RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/825,112 filed 28 March 2019, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 81583SequenceListing.txt, created on 24 March 2020, comprising 13,714 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of treating lipid- related disorders by using agents which increase the activity or amount of TREM-2 and, more particularly, but not exclusively, to activating antibodies of TREM-2.

The obesity pandemic has reached alarming magnitudes, with over 44 % of the adult world population estimated to be overweight. In addition to its widespread prevalence, obesity is considered a major risk factor for a number of metabolic diseases, including type II diabetes mellitus, non-alcoholic fatty liver disease, atherosclerosis, and ischemic cardiovascular disease. Furthermore, obesity is a predisposing factor for numerous other diseases that are not typically classified as metabolic/endocrine, such as cancer and neurodegeneration. Collectively, the global obesity pandemic has far-reaching consequences on life expectancy, quality of life, and healthcare costs.

The human tissue most strongly involved in the pathogenesis of obesity and its metabolic complications is the adipose tissue. Adipose tissue undergoes marked morphological changes during the development of obesity, including adipocyte hypertrophy and extensive vascularization. In addition, obese adipose tissue is characterized by a distinct repertoire of soluble mediators, typically referred to as adipokines, that influence both the tissue itself and several distal organ sites.

Major contributors to the adipose tissue secretome during obesity are tissue-resident immune cells (Mathis, 2013). Under both homeostatic and pathological conditions, adipose tissue is interspersed by a large range of immune cells, which dramatically increase in total abundance with greater adiposity. Adipose-resident immune cells display markedly distinct molecular characteristics compared to their circulating counterparts. Recently identified examples include regulatory T and B cells that interact with adipocytes (Feuerer et al., 2009; Nishimura et al., 2013), gd T and NKT cells that drive thermogenesis (Kohlgruber et al., 2018; Lynch et al., 2016), memory T cells protective against infection (Han et al., 2017), cytotoxic innate lymphoid cells (Boulenouar et al., 2017), dendritic cells and macrophages expressing the transcription factor PPARy (Cipolletta et al., 2012; Macdougall et al., 2018; Odegaard et al., 2007), as well as macrophages forming“crown-like” stmctures around large adipocytes that are suggested to preserve tissue integrity in face of massive adipocyte cell death (McNelis and Olefsky, 2014).

While inflammation is generally considered a driver of the metabolic derangements that accompany obesity, including insulin resistance and dyslipidemia (Winer et al., 2016), the molecular triggers, sensory receptors and signaling pathways of immune cell accumulation in adipose tissue remain incompletely understood. There is thus an urgent need to identify the regulatory mechanisms driving disease-associated immune cell behavior in obese adipose tissue and to understand their function in driving or protecting from obesity-related metabolic derangements.

Background art includes US Patent No. 20190010230, WO/2018/015573,

WO/2018/195506 and Park et al, 2015, Diabetes 64, 117-127.

SUMMARY OF THE INVENTION

According to an aspect of the present invention, there is provided a method of treating or preventing a lipid-related disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent which upregulates the amount and/or activity of triggering receptor expressed on myeloid cells 2 (TREM-2), thereby treating or preventing the lipid-related disorder.

According to an aspect of the present invention, there is provided a method of treating or preventing a lipid-related disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent which upregulates the amount and/or activity of CD9 ⁺ CD63 ⁺ macrophages in peripheral tissue, thereby treating or preventing the lipid- related disorder.

According to an aspect of the present invention, there is provided an agent which upregulates the amount and/or activity of TREM-2 for use in treating or preventing a lipid-related disorder. According to an aspect of the present invention, there is provided an agent which upregulates the amount and/or activity of CD9 ⁺ CD63 ⁺ macrophages in peripheral tissue for use in treating or preventing a lipid-related disorder.

According to an aspect of the present invention, there is provided a method of regulating the size and/or number of adipocytes of a subject comprising administering to the subject an agent which alters the amount and/or activity of triggering receptor expressed on myeloid cells 2 (TREM-2), thereby regulating the size and/or number of adipocytes of the subject, the subject not having a disease selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease and multiple sclerosis.

According to an aspect of the present invention, there is provided an article of manufacture comprising a weight-inducing medication and an agent which upregulates the amount and/or activity of triggering receptor expressed on myeloid cells 2 (TREM-2) and/or the amount and/or activity of CD9 ⁺ CD63 ⁺ macrophages in peripheral tissue.

According to an aspect of the present invention, there is provided a method of treating a disease associated with weight loss in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of an agent which downregulates the amount and/or activity of triggering receptor expressed on myeloid cells 2 (TREM-2) or an agent which downregulates the amount and/or activity of CD9 ⁺ CD63 ⁺ macrophages in peripheral tissue, thereby treating the disease.

According to embodiments of the present invention, the agent comprises CD9 ⁺ CD63 ⁺ macrophages.

According to embodiments of the present invention, the peripheral tissue comprises adipose tissue or liver tissue.

According to embodiments of the present invention, the subject is on a weight-inducing medication.

According to embodiments of the present invention, the subject does not have a disease selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease and multiple sclerosis.

According to embodiments of the present invention, the agent upregulates the amount and/or activity of TREM-2 on the macrophages.

According to embodiments of the present invention, the peripheral tissue comprises adipose tissue or liver tissue.

According to embodiments of the present invention, the regulating comprises down regulating. According to embodiments of the present invention, the subject is obese.

According to embodiments of the present invention, the subject is not obese.

According to embodiments of the present invention, the medication is selected from the group consisting of a steroid hormone and a psychoactive drug.

According to embodiments of the present invention, the agent binds specifically to TREM-2.

According to embodiments of the present invention, the agent is an activating antibody to TREM-2.

According to embodiments of the present invention, the agent is an agonist of TREM-2.

According to embodiments of the present invention, the agent is a small molecule.

According to embodiments of the present invention, the agent comprises a lipid.

According to embodiments of the present invention, the lipid is a phospholipid.

According to embodiments of the present invention, the lipid is selected from the group consisting of Cardiolipin (CL), Sphingomyelin (SM), Phosphatidylserine (PS), Phosphatidylcholine (PC) phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Sulfatide.

According to embodiments of the present invention, the agent comprises APOE or a cholesterol.

According to embodiments of the present invention, the activity of TREM-2 comprises an increase in phosphorylated spleen tyrosine kinase (stk).

According to embodiments of the present invention, the regulating comprises up- regulating.

According to embodiments of the present invention, the agent is an antibody antagonistic to TREM-2.

According to embodiments of the present invention, the agent is an antagonist of TREM-

2.

According to embodiments of the present invention, the agent is a small molecule.

According to embodiments of the present invention, the activity of TREM-2 comprises a change in the amount of phosphorylated spleen tyrosine kinase (stk).

According to embodiments of the present invention, the administering comprises locally administering into the adipose tissue of the subject.

According to embodiments of the present invention, the lipid-related disorder is selected from the group consisting of obesity, fatty liver disease, heart disease, stroke, atherosclerosis, diabetes, osteoarthritis, gout, sleep apnea and high blood pressure.

According to embodiments of the present invention, the -related disorder is obesity. According to embodiments of the present invention, the fatty liver disease is selected from the group consisting of hypertriglyceridemia, steatohepatitis, atherosclerosis and hypercholesterolemia.

According to embodiments of the present invention, the hypercholesterolemia is a familial hypercholesterolemia.

According to embodiments of the present invention, the disease is selected from the group consisting of cancer, hyperthyroidism and anorexia.

According to embodiments of the present invention, the agent is an antibody antagonistic to TREM-2.

According to embodiments of the present invention, the agent is an antagonist of TREM-

2.

According to embodiments of the present invention, the agent is a small molecule.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs.lA-F. Single-cell characterization of the adipose tissue immune niche during obesity progression. A. Schematic of experimental approach: single-cell RNA-seq pipeline of mouse and human adipose tissue immune cells during obesity. B. kNN graph of 21,210 QC-positive immune cells (244 metacells) from EAT of 20 mice fed a normal chow (NC) or high-fat diet (HFD). C. Shown are Log2 of average unique molecular identifier (UMI) count of selected genes across metacells, D. kNN graph of EAT immune cells of WT mice on HFD, down-sampled to 2,283 cells (in each condition), annotated as in (B). E-F. Immune cell type distribution of WT mice on HFD (E), and 7-week old db/db mice and WT littermates (F) of a total of 8,372 QC -positive single cells (75 metacells).

FIGs. 2A-H. Large changes during obesity in monocyte and macrophage subtypes are mainly characterized by the expansion of a distinct macrophage subset. A. kNN graph of 11,241 QC -positive immune cells (136 metacells) of the Monocyte/Macrophage compartment from Figure IB. B. Shown are Log2 average UMI count of selected genes across metacells of the Monocyte/Macrophage compartment. C. kNN graph of the Monocyte/Macrophage compartment of WT mice on HFD, down-sampled to 1,327 cells (in each condition), annotated as in (A). D, E. Cell type distribution within the Monocyte/Macrophage compartment of WT mice on HFD (D), and 15-week old db/db mice and WT littermates (E) of 4,988 QC-positive single cells (45 metacells). F. Log2 UMI count of Cd9 and Cd63 expression in single cells projected on the kNN graph in (A). G. Representative cell density FACS plots to enrich for macrophage (CDl lb+F4/80+) subpopulations using CD9 and CD63 markers. Left, 16-week old WT mouse on NC; right, WT mouse on HFD for 12 weeks. H. Representative immunofluorescence images of CD9 (green), F4/80 (cyan) and adipocytes (Perilipin-1, red) in EAT sections of 16- week old WT mice on NC (left), or after 12 weeks on HFD (right). Cell nuclei are stained with DAPI (blue). Scale bar, 20 pm.

FIGs. 3A-H. Conserved TREM-2 signature characterizes the obesity related macrophages in mice and humans. A. Gene-gene Pearson correlation heatmap of 200 most variable genes within the monocyte/macrophage compartment. B. Volcano plot showing the fold change of genes (log2 scale) between the HFD Mac3 to NC Mad (x axis) and their p-value significance (y axis, -loglog scale). Highly significant genes are indicated by a red dot. p-values were determined by Mann- Whitney U test with FDR correction. See Table 1. C. kNN graph of 15,150 QC-positive single cells (172 metacells) of human omental adipose tissue (OAT). D. Log2 UMI count of TREM-2 and CD9 expression in single cells projected on the kNN graph in (C). E. Volcano plot of LAM vs Mad macrophages fold change in the human OAT (x axis) and their p- value significance (y axis). Highly significant genes are indicated by a red dot. p-values were determined by Mann- Whitney U test with FDR correction. See Table 4. F. Scatterplot showing the average UMI counts (log2 scale) of human LAM (y axis) compared with the mouse LAM (x axis). G. KEGG pathway analysis of LAM genes shared between mouse and human. H. Pathway visualization for genes contributing to the KEGG annotation in (G).

FIGs. 4A-E. Conserved TREM-2 signature characterizes the obesity related macrophages in mice and humans. A. Projection of the monocyte/macrophage compartment onto the kNN graph of Figure 2A from a total of 10,042 QC -positive immune cells (133 metacells) from EAT of TREM-2 knock-out (KO) mice or WT littermate controls on HFD. Contour lines indicate the 2D density of projected cells, down-sampled to 2,289 cells (in each condition). B. Frequency of the EAT Mon/Mac subsets as defined in Figures 2A-H in each of four KO and four WT mice on HFD. C. Scatterplot showing the average molecule (UMI) count (log2 scale) of KO (x axis) compared with the WT (y axis) in 732 cells from each group, randomly down-sampled from the area of highest density in (A) for equal cell numbers. D. Expression level (UMI counts) of selected marker genes in the different TREM-2 genotypes at the three macrophage subsets found in EAT. E. Representative immunofluorescence images of CD9 (green), F4/80 (cyan) and adipocytes (Perilipin-1, red) in EAT sections from TREM-2 WT mice (left) or TREM-2 KO (right) 12 weeks on HFD. Cell nuclei are shown in blue (DAPI). Scale bar, 20 pm.

FIGs. 5A-I. TREM-2 prevents adipocyte hypertrophy and loss of systemic metabolic homeostasis. A. Representative images of hematoxylin and eosin (H&E) stain of fixated EAT sections. Left, EAT sections from WT and KO control mice. Right, Sections from mice on high fat diet. Scale bars, 100 pm, and 500 pm for HFD sections zoomed out areas. B. Area quantification of 500 adipocytes per genotype/diet tissue sections from photos taken to H&E sections. Bars indicate mean ± SEM. **** p < 0.0001 by one-way ANOVA. C. Percentage body fat content. Fat mass, lean mass and liquid mass of individual animals was measured by live non- invasive magnet resonance. Filled symbols, TREM-2 WT; open symbols, TREM-2 KO; squares, mice on normal chow (NC); circles, mice on high-fat diet (HFD). Bars indicate mean + SEM. * p

< 0.05; *** p < 0.001; n.s., non-significant. D-F. Total cholesterol (D), LDL (E), and HDL (F) levels in mouse serum from TREM-2 cohorts at week 12 on HFD or NC control. Bars indicate mean ± SEM. * p < 0.05; *** p < 0.01. G. Weight gain over time on HFD. Number of mice in each group is indicated next to each curve. Data are presented as mean ± SEM. * p < 0.05; ** p

< 0.01 by two-way ANOVA. H. Glucose tolerance test was performed at fasted mice on week 11 HFD. I. Area under the curve (AUC) as a measure of glucose intolerance, calculated for each individual mouse in (I). Bars indicate mean ± SEM. ** p < 0.01.

FIGs. 6A-H. Single-cell characterization of the adipose tissue immune niche during obesity progression. Related to Figures 1A-F. A-B. Weight gain (A) and body composition (B) of mice on high-fat diet (HFD) or normal chow (NC). Error bars indicate standard deviation. N=8. C. Confusion matrix of all metacells as shown in Figure IB. D. kNN graph of epididymal adipose tissue immune cells of wild-type mice on NC, down-sampled to each 2,187 cells, annotated as in Figure IB. E-F. Relative frequencies of higher abundant (E) and lower abundant (F) immune cell types of mice on HFD. Every dot represents a mouse. Stars marking significant p value of a Mann Whitney U test (*p < 0.05; **p < 0.01; ***p < 0.001, NS. not significant). G. Weight gain of mice on Db/Db mice and littermate wild-type controls with age. Error bars indicate standard deviation. N=6. H. kNN graph of epididymal adipose tissue immune cells of Db/Db mice and littermate wild-type controls with age, down-sampled to 1,985 cells each.

FIGs. 7A-E. Analysis of subsets of Monocytes and Macrophages of dbldb mice. Related to Figures 2A-H. A. Log2 of unique molecular identifier (UMI) count of selected genes in individual cells on the kNN graph of Figure 2A. B. kNN graph of Monocytes and Macrophages from epididymal adipose tissue of Db/Db mice. Subsets were obtained by hierarchical clustering based on similarity, homogenous groups were chosen. C. Log2 of average UMI count of selected genes across metacells within the Monocyte/Macrophage compartment. D. Log2 of UMI count of selected genes in individual cells on the kNN graph of (B).

FIGs. 8A-D. Conserved TREM-2 signature characterizes the obesity related macrophages in mice and humans. Related to Figures 3A-H. A. Volcano plot showing the fold change of genes (log2 scale) between Mac2 from wild-type mice on HFD to Mad from wild-type mice on NC (x axis) and their significance (y axis, -loglog scale). Highly significant genes are indicated by a red dot. p values were determined by Mann- Whitney U test with FDR correction. See Table 2. B. Volcano plot showing the fold change of genes (log2 scale) between Mac3 from wild-type mice on HFD to Mac2 from wild-type mice on HFD (x axis) and their significance (y axis, - loglog scale). Highly significant genes are indicated by a red dot. p values were determined by Mann-Whitney U test with FDR correction. See Table 3. C. Scatterplot comparing Z scores of log2 fold changes of the TREM-2 module genes between LAM versus Mad in WT mice (x axis) and DAM versus homeostatic microglia in AD mice (Keren-Shaul et al., 2017) (y axis). D. Scatterplot comparing Z scores of log2 fold changes of the TREM-2 module genes between LAM versus Mad in wild-type mice (x axis) versus dbldb (y axis). R indicates the Pearson correlation coefficient.

FIGs. 9A-I. Validation of LAM cells. Related to Figures 3A-H. A. kNN graph of subcutaneous adipose tissue immune cells of wild-type mice on NC and HFD, down-sampled to each 785 cells. B. Log2 of UMI count of selected genes in individual cells on the kNN graph of (A). C. kNN graph of 15,150 cells of human adipose tissue cells. D. Log2 of UMI count of selected genes in individual cells on the kNN graph of (C). E-F. GO-term analysis for top- expressed genes of mouse (E) and human (F) LAM. G-H. qPCR of bulk-sorted subsets of adipose tissue macrophages for discovered LAM marker genes (G) and TREM-2 module genes (H). I. Bodipy staining of bulk-sorted subsets of adipose tissue macrophages. FIGs. 10A-E. Characterization of adipose tissue immune cells in TREM-2 knock-out mice. Related to Figures 4A-E. A. Projection of the Monocyte/Macrophage compartment onto the kNN graph of Figure 2A from a total of 10,042 QC-positive immune cells (133 metacells) from EAT of TREM-2 knock-out (KO) mice or WT littermate controls on NC. Contour lines indicate the 2D density of projected cells, down-sampled to 453 cells. B-C. Relative frequencies of higher abundant (B) and lower abundant (C) immune cell types. Every symbol represents a mouse. Stars marking significant p value of a Mann Whitney U test (*p < 0.05; **p < 0.01; ***p < 0.001, NS. not significant). D. Frequency of the EAT Mon/Mac subsets as defined in Figures 2A-H in each of two KO and two WT mice on NC. E. Cell type distribution within the Monocyte/Macrophage compartment of KO and WT mice on HFD and NC.

FIGs. 11A-G Trem2 KO BM chimera display similar phenotype as full body KO. A. Percentage body fat content. Fat mass, lean mass and liquid mass of individual animals was measured by live non-invasive magnet resonance. Filled symbols, BM chimera with Trem2 WT; open symbols, BM chimera with Trem2 KO; Bars indicate mean + SEM. * p < 0.05; *** p < 0.001; n.s., non-significant. B-D. Total cholesterol (B), LDL (C), and HDL (D) levels in mouse serum from BM chimera mice at week 18. Bars indicate mean ± SEM. * p < 0.05; *** p < 0.01. E. Weight gain over time on HFD. Number of mice in each group is indicated next to each curve. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01 by two-way ANOVA. F. Glucose tolerance test was performed at fasted mice on week 18 HFD. G. Area under the curve (AUC) as a measure of glucose intolerance, calculated for each individual mouse in (I). Bars indicate mean ± SEM. ** p < 0.01.

FIG. 12. Percentage of LAM cell detected using single cell RNA-seq out of total immune cells in the visceral adipose tissue of 6 obese donors and 1 lean control.

FIGs. 13A-B: LAM cells accumulated in the liver of HFD mice. (A) kNN graph of mice immune cells from the liver of HFD and NC mice, log2 UMI count of Trem2 expression are highlighted. (B) quantification of LAM cells percentage in HFD mice vs. NC-mice.

FIG. 14. Trem2 activation assay. N9, N9 + Trem2 overexpression cell lines were incubated with liposome containing phosphatidylcholine (PC) or phosphatidylcholine- phosphatidylinositol (PC-PI) for 5 min and tested for pSyk levels using Flow Cytometry.

FIG. 15 is a graph illustrating the results of a glucose tolerance test in the presence and absence of a TREM2 agonist antibody. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of treating lipid related disorders by using agents which increase the activity or amount of TREM-2 and, more particularly, but not exclusively, to activating antibodies of TREM-2.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Immune cells residing in white adipose tissue have been highlighted as important factors contributing to the pathogenesis of metabolic diseases, but the molecular regulators that drive adipose tissue immune cell remodeling during obesity remain largely unknown. Using index and transcriptional single-cell sorting, the present inventors comprehensively mapped all adipose tissue immune populations in both mice and humans during obesity. They uncovered a novel and conserved TREM-2 ⁺ lipid-associated macrophage (LAM) subset and identified markers, spatial localization, and functional pathways associated with these cells.

Whilst reducing the present invention to practice, the present inventors performed genetic ablation of TREM-2 in mice so as to globally inhibit the downstream LAM molecular program during obesity. The present inventors showed that the absence of TREM-2, leads to adipocyte hypertrophy and both tissue-level and systemic hypercholesterolemia and glucose intolerance (Figures 4A-E, 5A-I and 10A-E. These findings highlight TREM-2 as a key sensor of metabolic pathologies across multiple tissues and a potential therapeutic target in metabolic and lipid- related diseases.

Thus, according to a first aspect of the present invention, there is provided a method of treating or preventing a lipid-related disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent which upregulates the amount and/or activity of triggering receptor expressed on myeloid cells 2 (TREM-2), thereby treating or preventing the lipid-related disorder.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. As used herein, the term“treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition.

The term “preventing” refers to substantially preventing the onset of clinical or aesthetical symptoms of a condition.

As used herein, the term“lipid-related disease” refers to a disease associated with excess lipids. Preferably, the lipid-related disease is one for which decreasing a size and/or number of adipocytes is beneficial.

Examples of such lipid-related diseases include but are not limited to obesity, fatty liver disease, heart disease, atherosclerosis, diabetes, osteoarthritis, gout, sleep apnea, metabolic syndrome and high blood pressure, homozygous familial hypercholesterolemia, heterozygous familial hypercholesterolemia, ischemic stroke, coronary artery disease, acute coronary syndrome, renal arterial stenosis, peripheral arterial disease, or atheroembolic renal disease.

According to a particular embodiment, the lipid-related disease is obesity.

It will be appreciated that agent may also be a suitable therapy to promote weight loss in a non-obese (but overweight) subject.

In one embodiment, the subject having the lipid-related disorder has a body mass index (BMI) of greater than 25, 26, 27, 28, 29 or 30.

In one embodiment, the subject having the lipid-related disorder has a body mass index (BMI) of between 25-35 or 26-35.

In one embodiment, the subject having the lipid-related disorder has a body mass index (BMI) of less than 25.

In yet another embodiment, the subject has no obesity-related co-morbidity.

The lipid-related disorder may also be a disorder of lipoprotein metabolism, including lipoprotein overproduction or deficiency. These disorders may be manifested by elevation of the serum total cholesterol, low-density lipoprotein (LDL) cholesterol and triglyceride concentrations, and a decrease in the high-density lipoprotein (HDL) cholesterol concentration and therefore include, for example, lipemia and hypercholesterolemia.

Lipemia is a condition in which an excess of fats or lipids is found in the blood of subject.

Hypercholesterolemia is a condition in which high levels of cholesterol are found in the blood of a subject. According to a particular embodiment, the subject treated for lipid-related disease does not have dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease and multiple sclerosis.

In another embodiment, the subject treated for lipid-related disease does not have a neurodegenerative disease.

According to a particular embodiment, the lipid-related disease is fatty liver disease.

As used herein, the term“fatty liver disease” refers to a disease or a pathological condition caused by, at least in part, abnormal hepatic lipid deposits. Fatty liver disease includes, e.g., alcoholic fatty liver disease, non-alcoholic fatty liver disease, and acute fatty liver of pregnancy. Fatty liver disease may be, e.g., macro-vesicular steatosis or micro-vesicular steatosis.

According to a particular embodiment, the disease is non-alcoholic fatty liver disease.

The non-alcoholic fatty liver disease may include simple steatosis, diabetes-related liver steatosis, non-alcoholic steatohepatitis, cholestasis and liver fibrosis and liver cirrhosis which result from the progression of such diseases.

The non-alcoholic fatty liver disease may be a primary or a secondary non-alcoholic fatty liver disease.

The non-alcoholic fatty liver disease may be either familial (e.g. inherited liver disease due to a mutation in the LDL receptor) or non-familial.

According to a particular embodiment, the familial fatty liver disease is familial hyperlipidemia.

In some embodiment, subjects with familial hyperlipidemia have mutations in the LDLR gene that encodes the LDL receptor protein, which normally removes LDL from the circulation, or apolipoprotein B (ApoB), which is the part of LDL that binds with the receptor.

According to this aspect of the present invention, the term“subject” (or“individual” which is interchangeably used herein) refers to an animal subject e.g., a mammal, e.g., a human being at any age who suffers from or is at risk of developing the pathology. Non-limiting examples of individuals who are at risk to develop the pathology of the present invention include individuals who are genetically predisposed to develop the pathology (e.g., individuals who carry a mutation or a DNA polymorphism which is associated with high prevalence of the pathology), and/or individuals who are at high risk to develop the pathology due to other factors such as environmental hazard or other pathologies.

Another example of individuals who are at risk of developing lipid-related disorders are those that are taking a weight-inducing medication, such as steroids (e.g. prednisone or birth control pills) anti-diabetic agents (e.g. insulin, thiazolidinediones, and sulfonylureas); antipsychotic agents including, but not limited to haloperidol, clozapine, risperidone, olanzapine, and lithium; antidepressant agents (e.g. amitriptyline, imipramine, paroxetine, and sertraline); anti-epileptic agents including, but not limited to valproate, carbamazepine, and gabapenti; and blood pressure-reducing medicines including beta-blockers such as propranolol and metoprolol.

As mentioned, the present invention contemplates treating lipid-related disorders with agents which upregulate the amount and/or activity of triggering receptor expressed on myeloid cells 2 (TREM-2, also referred to herein as Trem2). The present inventors have shown that down-regulation of TREM-2 increases the number and size of adipocytes. Accordingly, the present inventors contemplate that agents capable of upregulating the amount and/or activity of TREM-2 decrease the number and size of adipocytes and therefore can be used to reduce visceral fat in a subject.

TREM-2 proteins of the present disclosure include, without limitation, a mammalian TREM-2 protein including but not limited to human TREM-2 protein (Uniprot Accession No. Q9NZC2), mouse TREM-2 protein (Uniprot Accession No. Q99NH8), rat TREM-2 protein (Uniprot Accession No. D3ZZ89), Rhesus monkey TREM-2 protein (Uniprot Accession No. F6QVF2), bovine TREM-2 protein (Uniprot Accession No. Q05B59), equine TREM-2 protein (Uniprot Accession No. F7D6L0), pig TREM-2 protein (Uniprot Accession No. H2EZZ3), and dog TREM-2 protein (Uniprot Accession No. E2RP46).

TREM-2 is a 230 amino acid membrane protein. TREM-2 is an immunoglobulin-like receptor primarily expressed on myeloid lineage cells, including without limitation, macrophages, dendritic cells, osteoclasts, microglia, monocytes, Langerhans cells of skin, and Kupffer cells. In some embodiments, TREM-2 forms a receptor- signaling complex with DAP12. In some embodiments, TREM-2 phosphorylates and signals through DAP12 (an IT AM domain adaptor protein). In some embodiments TREM-2 signaling results in the downstream activation of PI3K. In some embodiments TREM-2 signaling results in the downstream phosphorylation of spleen tyrosine kinase (stk).

In some embodiments, an example of a human TREM-2 amino acid sequence is set forth below as SEQ ID NO: 1.

In some embodiments, the human TREM-2 is a preprotein that includes a signal peptide. In some embodiments, the human TREM-2 is a mature protein. In some embodiments, the mature TREM-2 protein does not include a signal peptide. In some embodiments, the mature TREM-2 protein is expressed on a cell. In some embodiments, TREM-2 contains a signal peptide located at amino acid residues 1-18 of human TREM-2 (SEQ ID NO: 1); an extracellular immunoglobulin-like variable-type (IgV) domain located at amino acid residues 29-112 of human TREM-2 (SEQ ID NO: 1); additional extracellular sequences located at amino acid residues 113-174 of human TREM-2 (SEQ ID NO: 1); a transmembrane domain located at amino acid residues 175-195 of human TREM-2 (SEQ ID NO: 1); and an intracellular domain located at amino acid residues 196-230 of human TREM-2 (SEQ ID NO: 1).

In some embodiments, the agent increases the amount and/or activity of TREM-2 which is expressed on macrophages.

According to one embodiment, the agent binds specifically to TREM-2 which is expressed on macrophages. The phrase "specifically bind(s)" or "bind(s) specifically" when referring to a binding molecule refers to a binding molecule which has intermediate or high binding affinity, exclusively or predominately, to a target molecule, such as to TREM-2. The phrase "specifically binds to" refers to a binding reaction which is determinative of the presence of a target protein (such as TREM-2) in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated assay conditions, the specified binding molecules bind preferentially to a particular target protein (e.g. TREM-2) and do not bind in a significant amount to other components present in a test sample. Specific binding to a target protein under such conditions may require a binding molecule that is selected for its specificity for a particular target protein. A variety of assay formats may be used to select binding molecules that are specifically reactive with a particular target protein. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore and Western blot may be used to identify binding molecules that specifically bind to TREM-2. Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background. Given that the binding molecule is an antibody, the phrase "specifically binds to" refers to a binding reaction that is determinative of the presence of the antigen (such as TREM-2) in a heterogeneous population of proteins and other biologies. Typically, an agent that specifically binds to an antigen binds the antigen with a dissociation constant (KD) of at least about 1 x 10 ⁶ to lxlO ⁷ , or about lxlO ⁸ to lxlO ⁹ M, or about lxlO ¹⁰ to lxlO ¹¹ or higher; and/or binds to the predetermined antigen (e.g. of TREM-2) with an affinity that is at least two-fold, five-fold, ten fold, twenty-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.

According to a particular embodiment, the agent which increases the amount and/or activity of TREM-2 is an activating antibody, also referred to herein as an agonist antibody.

The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, Fv or single domain molecules such as VH and VL to an epitope of an antigen. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen -binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule; and (6) Single domain antibodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.

In a particular embodiment, the TRFM-2 antibody is a monoclonal antibody.

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference and the Examples section which follows).

Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720] Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross- linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The stmctural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods 2: 97- 105 (1991); Bird et ah, Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a single complementarity determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

According to a particular embodiment, the agent capable of upregulating TREM-2 is a diabody. A diabody refers to small antibody fragment prepared by constructing sFv fragments with short linkers (about 5-10) residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the V _H nd VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161; Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48 (1993).

The antibody may be a bispecific antibody recognizing two different antigens, a multivarient antibody or a chimeric antibody.

The antigen binding proteins (e.g. antibodies) described herein may bind to TREM-2 with a KD of < 1 x 10 ⁷ M. In yet another embodiment, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 5 x 10 ⁸ M. In another embodiment, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 1 x 10 ⁸ M. In certain embodiments, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 5 x 10 ⁹ M. In other embodiments, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 1 x 10 ⁹ M. In one particular embodiment, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 5 x 10 ¹⁰ M. In another particular embodiment, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 1 x 10 ¹⁰ M. In another particular embodiment, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 1 x 10 ¹¹ M. In another particular embodiment, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 1 x 10 ¹² M. In another particular embodiment, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 1 x 10 ¹³ M. In another particular embodiment, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 1 x 10 ¹⁴ M. In another particular embodiment, the antigen binding proteins of the invention bind to human TREM-2 with a KD of < 1 x 10 ¹⁵ M.

Affinity is determined using a variety of techniques, an example of which is an affinity ELISA assay. In various embodiments, affinity is determined by a surface plasmon resonance assay (e.g., BIAcore®-based assay). Using this methodology, the association rate constant (ka) and the dissociation rate constant (kd) can be measured. The equilibrium dissociation constant (KD in M) can then be calculated from the ratio of the kinetic rate constants (kd/ka). In some embodiments, affinity is determined by a kinetic method, such as a Kinetic Exclusion Assay (KinExA) as described in Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008. Using a KinExA assay, the equilibrium dissociation constant (KD in M) and the association rate constant (ka in M'V ¹ ) can be measured. The dissociation rate constant (kd) can be calculated from these values (KD X ka). In other embodiments, affinity is determined by a bio-layer interferometry method, such as that described in Kumaraswamy et al., Methods Mol. Biol., Vol. 1278: 165-82, 2015 and employed in Octet ^® systems (Pall ForteBio). The kinetic (ka and kd) and affinity (KD) constants can be calculated in real-time using the bio-layer interferometry method. In some embodiments, the antigen binding proteins described herein exhibit desirable characteristics such as binding avidity as measured by kd (dissociation rate constant) for human TREM-2 of about 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ or lower (lower values indicating higher binding avidity), and/or binding affinity as measured by KD (equilibrium dissociation constant) for human TREM-2 of about 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ M or lower (lower values indicating higher binding affinity). In certain embodiments, the antigen binding proteins of the invention specifically bind to human TREM-2 with a KD from about 1 pM to about 100 nM as measured by bio-layer interferometry at 25° C. For instance, in some embodiments, the antigen binding proteins of the invention specifically bind to human TREM-2 with a KD less than 100 nM as measured by bio-layer interferometry at 25° C. In other embodiments, the antigen binding proteins of the invention specifically bind to human TREM-2 with a KD less than 50 nM as measured by bio-layer interferometry at 25° C. In yet other embodiments, the antigen binding proteins of the invention specifically bind to human TREM-2 with a KD less than 25 nM as measured by bio-layer interferometry at 25° C. In one particular embodiment, the antigen binding proteins of the invention specifically bind to human TREM-2 with a KD less than 10 nM as measured by bio-layer interferometry at 25° C. In another particular embodiment, the antigen binding proteins of the invention specifically bind to human TREM-2 with a KD less than 5 nM as measured by bio-layer interferometry at 25° C. In another particular embodiment, the antigen binding proteins of the invention specifically bind to human TREM-2 with a KD less than 1 nM as measured by bio-layer interferometry at 25° C.

The TREM-2 receptor is thought to require clustering on the cell surface in order to transduce a signal. Thus activating antibodies may have unique features to stimulate the TREM- 2 receptor. For example, they may have the correct epitope specificity that is compatible with receptor activation, as well as the ability to induce or retain receptor clustering on the cell surface. Such antibodies are disclosed in US Patent Application No. 20190010230, the contents of which are incorporated herein by reference.

In vivo, antibodies may cluster receptors by multiple potential mechanisms. Some isotypes of human antibodies such as IgG2 have, due to their unique structure, an intrinsic ability to cluster receptors, or retain receptors in a clustered configuration, thereby activating TREM-2 without binding to an Fc receptor (e.g., White et ah, (2015) Cancer Cell 27, 138-148).

Other antibodies cluster receptors (e.g., TREM-2) by binding to Fcg receptors on adjacent cells. Binding of the constant IgG Fc part of the antibody to Fcg receptors leads to aggregation of the antibodies, and the antibodies in turn aggregate the receptors to which they bind through their variable region (Chu et al (2008) Mol Immunol, 45:3926-3933; and Wilson et al., (2011) Cancer Cell 19, 101-113). Binding to the inhibitory Fcg receptor FcgR (FcgRIIB) that does not elicit cytokine secretion, oxidative burst, increased phagocytosis, and enhanced antibody-dependent, cell-mediated cytotoxicity (ADCC) is often a preferred way to cluster antibodies in vivo, since binding to FcgRIIB is not associated with immune adverse effects.

Other mechanisms may also be used to cluster TREM-2. For example, antibody fragments (e.g., Fab fragments) that are cross-linked together may be used to cluster receptors (e.g., TREM-2) in a manner similar to antibodies with Fc regions that bind Fcg receptors, as described above. Without wishing to be bound to theory, it is thought that cross-linked antibody fragments (e.g., Fab fragments) may function as agonist antibodies if they induce receptor clustering on the cell surface and bind an appropriate epitope on the target TREM-2.

Therefore, in some embodiments, antibodies that bind a TREM-2 protein include agonist antibodies that due to their epitope specificity bind TREM-2 and activate one or more TREM-2 activities. Without wishing to be bound to theory, such antibodies may bind to the ligand-binding site on the target antigen (e.g., TREM-2) and mimic the action of a natural ligand, or stimulate the target antigen to transduce signal by binding to one or more domains that are not the ligand binding sites. Such antibodies would not interfere with ligand binding and may act additively or synergistically with the natural ligands.

In one embodiment, activating antibodies bind the extracellular domain of TREM2, particularly the IgV domain (amino acid residues 29-112 of SEQ ID NO: 1), and through multimerization of receptors, such as IgG itself or NKp44, lead to activation. Thus these domains are rational targets for agonistic antibodies. Agonist anti-TREM2 antibodies can also be produced that target amino acid residues 99-115 of human TREM2.

In some embodiments, an antibody of the present disclosure is an agonist antibody that induces one or more TREM-2 activities. In certain embodiments, the one or more TREM-2 activities, are selected from TREM-2 binding to DAP 12; TREM-2 phosphorylation; PI3K activation; increased expression of one or more anti-inflammatory cytokines, increased expression of one or more anti-inflammatory mediators (e.g., cytokines) selected from IL-12p70, IL-6, and IL-10; reduced expression of one or more pro-inflammatory cytokines; reduced expression of one or more pro -inflammatory mediators selected from the group consisting of IFN-a4, IFN-b, IL-6, IL-12 p70, IL-lbeta, TNG, TNF-alpha, IL-10, IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, mCP-1, and CRP; reduced expression of TNF-alpha; reduced expression of IL-6; extracellular signal-regulated kinase (ERK) phosphorylation; increased expression of C-C chemokine receptor 7 (CCR7); induction of microglial cell chemotaxis toward CCL19 and CCL21 expressing cells; an enhancement, normalization, or both of the ability of bone marrow-derived dendritic cells to induce antigen- specific T-cell proliferation; induction of osteoclast production, increased rate of osteoclastogenesis, or both; increasing the survival and/or function of one or more of macrophages, microglial cells, Ml macrophages and/or microglial cells, activated Ml macrophages and/or microglial cells, M2 macrophages and/or microglial cells, monocytes, osteoclasts, Langerhans cells of skin, and Kupffer cells; induction of one or more types of clearance selected from apoptotic neuron clearance, nerve tissue debris clearance, non-nerve tissue debris clearance, bacteria or other foreign body clearance, disease-causing protein clearance, disease-causing peptide clearance, and disease -causing nucleic acid clearance; induction of phagocytosis of one or more of apoptotic neurons, nerve tissue debris, non-nerve tissue debris, bacteria, other foreign bodies, disease-causing proteins, disease-causing proteins, disease-causing peptides, or disease-causing nucleic acids; normalization of disrupted TREM- 2/DAP12-dependent gene expression; recruitment of Syk, ZAP70, or both to a DAP12/TREM-2 complex; Syk phosphorylation; increased expression of CD83 and/or CD86 on dendritic cells, macrophages, monocytes, and/or microglia; reduced secretion of one or more inflammatory cytokines; reduced secretion of one or more inflammatory cytokines selected from TNF-. alpha., IL-10, IL-6, MCP-1, FN-a4, IFN-b, IL-l.beta.. IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM- CSF, IL-11, IL-12, IL-17, and IL-18; reduced expression of one or more inflammatory receptors; increasing phagocytosis by macrophages, dendritic cells, monocytes, and/or microglia under conditions of reduced levels of MCSF; decreasing phagocytosis by macrophages, dendritic cells, monocytes, and/or microglia in the presence of normal levels of MCSF; increasing activity of one or more TREM-2-dependent genes (e.g., transcription factors of the nuclear factor of activated T-cells (NFAT) family of transcription factors).

In some embodiments, the agent that up-regulates TREM-2 activity induces spleen tyrosine kinase (Syk) phosphorylation after binding to a TREM-2 protein expressed in a cell. Spleen tyrosine kinase (Syk) is an intracellular signaling molecule that functions downstream of TREM-2 by phosphorylating several substrates, thereby facilitating the formation of a signaling complex leading to cellular activation and inflammatory processes.

An exemplary activating TREM-2 activating antibody is Ab21. The amino acid sequence of the heavy chain variable region of Ab21 is set forth in SEQ ID NO: 2. The CDRs are set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The amino acid sequence of the light chain variable region of Ab21 is set forth in SEQ ID NO: 10. The CDRs are set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

Another exemplary activating TREM-2 activating antibody is Ab52. The amino acid sequence of the heavy chain variable region of Ab52 is set forth in SEQ ID NO: 6. The CDRs are set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

The amino acid sequence of the light chain variable region of Ab52 is set forth in SEQ ID NO: 14. The CDRs are set forth in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.

In yet another embodiment, the antibody has the following CDRs:

CDR1 (heavy chain): GFTFTDFY (SEQ ID NO: 18)

CDR2 (heavy chain): IRNKTKGYTT (SEQ ID NO: 19)

CDR3 (heavy chain): ARIGVNNGGSLDYWG (SEQ ID NO: 20)

CDR1 (light chain): QSLLYSENNQDY (SEQ ID NO: 21)

CDR2 (light chain): GAS(SEQ ID NO: 22)

CDR3 (light chain): EQTYSYPYT (SEQ ID NO: 23).

In yet another embodiment, the antibody has the following CDRs:

CDR1 (heavy chain): GFTFTDFY (SEQ ID NO: 24)

CDR2 (heavy chain): IRNKANGYTT (SEQ ID NO: 25)

CDR3 (heavy chain): ARIGINN GGS LD YW G (SEQ ID NO: 26)

CDR1 (light chain): QSLLYSEKNQDY (SEQ ID NO: 27)

CDR2 (light chain): GAS(SEQ ID NO: 28)

CDR3 (light chain): EQTYSYPYT (SEQ ID NO: 29).

In yet another embodiment, the antibody has the following CDRs:

CDR1 (heavy chain): GFTFTDFY (SEQ ID NO: 30)

CDR2 (heavy chain): IRNKAYGYTT (SEQ ID NO: 31)

CDR3 (heavy chain): ARIGINY GGS LD YW G (SEQ ID NO: 32)

CDR1 (light chain): QSLLYSESNQDY (SEQ ID NO: 33)

CDR2 (light chain): GAS(SEQ ID NO: 34)

CDR3 (light chain): EQTYSYPYT (SEQ ID NO: 35)

According to another embodiment, the agent that upregulates the amount and/or activity of TREM-2 is one which is able to inhibit TREM-2 ectodomain shedding (i.e. cleavage). It has been shown that TREM-2 ectodomain shedding (i.e. TREM-2 cleavage) takes place at Hisl57 of TREM-2. Thus, the cleavage enzyme (e.g. ADAM 10, ADAM17 or matrix metalloproteinases) has to have access to this amino acid for cleaving TREM-2. Accordingly, it is conceived that a binding molecule (e. antibody) blocking His 157 can successfully inhibit cleavage of TREM-2. Access of the cleavage enzyme to Hisl57 may be blocked by directly binding to Hisl57. In addition or alternatively, access of the cleavage enzyme to His 157 may be sterically blocked by binding to an amino acid that is located in close proximity (e.g. having a distance of up to 10 amino acids) to His 157. For example, an antibody or a small molecule binding to an amino acid that is located in close proximity to His 157 may sterically block access of the cleavage enzyme to Hisl57, thereby inhibiting TREM-2 cleavage at this site.

Thus, the present invention contemplates a binding molecule (e.g. antibody) that inhibits (preferably prevents) TREM-2 cleavage. Examples of such antibodies are disclosed in WO2018015573 (the contents of which are incorporated herein by reference).

Preferably, the agent capable of up-regulating TREM-2 cleavage is not an ADAM 10, ADAM 17 or matrix metalloproteinase inhibitor.

Additional TREM-2 activating antibodies are disclosed in WO2018195506, the contents of which are incorporated herein by reference.

In one embodiment, the agent which increases the activity of TREM-2 is one which activates DAP12. Such agents are disclosed in US Patent Application No. 20190010230.

Other agents known to increase the activity of TREM-2 include lipids.

In one embodiment, the lipid is a phospholipid. Exemplary lipids contemplated by the present invention include, but are not limited to Cardiolipin (CL), Sphingomyelin (SM), Phosphatidylserine (PS), Phosphatidylcholine (PC) phosphatidylethanolamine (PE), Phosphatidylinositol (PI) and Sulfatide.

According to still another embodiment, the agent comprises APOE or a cholesterol.

Preferably, the agents of the present invention preferably do not cross the blood brain barrier.

According to still another aspect of the present invention there is provided a method of regulating the size and/or number of adipocytes of a subject comprising administering to the subject an agent which alters the amount and/or activity of triggering receptor expressed on myeloid cells 2 (TREM-2), thereby regulating the size and/or number of adipocytes of the subject, the subject not having a disease selected from the group consisting of dementia, frontotemporal dementia, Alzheimer’s disease, Nasu-Hakola disease and multiple sclerosis.

In one embodiment, the agents reduce the size and/or number of adipocytes in a subject. In this case the agents increase the amount and/or activity of TREM-2. Such agents can be used to treat lipid related disorders, as disclosed herein above.

Preferably, such agents decrease the mean size (e.g. diameter) of an adipocyte by at least 10 %, 20 %, 30 %. 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more. Preferably, such agents decrease the number of adipocytes in the body by at least 10 %, 20 %, 30 %. 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more.

Preferably, such agents decrease the amount of visceral fat in the body by at least 10 %, 20 %, 30 %. 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more.

In another embodiment, the agents increase the size and/or number of adipocytes in a subject. In this case the agents decrease the amount and/or activity of TREM-2. Such agents can be used to treat diseases associated with weight loss, including but not limited to body wasting associated with cancer and/or chemotherapeutic agents, hyperthyroidism, bulimia and anorexia.

Preferably, such agents increase the mean size (e.g. diameter) of an adipocyte by at least 10 %, 20 %, 30 %. 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more.

Preferably, such agents increase the number of adipocytes in the body by at least 10 %, 20 %, 30 %. 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more.

Preferably, such agents increase the amount of visceral fat in the body by at least 10 %, 20 %, 30 %. 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more.

Agents that down-regulate TREM-2 include antagonistic antibodies such as those disclosed in US Application No. 20190010230. Other examples include TREM-2 antagonists including small molecules and the like.

According to still another aspect of the present invention there is provided a method of treating or preventing a lipid-related disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent which upregulates the amount and/or activity of CD9 ⁺ CD63 ⁺ macrophages in peripheral tissue (for example in adipose tissue of the periphery, in the liver or in the arteries of the periphery), thereby treating or preventing the lipid-related disorder.

Preferably, the agent of this aspect of the present invention increases peripheral CD9 ⁺ CD63 ⁺ macrophages.

The agent may upregulate the amount and/or activity of CD9 ⁺ CD63 ⁺ macrophages in other tissue such as in liver tissue.

Preferably, the agent does not upregulate the amount and/or activity of CD9 ⁺ CD63 ⁺ macrophages in the brain.

The macrophages of this aspect of the present invention are preferably positive for both CD9 and CD63. Positive is also abbreviated by (+). Positive for a marker means that at least about 70 %, 80 %, 85 %, 90 %, 95 %, or 100 % of the cells in the population present detectable levels of the marker assayed by a method known to those of skill in the art. Thus, for example, the cells stain positively with anti CD9 antibody as determined using FACS or stained positive by immunofluorescence or immunohistochemistry using an anti CD9 antibody. The cells also stain positively with anti CD63 antibody as determined using FACS or stained positive by immunofluorescence or immunohistochemistry using an anti CD63 antibody. In another embodiment, the cells are also positive for TREM-2 - i.e. they also stain positively with anti TREM-2 antibody as determined using FACS or stained positive by immunofluorescence or immunohistochemistry using an anti TREM-2 antibody.

The agent of this aspect of the present invention may increase the mobilization and/or infiltration of such cells into adipose tissue or other such tissue (e.g. liver or arteries).

Alternatively, the agent may act on tissue resident macrophages and convert them into CD9 ⁺ CD63 ⁺ macrophages.

The agent of this aspect of the present invention may increase the differentiation of peripheral blood monocytes to CD9+CD63+ macrophages.

Examples of such agents include agents that bind to TREM-2 as described herein above (e.g. activating antibody and/or lipids).

Other examples of such agents are the cells themselves. According to this embodiment, bone marrow cells may be treated ex vivo to obtain the CD9 ⁺ CD63 ⁺ profile and subsequently administered to the subject.

In one embodiment, the agent of this aspect of the present invention is a non-caloric agent (e.g. not a food).

In another embodiment, the agent of this aspect of the present invention is a protein.

It will be appreciated that the agent of the present invention (e.g., the antibody) can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the agent of the present invention (e.g., the antibody) which is accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, neurosurgical strategies (e.g., intracerebral injection, intrastriatal infusion or intracerebroventricular infusion, intra spinal cord, epidural), transmucosal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in a local rather than a systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient (e.g. adipose tissue).

According to a preferred embodiment, the agents are not administered into the brain of the subject.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.

Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose (e.g. reduction of number or size of adipocytes, or decrease in the amount of visceral fat).

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-1)·

Dosage amount and interval may be adjusted individually to provide tissue levels of the active ingredient that are sufficient to decrease the number or size of adipocytes or decrease visceral fat (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription dmgs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

According to another aspect of the present invention, there is provided an article of manufacture comprising a weight-inducing medication and an agent which upregulates the amount and/or activity of triggering receptor expressed on myeloid cells 2 (TREM-2).

As used herein the term“about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term“consisting of’ means“including and limited to”. The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-PI Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);“Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et ak, "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

MATERIALS AND METHODS

Mice: Wild-type (WT) mice (C57B1/6) were purchased from Harlan and housed in the Wei 7 m an n Institute animal facility. Only male mice were used. All mice were provided with normal chow and water ad libitum, and housed under a strict 12-hour light-dark cycle. At age 8-9 weeks, for some mice the normal chow was replaced with a high fat diet (HFD; irradiated Rodent Diet With 60 kcal% Fat, D12492i Research Diets Inc., New Brunswick, NJ). TREM-2 ⁷ knock-out (KO) mice were provided by Prof. Marco Colonna (Turnbull et ak, 2006). Founding KO breeders were crossed with WT mice at the Weizmann Institute animal facility to produce second-generation cohorts of WT and KO littermates. FI offspring was bred to produce homozygous WT or KO. Heterozygous F2 mice were not used for experiments. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC).

Human samples: Biopsies from visceral adipose tissue from the omental depot (OAT) were obtained from obese individuals with participant informed consent obtained after the nature and possible consequences of the studies were explained under protocols approved by the Institutional Review Boards of the Perelman School of Medicine at the University of Pennsylvania and the Children’s Hospital of Philadelphia. OAT samples were placed in 1 mL of DMEM, and finely minced under sterile conditions before digestion in 50 mL of DMEM with 3 mg/1 mL collagenase IV (Gibco). Samples were incubated at 37 °C in a rotating oven for 60 min. Adipocyte and stromal vascular fractions (SVF) were separated by centrifugation, and red blood cells (RBCs) were removed from the SVF by histopaque gradient (Sigma). Single-cell RNA-sequencing libraries were prepared using the Chromium platform (lOx genomics), and sequenced on the HiSeq 2500 Sequencing System (Illumina).

Isolation of adipose tissue-derived leukocytes: Mice were sacrificed by cervical dislocation and perfused immediately through the left ventricle of the heart with 20 ml of phosphate-buffered saline (PBS) to remove circulating leukocytes from the tissue. The epididymal (visceral) adipose tissue (EAT) was readily located and excised right above the epididymis. The tissue was placed in a 50 ml tube with 10 ml DMEM without phenol red at room temperature, cut into tiny bits with scissors, incubated with collagenase II at 37°C for 20 min while gently agitating, filtered through a 100-pm cell strainer, and spun down at 500g for 10 min with low acceleration/brake, beginning at room temperature and cooling to 4°C. Cells were resuspended in 500 pi RBC lysis solution (Sigma) and incubated on ice for 2-5 min (depending on the initial amount of tissue) and washed. The resulting cell suspension was incubated with anti-CD45 or a cocktail of antibodies for selected markers.

Flow cytometry and single-cell capture: After staining, cells were washed and resuspended in cold FACS buffer (0.5% BSA and 2 inM EDTA in PBS), stained with fluorophore-conjugated anti-mouse CD45 antibody, and filtered through a 40-pm strainer. Right before sorting, cells were stained with propidium iodide to exclude dead/dying cells. Cell sorting was performed using a BD FACSAria Fusion flow cytometer (BD Biosciences), gating for CD45+ cells (leukocytes) after exclusion of dead cells and doublets. Single cells were sorted into 384-well capture plates containing 2 pi of lysis solution and barcoded poly(T) reverse- transcription (RT) primers for scRNA-seq as described previously (Jaitin et al., 2014). Immediately after sorting, plates were spun down to ensure cell immersion into the lysis solution, snap-frozen on dry ice and stored at -80 °C until further processing.

For marker-based validations, samples were stained using the following antibodies: APC- conjugated CD45 (leukocytes), eFluor450-conjugated CD3, CD19, NK1.1, and Ly6G (Fineage negative to exclude T, B, NK and neutrophils), PE-conjugated CDl lb (myeloid cells), APC/Cy7-conjugated Fy6C (monocytes), PerCP/Cy5.5-conjugated F4/80 (macrophages), FITC- conjugated CD9 and PE/Cy7-conjugated CD63, purchased from eBioscience or Biolegend, and DAPI (in the same channel with Fineage staining), for live/dead cell detection. Cell populations from selected gates (Figures 2G and 7E) were sorted for RT-qPCR validation (Figures 9G and 9H). Cells were analyzed using BD FACSDIVA software (BD Bioscience) and FlowJo software (Flow Jo, EEC).

Single cell library preparation: Single cell libraries were prepared with Massively Parallel Single-Cell RNA-seq method (MARS-seq) (Jaitin et al., 2014). In brief, mRNA from single cells sorted into cell capture plates was barcoded, converted into cDNA and pooled using an automated pipeline. Subsequently, the pooled sample was linearly amplified by T7 in vitro transcription, and the resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with pool barcodes and Illumina sequences during ligation, reverse transcription, and PCR. Each pool of cells was tested for library quality and library concentration was assessed.

Immunofluorescence: Immunostaining of frozen adipose tissue were performed as described previously with some modifications (Honvo-Houeto and Truchet, 2015). Mice were sacrificed and epididymal white adipose tissues harvested and chopped into small pieces, fixed in 4% Paraformaldehyde solution in PBS (Santa Cruz Biotechnology) overnight at 4 °C, transferred to 10-30 % sucrose in PBS at 4 °C for 3 days, then embedded in OCT (optimal cutting temperature compound), frozen on dry ice and stored at -80 °C until further processing. Immunofluorescent staining was carried out on 35-pm thick sections. Samples were permeabilized with 0.3 % Triton X-100 for 15 min. After washing three times with PBS, sections were blocked with a solution containing 5 % FBS and 1 % BSA in PBS pH 7.4 for 2 hours at room temperature. The blocking solution was then replaced with a cocktail of primary antibodies, including rabbit anti-CD9 (1:200, Abeam), rat anti-F4/80 (1:200, Abeam), goat anti- Perilipin-1 (1:200, Abeam) diluted in blocking solution, and incubated overnight at 4°C. The secondary antibody mixture included 1:500 dilutions of the following antibodies: DyLight550 Donkey anti-Goat IgG-heavy and light (Bethyl), Donkey anti-Rat IgG-heavy and light chain cross-adsorbed Antibody DyLight594 Conjugated (Bethyl), and Alexa Fluor 647-AffiniPure Donkey Anti-Rabbit IgG (H+L; Jackson ImmunoResearch Labs) and was diluted in 1% BSA in PBS and incubated for 1 hour at room temperature and followed DAPI staining. All sections were imaged on a Leica STED confocal microscope with 63X objective (Leica, Germany). Images were analyzed using Fiji (Schindelin et al., 2012). Single adipocytes were manually demarcated using the selection tool and these areas were tracked using the ROI Manager. Only complete adipocytes in the field of view were considered. The relative area of the demarcated adipocytes was then quantified (Figure 5B).

Histology: Adipose tissues were fixed in in a 4% paraformaldehyde solution in PBS for two days and embedded in paraffin (Laica). Four pm-thick sections were stained with hematoxylin and eosin.

Glucose tolerance test: Mice were fasted for 14 h and subsequently given 200 pi of a O.l g/ml (10%) glucose solution (JT Baker) by intraperitoneal injection. Blood glucose was determined at 0, 15, 45, 30, 60, 90 and 120 min post injection (Contour blood glucose meter, Bayer).

Body composition measurements: Lean, fluid and fat mass of mice was determined with a Minispec LF50 body composition device (Bruker).

Lipid profiting: Mice were fasted for 14h and subsequently anesthetized by intraperitoneal injection of 200 microliter of 10 vol. % ketamine. Blood was obtained from the eye. Measurements of concentrations of triglycerides, HDL, and cholesterol from blood plasma were performed with the Kenshin-2 kit (Medtechnica) at a SPOTCHEM EZ sp-4430 (Arkray).

Single cell RNA-sequencing analyses:

MARS-seq processing: scRNA-seq libraries (pooled at equimolar concentration) were sequenced on an Illumina NextSeq 500 at a median sequencing depth of -40,000 reads per cell. Sequences were mapped to the mouse (mmlO) and human (hg38) genome, respectively. Demultiplexing and filtering was performed as previously described (Jaitin et ah, 2014), with the following adaptations: Mapping of reads was performed using HIS AT (version 0.1.6); reads with multiple mapping positions were excluded. Reads were associated with genes if they were mapped to an exon, using the ensembl gene annotation database (embl release 90). Exons of different genes that shared a genomic position on the same strand were considered as a single gene with a concatenated gene symbol. The level of spurious unique molecular identifiers (UMIs) in the data were estimated by using statistics on empty MARS-seq wells, and excluded rare cases with estimated noise > 5% (median estimated noise over all experiments was 2%).

Metacell modeling: We used the R package“MetaCell” (Baran et al., 2018) with the following specific parameters (complete script reproducing all analyses from raw data will be available). We removed specific mitochondrial genes, immunoglobulin genes, and genes linked with poorly supported transcriptional models (annotated with the prefix“Rp-”). We then filtered cells with less than 400 UMIs. Gene features were selected using the parameter Tvm=0.3 and a minimum total UMI count > 50. We subsequently performed hierarchical clustering of the correlation matrix between those genes (filtering genes with low coverage and computing correlation using a down- sampled UMI matrix) and selected the gene clusters that contained anchor genes.

The gene selection strategy discussed above retained a total of 425 marker gene features for the computation of the MetaCell balanced similarity graph. We used K = 150, 500 bootstrap iterations and otherwise standard parameters. Metacells were annotated as Monocytes /Macrophages or others, by applying a straightforward analysis of known cell type marker genes (e.g. Ear2, Fnl, Ccr2, Mrcl , Cd3d, Cd79b, and more). Subsets of Monocytes and Macrophages were obtained by hierarchical clustering of the confusion matrix (Figure 6C) and supervised analysis of enriched genes in homogeneous groups of metacells.

kNN projection of cells on the graph of Figure 2A: To project the adipose tissue Monocytes/Macrophages obtained from TREM-2 knock-out mice and their littermate wild-type controls onto the kNN graph of Figure 2A, we generated a UMI matrix of the 200 most variable genes of the Monocytes/Macrophages from Figure 2A. As query data, we utilized the UMI matrix of those genes of the Monocytes/Macrophages from the TREM-2 knock-out experiment. To obtain the K=6 nearest neighbors of Pearson correlation to every cell in the query data, we applied the knn.index.dist function of the R package“KernelKnn” (version 1.0.8). We averaged the x- and y-coordinates of the nearest neighbors to calculate the position of every cell from the TREM-2 knock-out experiment on the graph of Figure 2A.

Defining a module gene signature: To define a module gene signature for Monocytes/Macrophages (Figure 3A), we identified a group of 33 genes (incl. TREM-2, Cd9, Lpl ) that exhibited a strong Pearson correlation across the metacells’ log2 footprint expression of the 200 most variable genes excluding genes associated with cell cycle and stress that were filtered from these lists in advance.

Human-mouse gene comparison: To compare the gene expression of LAM and other macrophages between human and mouse (Figure 3F), we used the orthologous gene annotation from ensembl 95 of the BioMart browser. We only plotted genes for which an orthologue between mouse and human existed.

Statistical Analyses: Differential gene expression analysis was performed upon down- sampling of the UMI matrix as part of the MetaCell package on molecules/ 1,000 UMIs by Mann Whitney U test with false-discovery rate (FDR) correction. Metabolic parameters in Figures 5A- I were compared by one- or two-way ANOVA. Relative frequencies of cell types in Figures 6E and 6F and 10B and IOC were analyzed by Mann Whitney U test.

EXAMPLE 1

Lipid-associated macrophages control metabolic homeostasis in a Treml-dependent manner

RESULTS

Time-resolved single-cell characterization of adipose tissue immune cells in obesity

To identify key factors in adipose tissue immune cell remodeling during metabolic disease, we first studied a well-established diet-induced obesity model using mice fed on a high- fat diet (HFD) to trigger weight gain. We isolated epididymal visceral adipose tissue (EAT) at regular intervals after 6, 12, and 18 weeks of HFD feeding, to cover the entire timespan from the lean state to morbid adiposity, followed by massively-parallel single-cell RNA-sequencing (MARS-seq) of tissue-resident CD45 ⁺ immune cells (Figure 1A) (Jaitin et al., 2014). At each time point we compared all animals to aged matched littermates fed on a normal chow (NC) diet. Expectedly, HFD feeding induced weight gain and adiposity over the course of the experiment (Figures 6A and 6B). We profiled a total of 21,210 quality control (QC)-positive cells sampled from 20 mice at different time points. We applied the MetaCell algorithm (Baran et al., 2018) to identify homogeneous and robust groups of cells (“metacells”) from single-cell RNA-seq data, resulting in a detailed map of 244 metacells organized into 15 broad immune cell types (Figure IB). We classified the different groups based on expression levels of the most variable genes using the MetaCell similarity matrix (Figure 6C) and used the differentially expressed genes to annotate broad myeloid and lymphoid cell types (Figures IB and 1C). Following this approach, we detected a massive reorganization of the immune cell population in visceral adipose tissue between 6 and 12 weeks on HFD (Figure ID), while NC diet did not induce alterations in the immune cell compartment over time (Figure 6D). The most prominent changes induced by HFD included an expansion of adipose tissue macrophages and a reduction of regulatory T cells and type 2 innate lymphoid cells (ILCs) (Figures IE, 6E and 6F), in line with previous reports (Biswas and Mantovani, 2012; Cipolletta et al., 2012; Feuerer et al., 2009; Kanneganti and Dixit, 2012; Mathis, 2013; McNelis and Olefsky, 2014).

To determine whether this global rearrangement of the immune cell population was induced by the dietary conditions or associated with obesity per se, we used a genetic model of obesity, the db/db mouse (Bahary et al., 1990), which harbors a mutation in the leptin receptor gene that causes hyperphagia and massive adiposity even on NC diet (Figure 6G). We profiled a total of 8,372 QC-positive cells sampled from two seven-week old, two 15-week old db/db, and four wild-type littermate control mice. The db/db mice underwent similar remodeling of the immune cell compartment as observed in mice fed on HFD (Figures IF and 6H), indicating that these changes occur across different dietary conditions.

Macrophage and monocyte compartments undergo dramatic remodeling during obesity

By far the most significant cell population changes during obesity development occurred in the monocyte/macrophage compartment, characterized by two monocyte and three macrophage subgroups comprising of 136 metacells (Figure 2A). The two monocyte subsets, Monl and Mon2, were distinguished by the expression of Retnla, Fnl (Monl), Plac8, and Clec4e (Mon2), among other differentially expressed genes (Figures 2B and 7A). One macrophage population (Macl) highly expressed Retnla, Cdl63, Lyvel, and Cd209f, a signature partially overlapping with the classical definition of M2 macrophages (Figures 2B and 7A). Two additional macrophage populations (Mac2 and Mac3) were characterized by graduated expression of genes, such as Cd9 and Ncehl, in line with the recently described CD9 ⁺ subset of adipose tissue macrophages (Hill et al., 2018). Among the differentially expressed genes between these macrophage subsets was the osteopontin-encoding gene Sppl (Figures 2B and 7 A). Both Mac2 and Mac3 emerged only under obese conditions and represented more than 75% of the myeloid compartment after 18 weeks of HFD (Figures 2C and 2D). Similar dynamic changes in the monocyte/macrophage compartment were observed in db/db mice, with the Mac2 and Mac3 subsets being strongly expanded in obese mice compared to littermate controls (Figures 2E and 7B-7D). We validated these findings on the protein level by flow cytometry, which confirmed the emergence of CD9 ⁺ CD63 ⁺ macrophages during obesity, as predicted by single-cell RNA-seq (Figures 2F, 2G and 7E). In addition, immunofluorescence staining revealed that these CD9-expressing macrophages accumulated in “crown-like” structures surrounding adipocytes of HFD fed mice (Figure 2H) (Hill et al., 2018). Together, these data provide a time-resolved single-cell map of the immune cell compartment in visceral adipose tissue and highlight a stereotypical change in specific monocyte and macrophage subsets during obesity.

Lipid-associated macrophages are characterized by TREM-2 expression in mice and humans

Given its strong expansion under obese conditions, we next sought to functionally characterize the Mac3 subset. Analysis of gene modules most highly associated with this subset revealed a transcriptional signature of TREM-2, Lipa, Lpl, Ctsb, Ctsl, Fabp4, Fabp5 and Cd36 (Figures 3A and 3B), a lipid metabolism signature reminiscent of“disease-associated microglia” (DAM) found in the context of Alzheimer’s disease (Keren-Shaul et al., 2017). With the exception of Fabp4, these“lipid-associated macrophages” (LAM) in adipose tissue expressed a highly similar gene profile as DAM cells (Figure 8A). In contrast, the Mac2 population did not express the full signature (Figures 8B and 8C). LAM cells were not unique to the HFD model, since the same gene signature emerged in Mac3 cells in EAT harvested from 15-week-old db/db mice (Figure 8D). LAM cells were likewise present in inguinal subcutaneous adipose tissue (SAT) sampled from mice on HFD (Figures 9A and 9B).

Given the unexpected finding that LAM cells expressed TREM-2, a cell surface receptor primarily investigated for its role in microglia during neurodegeneration (Ulland et al., 2017; Wang et al., 2015), we sought to confirm their existence in the context of human obesity. To this end, we analyzed the stromal-vascular fraction of visceral adipose tissue from an obese human donor (BMI 46) by single-cell RNA-seq of 15,150 QC-positive cells, resulting in 172 metacells (Figures 3C, 9C, 9D). Indeed, TREM-2-ex pressing human LAM cells constituted a defined cluster (Figure 3D), which was characterized by a highly conserved gene signature compared to what we had observed in mice, including LIPA, CTSB, CTSL, FABP4, FABP5, and CD36 (Figure 3E). In addition to the conserved gene signature, human LAM cells expressed a small number of unique genes, including the metallopeptidase inhibitors TIMP1 and TIMP3 as well as the aldolase A gene AFDOA (Figures 3E and 3F).

Next, we investigated the pathways activated in human and mouse LAM cells. We ranked all genes by their relative enrichment in the TREM-2-expressing subsets, followed by functional classification of the most characteristic genes according to KEGG pathways. This approach revealed a strong enrichment in pathways related to phago- and endocytosis as well as lipid metabolism (Figures 9E and 9F), many of which were shared between mouse and human (Figure 3G), in line with previous reports on the function of TREM-2 as a lipid receptor (Coats et al., 2017; Ulland et al., 2017; Wang et al., 2015; Xu et ah, 2013), and with the notion of “metabolically-activated” adipose tissue macrophages (Hill et al., 2018; Kratz et al., 2014; Xu et al., 2013). The enriched gene signature was indicative of a highly active pathway initiated by phago- and endocytosis, coupled to lipid metabolism and oxidative phosphorylation (Figure 3H). To functionally validate these findings, we isolated mouse LAM cells by flow cytometry based on the expression of CD9 and CD63 (Figure 2G). We first confirmed the presence of the LAM gene signature by qPCR in sorted CD9 ⁺ CD63 ⁺ macrophages (Figures 9G and 9H). Indeed, this subset was characterized by the expression of TREM-2 and the same gene module we had identified for TREM-2 ⁺ cells (Figure 9H). Bodipy staining confirmed the presence of intracellular lipids specifically in the CD9 ⁺ CD63 ⁺ subset (Figure 91), further emphasizing the functional role of LAM cells in lipid metabolism (Hill et al., 2018). These data identify TREM- 2 ⁺ LAM cells as a conserved cell type in both mouse and human visceral adipose tissue under obese conditions.

TREM-2 is essential for adipose tissue macrophage remodeling during obesity

We next determined the functional importance of TREM-2 for macrophage remodeling in obese adipose tissue. To this end, we used TREM-2-deficient mice and littermate controls and analyzed the adipose tissue immune cell compartment after 12 weeks of HFD feeding. We profiled a total of 10,042 QC-positive cells by single-cell RNA-seq, resulting in 133 metacells. Remarkably, the monocyte/macrophage compartment of TREM-2 ¹ mice did not fully progress towards the HFD-associated state observed in wild-type littermates. Instead, these cells retained multiple features of NC controls (Figure 4A). In particular, LAM cells failed to accumulate in the absence of TREM-2, indicating that this surface receptor is not merely a characteristic marker, but an essential driver of the LAM cell molecular program (Figure 4B). TREM-2 ¹ macrophages lacked the majority of the LAM gene signature, featuring markedly reduced levels of Lipa, Lpl, Ctsb, Ctsl, Fabp4, Fabp5, and Cd36 (Figures 4C and 4D). This function of TREM- 2 was specific to the metabolically-challenged condition, since TREM-2-deficient mice on NC did not show any abnormalities in their adipose tissue immune cell population (Figures 10A and 10B). Notably, the strong expansion of the monocyte/macrophage compartment upon HFD feeding equally took place in TREM-2-deficient mice; however, instead of accumulating LAM cells, macrophages in TREM-2-deficient mice retained the Mad and partially Mac2 gene expression signature (Figures 4B, IOC and 10D). Notably, TREM-2 was also required for the formation of LAM cell-rich crown-like structures in obese adipose tissue, as accumulation of LAM cells surrounding adipocytes under HFD conditions was substantially reduced in TREM-2 (Figures 4E and 5A). Other HFD-associated changes in the immune cell compartment were not affected by TREM-2 deficiency, with the exception of a modest reduction in number of cDCl and Mast cells (Figure 10E). Together, these data indicate that TREM-2 is a critical checkpoint for the response of adipose tissue myeloid cells to obese conditions and highlight its importance for macrophage remodeling, LAM cell formation, and the assembly of crown-like structures during obesity.

TREM-2 prevents adipocyte hypertrophy and loss of systemic metabolic homeostasis

Finally, we sought to determine the physiological importance of LAM cells. Histological analysis of visceral adipose tissue revealed massive adipocyte hypertrophy in the absence of TREM-2 (Figures 5A and 5B). This inability to control adipocyte size in the absence of TREM-2 was not observed under NC conditions, indicating that LAM cells are required to prevent adipocyte expansion upon loss of adipose tissue homeostasis. Additionally, upon HFD feeding, TREM-2-deficient mice featured enhanced body fat accumulation (Figure 5C), hypercholesterolemia (Figure 5D), elevated levels of LDL and HDL cholesterol (Figures 5E and 5F), accelerated weight gain (Figure 5G), and marked glucose intolerance (Figures 5H and 51). Together, these results suggest a new role for TREM-2-expressing lipid-metabolizing LAM cells in the local containment of adipocytes and prevention of metabolic derangements upon loss of adipose tissue homeostasis. Table 1

Table 2

Table 3

Table 4

EXAMPLE 2

Trem2 KO phenotype is immune specific

As the Trem2 KO mice have a global deletion of Trem2, the present inventors sought to confirm that the deficiency of Trem2 on macrophages and the absence of LAM cells drives the phenotypes of increased cholesterol and glucose intolerance, and not the deficiency of Trem2 on adipocytes or endothelial cells. For this purpose, they transplanted BM cells from Trem2-/- or Trem2+/+ mice to a lethally irradiated WT recipients. After 9 weeks of recovery chimeric mice were fed with HFD for 18 weeks, Trem2-/- chimeric mice featured increased body fat accumulation, higher LDL levels and glucose intolerance (Figures 11A-G). These results indicate that KO effect is immune specific and not due to Trem2 absence in adipocytes, endothelial cells or any other non-hematopoietic cells.

EXAMPLE 3

Percentage of human LAM cells increases with BMI

Given the unexpected finding that LAM cells expressed Trem2, a cell surface receptor primarily investigated for its role in microglia during neurodegeneration (Ulland et al., 2017; Wang et al., 2015), the present inventors sought to confirm their existence in the context of human obesity. To this end, they analyzed the stromal-vascular fraction of visceral adipose tissue from 6 obese human donors (BMI 37-46) and one lean donor (BMI 23) by single-cell RNA-seq. Indeed, TREM2-expressing human LAM cells constituted a defined cluster (Figure 3D), which was characterized by a highly conserved gene signature compared to what we had observed in mice, including LIPA, CTSB, CTSL, FABP4, FABP5, CD9 and CD36 (Figure 3E). In addition to the conserved gene signature, human LAM cells expressed a small number of unique genes, including the metallopeptidase inhibitors TIMP1 and TIMP3 as well as the aldolase A gene ALDOA (Figures 3E and 3F). Finally, comparing the percentage of LAM cells out of the total CD45+ cells to the BMI of the donors revealed a striking positive correlation (Figure 12).

EXAMPLE 4

LAM cells are not restricted to adipose tissue and are found in the liver of obese mice To test if LAM cells are restricted to adipose tissue or accumulate in various tissue during obesity we sequenced CD45+ from liver of mice 12 weeks on HFD or NC. Metacell analysis revealed Trem2+ subsets of cells expressing LAM markers such as Lpl, Lipa, Apoe, Sppl, Fabp5 and more (Figure 13A). Quantification of LAM cell percentage per diet showed 6- fold expansion of LAM population after 12 weeks on HFD compared to normal diet (Figure 13B).

EXAMPLE 5

TREM-2 activation assay

MATERIALS AND METHODS

Assay to probe Trem.2 signaling activity after exposure to different ligand: For overexpression, Raw and N9 cells were infected with Lentivirus expressing mouse Trem2 under CMV promoter for constitutive expression. For KO cell line, Raw and N9 cells were infected with Lentivirus expressing Cas9 and Trem2 guide RNA for knockout of Trem2 gene. Stable expressing clones were selected by puromycin and the cell surface TREM2 expression was evaluated by flow cytometry. The highest Trem2 expressing single cell clone was selected for expansion.

Trem.2 agonist for activation assay: Phospholipids liposomes, Phospholipidcholin, Phospholipidserin, Phospholipidinositol, Sphingolipids, Exosomes perigonadal adipose tissue

Phospho-Syk screen assay: Activation of Trem2-dependent phosphoSyk signaling was analyzed using Flow cytometry. Stable cell lines were treated with Trem2 agonists following fixation-permeabilization and staining with phospho-Syk antibody.

RESULTS

The results are illustrated in Figure 14. Both phosphatidylcholine- and phosphatidylinositol (PC-PI) activated the TREM2 receptor.

EXAMPLE 6

Glucose tolerance test

MATERIALS AND METHODS

Mice on high fat diet (HFD) for 8 weeks were treated with 1 mg/kg of Trem2 agonist antibody (as described in Schlepckow et al, EMBO Molecular Medicine (2020) el 1227, the contents of which are incorporated herein by reference), or IgG control (3 mice per group) intraperitoneal once per week for a 10 week period. Glucose tolerant test (GTT) was performed after 8 weeks.

RESULTS

As illustrated in Figure 15, Trem2 agonist antibody reduced the amount of glucose in the blood for more than 120 minutes.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.