that were TREM2 relevant, and these gene expression changes translated to changes in protein levels.

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

Iluzanebart induces proliferation of microglia in vivo

Finally, equal numbers of adult male and female hTREM2-CV mice were dosed intraperitoneally with either iluzanebart at 200 mpk, IgG at 200 mpk, or an equal volume of sterile saline solution, and 48 h later, whole brain tissue was harvested for flow cytometric analysis. Following tissue dissociation and myelin removal, cells were sorted by CD45 and CD68 expression to identify microglial populations, and then analyzed for human IgG for target engagement, Ki67 as a marker of proliferation, and CSF1R. Samples without detectable human IgG, and therefore without measurable iluzanebart or IgG, were excluded from the analysis (Supplemental Fig. 5). As seen in Fig. 10, iluzanebart treatment significantly increased the number of Ki67 positive microglia in vivo by 192.3%. While the IgG control resulted in a smaller increase in Ki67 positive microglia, this increase (69.7%) was not statistically significant (Fig. 10A). This confirms that the transcriptional signature for proliferation observed in vitro translates in vivo to proliferation of microglia. Additionally, CSF1R levels were analyzed, and a representative histogram is seen in Fig. 10B. Strikingly, a significant increase in microglia with high CSF1R levels is observed (Blue circle). When quantified (Fig. 10C), this translates to a significant, 60.1% increase in MFI in the iluzanebart treated group, whereas no change was observed in the IgG control treated group. These data demonstrate that not only does the transcriptional reprogramming observed in iluzanebart treated mice translate to proliferation of microglia, but this also translates to a population of microglia with increased CSF1R levels; both being critical to rescue loss of microglia and hypofunction of CSF1R signaling seen in CSF1R-ALSP individuals.

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