WO2020194317A1 - Méthode de traitement de troubles liés aux lipides - Google Patents

Méthode de traitement de troubles liés aux lipides Download PDF

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Publication number
WO2020194317A1
WO2020194317A1 PCT/IL2020/050375 IL2020050375W WO2020194317A1 WO 2020194317 A1 WO2020194317 A1 WO 2020194317A1 IL 2020050375 W IL2020050375 W IL 2020050375W WO 2020194317 A1 WO2020194317 A1 WO 2020194317A1
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agent
trem
subject
lipid
disease
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PCT/IL2020/050375
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English (en)
Inventor
Ido Amit
Eran Elinav
Diego Jaitin
Assaf WEINER
Hagit SHAPIRO
Christoph Alexander THAISS
Lorenz Kurt ADLUNG
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Yeda Research And Development Co. Ltd.
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Publication of WO2020194317A1 publication Critical patent/WO2020194317A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • 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.
  • 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.
  • obesity is a predisposing factor for numerous other diseases that are not typically classified as metabolic/endocrine, such as cancer and neurodegeneration.
  • the global obesity pandemic has far-reaching consequences on life expectancy, quality of life, and healthcare costs.
  • adipose tissue 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.
  • 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.
  • tissue-resident immune cells 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.
  • 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.
  • 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.
  • 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.
  • an agent which upregulates the amount and/or activity of TREM-2 for use in treating or preventing a lipid-related disorder 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.
  • 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.
  • a disease selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease and multiple sclerosis.
  • 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.
  • TRM-2 myeloid cells 2
  • a method of treating a disease associated with weight loss in a subject in need thereof 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.
  • 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.
  • the agent comprises CD9 + CD63 + macrophages.
  • the peripheral tissue comprises adipose tissue or liver tissue.
  • the subject is on a weight-inducing medication.
  • 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.
  • the agent upregulates the amount and/or activity of TREM-2 on the macrophages.
  • the peripheral tissue comprises adipose tissue or liver tissue.
  • the regulating comprises down regulating.
  • the subject is obese.
  • the subject is not obese.
  • the medication is selected from the group consisting of a steroid hormone and a psychoactive drug.
  • the agent binds specifically to TREM-2.
  • the agent is an activating antibody to TREM-2.
  • the agent is an agonist of TREM-2.
  • the agent is a small molecule.
  • the agent comprises a lipid.
  • the lipid is a phospholipid.
  • the lipid is selected from the group consisting of Cardiolipin (CL), Sphingomyelin (SM), Phosphatidylserine (PS), Phosphatidylcholine (PC) phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Sulfatide.
  • CL Cardiolipin
  • PS Phosphatidylserine
  • PC Phosphatidylcholine
  • PE phosphatidylethanolamine
  • PI Phosphatidylinositol
  • the agent comprises APOE or a cholesterol.
  • the activity of TREM-2 comprises an increase in phosphorylated spleen tyrosine kinase (stk).
  • the regulating comprises up- regulating.
  • the agent is an antibody antagonistic to TREM-2.
  • the agent is an antagonist of TREM-
  • the agent is a small molecule.
  • the activity of TREM-2 comprises a change in the amount of phosphorylated spleen tyrosine kinase (stk).
  • the administering comprises locally administering into the adipose tissue of the subject.
  • 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.
  • the -related disorder is obesity.
  • the fatty liver disease is selected from the group consisting of hypertriglyceridemia, steatohepatitis, atherosclerosis and hypercholesterolemia.
  • the hypercholesterolemia is a familial hypercholesterolemia.
  • the disease is selected from the group consisting of cancer, hyperthyroidism and anorexia.
  • the agent is an antibody antagonistic to TREM-2.
  • the agent is an antagonist of TREM-
  • the agent is a small molecule.
  • 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.
  • FIGs. 3A-H conserveed 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 kNN graph of 15,150 QC-positive single cells (172 metacells) of human omental adipose tissue
  • FIGs. 4A-E conserveed 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 Frequency of the EAT Mon/Mac subsets as defined in Figures 2A-H in each of four KO and four WT mice on HFD.
  • 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
  • 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
  • FIGs. 6A-H Single-cell characterization of the adipose tissue immune niche during obesity progression.
  • 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.
  • FIGs. 7A-E Analysis of subsets of Monocytes and Macrophages of dbldb mice.
  • 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.
  • 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).
  • 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.
  • 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 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
  • FIGs. 10A-E Characterization of adipose tissue immune cells in TREM-2 knock-out mice.
  • 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.
  • 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.
  • 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.
  • PC phosphatidylcholine
  • PC-PI phosphatidylcholine- phosphatidylinositol
  • 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.
  • 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.
  • 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.
  • LAM lipid-associated macrophage
  • TREM-2 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.
  • 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.
  • 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.
  • 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.
  • the term“treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition.
  • preventing refers to substantially preventing the onset of clinical or aesthetical symptoms of a condition.
  • lipid-related disease refers to a disease associated with excess lipids.
  • the lipid-related disease is one for which decreasing a size and/or number of adipocytes is beneficial.
  • 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.
  • the lipid-related disease is obesity.
  • agent may also be a suitable therapy to promote weight loss in a non-obese (but overweight) subject.
  • the subject having the lipid-related disorder has a body mass index (BMI) of greater than 25, 26, 27, 28, 29 or 30.
  • BMI body mass index
  • the subject having the lipid-related disorder has a body mass index (BMI) of between 25-35 or 26-35.
  • BMI body mass index
  • the subject having the lipid-related disorder has a body mass index (BMI) of less than 25.
  • BMI body mass index
  • 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.
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • 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.
  • the subject treated for lipid-related disease does not have dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease and multiple sclerosis.
  • the subject treated for lipid-related disease does not have a neurodegenerative disease.
  • the lipid-related disease is fatty liver disease.
  • 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.
  • 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.
  • the familial fatty liver disease is familial hyperlipidemia.
  • 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.
  • LDL receptor protein which normally removes LDL from the circulation
  • ApoB apolipoprotein B
  • the term“subject” 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.
  • 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
  • blood pressure-reducing medicines including beta-blockers such as propranolol and metoprolol.
  • 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).
  • TREM-2 myeloid cells 2
  • Trem2 myeloid cells 2
  • 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).
  • human TREM-2 protein Uniprot Accession No. Q9NZC2
  • mouse TREM-2 protein Uniprot Accession No. Q99NH8
  • rat TREM-2 protein Uniprot Accession No.
  • 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.
  • TREM-2 forms a receptor- signaling complex with DAP12.
  • TREM-2 phosphorylates and signals through DAP12 (an IT AM domain adaptor protein).
  • TREM-2 signaling results in the downstream activation of PI3K.
  • TREM-2 signaling results in the downstream phosphorylation of spleen tyrosine kinase (stk).
  • an example of a human TREM-2 amino acid sequence is set forth below as SEQ ID NO: 1.
  • 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.
  • 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).
  • SEQ ID NO: 1 signal peptide located at amino acid residues 1-18 of human TREM-2
  • IgV immunoglobulin-like variable-type
  • the agent increases the amount and/or activity of TREM-2 which is expressed on macrophages.
  • 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.
  • the specified binding molecules bind preferentially to a particular target protein (e.g.
  • TREM-2 TREM-2
  • 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.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background.
  • 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.
  • 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.
  • KD dissociation constant
  • TREM-2 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.
  • a non-specific antigen e.g., BSA, casein
  • the agent which increases the amount and/or activity of TREM-2 is an activating antibody, also referred to herein as an agonist antibody.
  • 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.
  • 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
  • the TRFM-2 antibody is a monoclonal antibody.
  • 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.
  • 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.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • 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.
  • sFv single-chain antigen binding proteins
  • 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.
  • 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.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • 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.
  • 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)].
  • Fc immunoglobulin constant region
  • 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.
  • 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.
  • 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)].
  • 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.
  • 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.
  • the antigen binding proteins of the invention bind to human TREM-2 with a KD of ⁇ 1 x 10 9 M.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • antibody fragments e.g., Fab fragments
  • TREM-2 cluster receptors
  • cross-linked antibody fragments e.g., Fab fragments
  • cross-linked antibody 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.
  • 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.
  • 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.
  • TREM-2 target antigen
  • Such antibodies would not interfere with ligand binding and may act additively or synergistically with the natural ligands.
  • 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.
  • IgV domain amino acid residues 29-112 of SEQ ID NO: 1
  • receptors such as IgG itself or NKp44
  • Agonist anti-TREM2 antibodies can also be produced that target amino acid residues 99-115 of human TREM2.
  • an antibody of the present disclosure is an agonist antibody that induces one or more TREM-2 activities.
  • 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,
  • 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).
  • TREM-2-dependent genes e.g., transcription factors of the nuclear factor of activated T-cells (NFAT) family of transcription factors.
  • 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.
  • 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.
  • the antibody has the following CDRs:
  • CDR2 (heavy chain): IRNKTKGYTT (SEQ ID NO: 19)
  • CDR3 (heavy chain): ARIGVNNGGSLDYWG (SEQ ID NO: 20)
  • CDR2 (light chain): GAS(SEQ ID NO: 22)
  • CDR3 (light chain): EQTYSYPYT (SEQ ID NO: 23).
  • the antibody has the following CDRs:
  • CDR2 (heavy chain): IRNKANGYTT (SEQ ID NO: 25)
  • CDR1 (light chain): QSLLYSEKNQDY (SEQ ID NO: 27)
  • CDR2 (light chain): GAS(SEQ ID NO: 28)
  • CDR3 (light chain): EQTYSYPYT (SEQ ID NO: 29).
  • the antibody has the following CDRs:
  • CDR1 (light chain): QSLLYSESNQDY (SEQ ID NO: 33)
  • CDR2 (light chain): GAS(SEQ ID NO: 34)
  • CDR3 (light chain): EQTYSYPYT (SEQ ID NO: 35)
  • 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.
  • the cleavage enzyme e.g. ADAM 10, ADAM17 or matrix metalloproteinases
  • 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.
  • 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.
  • 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.
  • the present invention contemplates a binding molecule (e.g. antibody) that inhibits (preferably prevents) TREM-2 cleavage.
  • a binding molecule e.g. antibody
  • Examples of such antibodies are disclosed in WO2018015573 (the contents of which are incorporated herein by reference).
  • the agent capable of up-regulating TREM-2 cleavage is not an ADAM 10, ADAM 17 or matrix metalloproteinase inhibitor.
  • TREM-2 activating antibodies are disclosed in WO2018195506, the contents of which are incorporated herein by reference.
  • the agent which increases the activity of TREM-2 is one which activates DAP12.
  • DAP12 Such agents are disclosed in US Patent Application No. 20190010230.
  • TREM-2 Other agents known to increase the activity of TREM-2 include lipids.
  • 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.
  • the agent comprises APOE or a cholesterol.
  • the agents of the present invention preferably do not cross the blood brain barrier.
  • 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.
  • a disease selected from the group consisting of dementia, frontotemporal dementia, Alzheimer’s disease, Nasu-Hakola disease and multiple sclerosis.
  • the agents reduce the size and/or number of adipocytes in a subject.
  • 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.
  • 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.
  • such agents decrease the number of adipocytes in the body by at least 10 %, 20 %, 30 %. 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more.
  • 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.
  • the agents increase the size and/or number of adipocytes in a subject.
  • 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.
  • 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.
  • such agents increase the number of adipocytes in the body by at least 10 %, 20 %, 30 %. 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or more.
  • 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.
  • TREM-2 down-regulate TREM-2
  • antagonistic antibodies such as those disclosed in US Application No. 20190010230.
  • Other examples include TREM-2 antagonists including small molecules and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • agents that bind to TREM-2 as described herein above include agents that bind to TREM-2 as described herein above (e.g. activating antibody and/or lipids).
  • bone marrow cells may be treated ex vivo to obtain the CD9 + CD63 + profile and subsequently administered to the subject.
  • the agent of this aspect of the present invention is a non-caloric agent (e.g. not a food).
  • the agent of this aspect of the present invention is a protein.
  • agent of the present invention e.g., the antibody
  • the agent of the present invention can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • 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.
  • active ingredient refers to the agent of the present invention (e.g., the antibody) which is accountable for the biological effect.
  • 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.
  • 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.
  • 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.
  • 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.
  • a tissue region of a patient e.g. adipose tissue
  • the agents are not administered into the brain of the subject.
  • 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.
  • 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.
  • 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.
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • 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).
  • 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.
  • 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.
  • 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.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • 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.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • 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.
  • compositions 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.
  • 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.
  • 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.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions 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.
  • 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).
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • 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).
  • MEC minimum effective concentration
  • 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.
  • 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.
  • compositions 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.
  • 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).
  • compositions, methods 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.
  • Wild-type 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).
  • HFD high fat diet
  • 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).
  • Biopsies from visceral adipose tissue from the omental depot 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 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).
  • 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.
  • PBS phosphate-buffered saline
  • EAT epididymal adipose tissue
  • 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.
  • BD FACSAria Fusion flow cytometer BD Biosciences
  • 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.
  • RT reverse- transcription
  • 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.
  • 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
  • Single cell library preparation Single cell libraries were prepared with Massively Parallel Single-Cell RNA-seq method (MARS-seq) (Jaitin et al., 2014).
  • MERS-seq Massively Parallel Single-Cell RNA-seq method
  • mRNA from single cells sorted into cell capture plates was barcoded, converted into cDNA and pooled using an automated pipeline.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • Lipid-associated macrophages control metabolic homeostasis in a Treml-dependent manner
  • Mac2 and Mac3 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).
  • Lipid-associated macrophages are characterized by TREM-2 expression in mice and humans
  • 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).
  • 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).
  • 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).
  • 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).
  • Figures 9G and 9H We first confirmed the presence of the LAM gene signature by qPCR in sorted CD9 + CD63 + macrophages. 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
  • TREM-2 for macrophage remodeling in obese adipose tissue.
  • 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).
  • 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).
  • 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).
  • 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
  • Trem2 KO phenotype is immune specific
  • 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.
  • Trem2-/- chimeric mice featured increased body fat accumulation, higher LDL levels and glucose intolerance ( Figures 11A-G).
  • 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.
  • 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).
  • 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).
  • Figures 3E and 3F 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).
  • 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).
  • 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.
  • 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.
  • Trem2 agonist antibody reduced the amount of glucose in the blood for more than 120 minutes.

Abstract

L'invention concerne une méthode permettant de traiter ou de prévenir un trouble lié aux lipides chez un sujet nécessitant un tel traitement. La méthode consiste à administrer au sujet une quantité thérapeutiquement efficace d'un agent qui régule à la hausse le taux et/ou l'activité de déclenchement du récepteur exprimé sur les cellules myéloides 2 (TREM-2).
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