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WO2024263639A1 - Fragments de trem2 pour le traitement de la nafld, de la nash et de maladies et de troubles hépatiques - Google Patents

Fragments de trem2 pour le traitement de la nafld, de la nash et de maladies et de troubles hépatiques Download PDF

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WO2024263639A1
WO2024263639A1 PCT/US2024/034618 US2024034618W WO2024263639A1 WO 2024263639 A1 WO2024263639 A1 WO 2024263639A1 US 2024034618 W US2024034618 W US 2024034618W WO 2024263639 A1 WO2024263639 A1 WO 2024263639A1
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strem2
nash
vector
trem2
sequence
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Debanjan Dhar
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University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/055—Fusion polypeptide containing a localisation/targetting motif containing a signal for localisation to secretory granules (for exocytosis)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • NAFLD Non-alcoholic Fatty Liver Disease
  • NASH may progress to cirrhosis and liver failure. NASH is a major risk factor for hepatocellular carcinoma (HCC), the 2nd leading cause of cancer deaths worldwide. NASH is also the second leading cause of liver transplants in the US, making it a significant driver of healthcare costs. Apart from lifestyle changes and bariatric surgery there are no approved treatments for NASH.
  • HCC hepatocellular carcinoma
  • TAM Triggering Receptor Expressed on Myeloid cells
  • sTREM2 soluble form
  • DAP12 DAP12
  • sTREM2 soluble form
  • the disclosure provides novel evidence that sTREM2 protects the liver from NASH/fibrosis development and therefore, can be developed as a therapeutic molecule.
  • the disclosure demonstrates that introduction of sTREM2 in mice (using AAV8 gene therapy vector) that already has advanced NASH and fibrosis significantly improved NASH pathology (therapeutic intervention).
  • the disclosure provides for methods of treatment of NASH, fibrosis and HCC patients with recombinant sTREM2 or with sTREM2 expressing viral and non-viral vectors can be used as a therapeutic and preventative approach.
  • the disclosure provides a pharmaceutical composition comprising an extracellular fragment of TREM2 that binds a ligand selected from the group consisting of phospholipids, lipopolysaccharide (LPS), apoptotic cell components, and lipoproteins, and a pharmaceutically acceptable carrier.
  • the extracellular domain is a soluble fragment or TREM2.
  • the soluble fragment is about 130 to 155 amino acids in length and is at least 80% identical to SEQ ID NO:2 from amino acid 19 to 157.
  • the composition is formulated for parenteral delivery.
  • the composition comprises and enteric coating such that it is delivered to the small intestine.
  • the composition is formulated for delivery to the hepatic artery or hepatic portal vein.
  • the disclosure also provides an isolated polynucleotide encoding a soluble polypeptide having a sequence that is about 130 to 155 amino acids in length and comprises a sequence that is at least 80% identical to SEQ ID NO:2 from amino acid 19 to 157.
  • the polynucleotide comprises coding sequence for a secretory signal sequence operably linked to the 5’ end of the polynucleotide.
  • the secretory signal sequence comprises amino acid 1-18 of SEQ ID NO:2 or a sequence Attorney Docket No.00015-428WO1 that is 80% identical thereto.
  • the polynucleotide comprises or consist of a sequence that 80% identical to SEQ ID NO:1 from nucleotide 55 to 471.
  • the disclosure also provides a recombinant host cell comprising the polynucleotide [0010]
  • the disclosure also provides a vector comprising a polynucleotide of the disclosure.
  • the vector is an expression vector.
  • the vector is a viral vector.
  • the viral vector comprises a retroviral vector.
  • the viral vector is an adeno-associated viral (AAV) vector.
  • the AAV vector is AAV8.
  • the disclosure also provide a recombinant host cell comprising a vector of the disclosure.
  • the disclosure also provides a method of producing an sTREM2 polypeptide comprising culturing a host cell of the disclosure under condition such that the polynucleotide is expressed and isolating an sTREM2 polypeptide.
  • the disclosure also provides a method of treating a subject suffering from a disease or disorder selected from the group consisting of alcoholic liver disease (ALD), non-alcoholic fatty liver (NAFL), cirrhosis, non-alcoholic steatohepatitis (NASH), liver cancer, and liver fibrosis comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the disclosure or a polynucleotide of the disclosure or a vector containing the polynucleotide of the disclosure.
  • the disease or disorder is a fatty liver disease.
  • the fatty liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolman’s disease, acute fatty liver of pregnancy, and lipodystrophy.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • fatty liver disease resulting from obesity fatty liver disease resulting from diabetes
  • fatty liver disease resulting from insulin resistance fatty liver disease resulting from hypertriglyceridemia
  • Abetalipoproteinemia glycogen storage diseases
  • Weber-Christian disease Wolman’s disease
  • acute fatty liver of pregnancy and lipodystrophy.
  • the disclosure also provides a method of reducing fibrosis or fat content or fat accumulation in the liver associated with non-alcoholic fatty liver disease (NAFLD) comprising administering a therapeutically effective amount of a pharmaceutical composition, polynucleotide or vector of the disclosure.
  • NALFD comprises NASH.
  • the disclosure also provide a method for treating fatty liver disease in a patient in need thereof, the method comprising administering an effective amount of an sTREM2 polypeptide or oligomer thereof.
  • the sTREM2 polypeptide is selected from the group consisting of (i) an extracellular domain of TREM2; (ii) a polypeptide having a sequence from about amino acid 19-157 of SEQ ID NO:2; (iii) a fragment of a polypeptide having a sequence of SEQ ID NO:2 from about amino acid 19-174 that can (a) bind to a ligand such as LPS and/or (b) inhibit the progression of liver disease in a subject or mouse model; and (iv) a polypeptide of any one of (i)-(iii) that is at least 70% identical to SEQ ID NO:2 from amino acid 19-157 and which (a) bind to a ligand such as LPS and/or (b) inhibit the progression of liver disease in a subject or mouse model.
  • the sTRREM2 polypeptide is a fusion protein comprising a constant domain of an immunoglobulin.
  • the sTREM2 polypeptide is fused to an Fc portion of an immunoglobulin.
  • the immunoglobulin is a human IgGl.
  • RNA isolated from the liver of indicated mice was subjected to qRT-PCR analyses for Trem2.
  • B-D TREM2 expression analyses in human samples.
  • C Majority of TREM2+ cells coexpress Cd11b marker and concentrate near fibrotic scar.
  • D Healthy and NASH liver samples (with F1 and F3 fibrosis) were co-stained with anti-Type-1 Collagen (Red) and TREM2 (Green) antibodies. Images were captured with Zeiss 880 airyscan confocal microscope.
  • Figure 3A-D shows differences in ‘gene expression’ in TREM2+ vs TREM2-M ⁇ in NASH livers.
  • A-C scRNA-seq analyses of purified mouse NPCs.
  • C Cartoon summarizing the pathways enriched in genes which are positively correlated (Red) or negatively correlated (Blue) with Trem2 in Foz NASH M ⁇ . Only significant pathways were considered (adj.
  • TREM2 deficient M ⁇ have functional defects in phagocytosis.
  • Phagocytosis activity in WT and Trem2 BMDM ( ⁇ LPS) was analyzed in Incucyte system (a real-time live-cell imaging and Attorney Docket No.00015-428WO1 analysis platform) using pHrodo green E. coli particles. Phagocytosis was quantified every 30 min for a total time of 12hr and plotted as mean intensity of green object as a measure of phagocytic activity. Cells treated with cytochalasin D, an inhibitor of actin polymerization, is the negative control.
  • Figure 4A-D shows Trem2 deletion enhances NASH and HCC.
  • A-B Foz/Foz and Foz::Trem2 mice were fed WD for 8-12w and livers were analyzed as indicated.
  • A Sirius red stain and quantification for liver fibrosis.
  • B qRT-PCR of indicated fibrogenic genes.
  • C WT and Trem2 mice were fed WD and 30% fructose in drinking water for 16w. Liver fibrosis was measured by sirius red stain and quantified.
  • D Trem2 mice progressed to HCC by 34w on WD+fructose diet. Liver photographs showing visible tumors. Bar graphs indicate tumor incidence, average number of tumors/liver and the diameter of the largest tumor.
  • FIG. 5A-D shows TREM2 regulates NLRP3 inflammasome in M ⁇ .
  • A Livers from Foz+WD 8w and Foz::Trem2 +WD were subjected to immunoblot analyses with the indicated antibodies.
  • B, C WT and Trem2 BMDMs were isolated and key signaling events were analyzed after stimulation with LPS and NLRP3 activators.
  • B Schematics of the experimental design.
  • C WT and Trem2 BMDMs were either stimulated with LPS alone or LPS+ATP to activate inflammasome. Cell lysates were subjected immunoblot analyses with the indicated antibodies.
  • D Media supernatant was subjected to IL1 ⁇ ELISA.
  • FIG. 6A-C shows TREM2 Shedding.
  • A Schematic representation of TREM2/DAP12 intracellular signaling. Upon ligand binding, the extracellular domain of TREM2 is cleaved by ADAM proteases which is released into circulation.
  • B-C Systemic sTREM2 increases with NASH.
  • B sTREM2 was detected by ELISA in the plasma of indicated mice.
  • Figure 7A-H shows sTREM2 expression reverses NASH and liver fibrosis.
  • Trem2 NASH mice that are already fed Fructose+WD for 6m were injected with either vehicle or AAV8-sTREM2 (1x10 gc/Kg). Tissues were harvested 2 weeks later.
  • E sTREM2 ELISA showing successful and sustained release of sTREM2 into circulation.
  • F Serum ALT was measured.
  • G FFPE liver sections were subjected to H&E and Sirius red stain. Bar graphs showing steatosis and sirius red quantification by Image J software.
  • H Total RNA was extracted from the livers and QRTPCR was performed for the indicated NASH, fibrosis and HCC markers. WT+Chow liver tissues were analyzed to show baseline gene expression.
  • FIG 8A-C shows TREM2::DAP12 Chimeric Receptors.
  • A Schematics of various TREM2::DAP12 chimera.
  • B Retroviral vector map that express the chimeric proteins along with GFP marker.
  • C DAP12 deleted RAW cells expressing either TREM2::DAP12 chimera or not were stimulated with either anti-TREM2 agonistic antibody or IgG control antibody for 10min. The cell lysates were subjected to immunoblot analysis with indicated antibodies.
  • Figure 9A-B shows NASH in a dish spheroid culture.
  • FIG. 12 shows that sTREM2 improves LPS septic shock survival.
  • Figure 13 provides an AAV construct (SEQ ID NO:3) containing a murine sTREM2 domain at nucleotides 648-1139.
  • the murine domain can be replaced with a human domain as provided in SEQ ID NO:2.
  • Figure 14 shows that Trem2 cleavage is pronounced in NASH compared to ALD.
  • Figure 15 shows that the absence of Trem2 leads to pronounced steatosis and liver damage as indicated by an increase in ALT levels and fibrogenic markers compared to pair fed mice. This supports the hypothesis that sTREM2 treatment can improve ALD pathogenesis.
  • Figure 16 demonstrates that exogenous sTREM2 improves ALD. Utilizing the modified Leiber Decarli model to administer alcohol to mice injected with and AAV8-strem2 vector and control AAV8-gfp vector.
  • M ⁇ macrophage
  • TREM Triggering Receptor Expressed on Myeloid cells
  • TREMs bind ligands, but lack downstream signaling components.
  • TREMs associate with TYRO Protein Tyrosine Kinase Binding Protein (TyroBP), also known as DAP12, that contains an immuno receptor tyrosine-based activation motif (ITAM).
  • TyroBP TYRO Protein Tyrosine Kinase Binding Protein
  • DAP12 TYRO Protein Tyrosine Kinase Binding Protein
  • ITAM immuno receptor tyrosine-based activation motif
  • Src kinases phosphorylate the tyrosine residues in the DAP12 ITAM, forming a 'docking site' for the protein tyrosine kinase Syk, thereby activating a downstream signaling cascade.
  • TREM-2 ligands include various phospholipids, Attorney Docket No.00015-428WO1 lipopolysaccharide (LPS), apoptotic cell components, and lipoproteins, that may be crucial in NAFL to NASH progression.
  • LPS lipopolysaccharide
  • the disclosure demonstrates the upregulation of TREM2 (but not TREM1 and TREM3) and DAP12 in both mouse and human NASH livers. Even though liver TREM2 expression increases during NASH and metabolic disorders its role has remained unclear.
  • Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed by myeloid cells such as macrophages, microglia, dendritic cells, and osteoclasts.
  • TREM2 is a transmembrane receptor that contains binding domain for a ligand in the extracellular region and a cytoplasmic domain that lacks signal transduction domain(s).
  • Ligands of TREM2 encompass a group of molecules, such as LDL and apolipoprotein E (apoE) and lipids.
  • apoE apolipoprotein E
  • TREM2 has been shown to be cleaved in certain circumstances releasing a soluble extracellular domain (sTREM2).
  • sTREM2 soluble extracellular domain
  • the role of sTREM2 in the pathogenesis of liver disease including alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) is being investigated by a number of groups.
  • TREM2 is about 230 amino acids in length and includes 3 domain: an extracellular binding domain from about amino acid 19-174, a transmembrane domain from about amino acids 175 to 195 and a cytoplasmic domain from about amino acid 196 to 230 of SEQ ID NO:2.
  • the disclosure provides recombinant soluble domains or fragments of TREM2.
  • a TREM2 fragments is a soluble sTREM2 polypeptides comprising all or part of the extracellular domain, but lacking the transmembrane region that would cause retention of the polypeptide on a cell membrane.
  • Soluble sTREM2 polypeptides are capable of being secreted from the cells in which they are expressed.
  • a heterologous signal peptide is fused to the N-terminus such that the soluble sTREM2 is secreted upon expression.
  • Such soluble domains can Attorney Docket No.00015-428WO1 comprise or consist of amino acids 19-174 or any fragment thereof that can bind to a ligand such as LPS and/or inhibit the progression of liver disease in a subject or mouse model.
  • sTREM2 refers to (i) an extracellular domain of TREM2; (ii) a polypeptide having a sequence from about amino acid 19-174 (e.g., 19 to 157, to 158, to 159, to 160, to 161, to 162, to 163, to 164, to 165, to 166, to 167, to 168, to 169, to 170, to 171, to 172, to 173, to 174, or 19 to 175) of SEQ ID NO:2; (iii) a fragment of a polypeptide having a sequence of SEQ ID NO:2 from about 19-174 that can (a) bind to a ligand such as LPS and/or (b) inhibit the progression of liver disease in a subject or mouse model; and (iv) a polypeptide of any one of (i)-(iii) that is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO:2
  • an sTREM2 polypeptide comprises an enzymatic product resulting from treating TREM2 with ADAM10 and/or ADAM17.
  • sTREM2 fragments including soluble polypeptides, may be prepared by any of a number of conventional techniques.
  • a DNA sequence encoding a desired sTREM2 fragment may be subcloned into an expression vector for production of the sTREM2 fragment.
  • the sTREM2-encoding DNA sequence advantageously is fused to a sequence encoding a suitable leader or signal peptide.
  • the desired sTREM2- encoding DNA fragment may be chemically synthesized using known techniques.
  • DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels. If necessary, oligonucleotides that reconstruct the 5' or 3' terminus to a desired point may be ligated to a DNA fragment generated by restriction enzyme digestion. Such oligonucleotides may additionally contain a restriction endonuclease cleavage site upstream of the desired coding sequence, and position an initiation codon (ATG) at the N-terminus of the coding sequence.
  • TREM2 coding sequence SEQ ID NO:1
  • PCR polymerase chain reaction
  • Oligonucleotides that define the desired termini of the DNA fragment are employed as 5' and 3' primers.
  • the oligonucleotides may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified DNA fragment into an expression vector.
  • transmembrane region of TREM2 discussed above is identified in accordance with conventional criteria for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane region may vary slightly (most likely by no more than five amino acids on either end) from those presented above.
  • Certain embodiments of the disclosure provide isolated DNA comprising a nucleotide sequence selected from the group consisting of nucleotides 1 to 693 of SEQ ID NO:1 (human TREM2 coding region); nucleotides 55 to 522 of SEQ ID NO:1 (encoding the human sTREM2 extracellular domain). DNAs encoding biologically active fragments of the proteins of SEQ ID NOS:2 are also provided. [0048] Also provided herein are purified sTREM2 polypeptides, both recombinant and non-recombinant. Variants and derivatives of sTREM2 proteins that retain a desired biological activity are within the scope of the disclosure.
  • the biological activity of an sTREM2 variant is essentially equivalent to the biological activity of a native extracellular sTREM2 domain.
  • One desired biological activity of sTREM2 is the ability to bind LPS.
  • sTREM2 variants may be obtained by mutagenesis.
  • a sTREM2 variant, as referred to herein, is a polypeptide substantially homologous to SEQ ID NO:2 from amino acid 19 to 174, but which has an amino acid sequence that differs due to one or more of deletions, insertions or substitutions.
  • Attorney Docket No.00015-428WO1 [0050] Alterations of the native amino acid sequence may be accomplished by any of a number of known techniques.
  • Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.
  • Variants may comprise conservatively substituted sequences, meaning that one or more amino acid residues of a wild- type sTREM2 polypeptide are replaced by different residues, but that the conservatively substituted sTREM2 polypeptide retains a desired biological activity that is essentially equivalent to that of a wild-type sTREM2 polypeptide.
  • conservative substitutions include substitution of amino acids that do not alter the secondary and/or tertiary structure of sTREM2.
  • Other examples involve substitution of amino acids outside of the ligand binding domain.
  • a given amino acid may be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn).
  • substituting one aliphatic residue for another such as Ile, Val, Leu, or Ala for one another
  • substitution of one polar residue for another such as between Lys and Arg; Glu and Asp; or Gln and Asn.
  • Other such conservative substitutions e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • sTREM2 polypeptides comprising conservative amino acid substitutions may be tested in one of the assays described herein to confirm that a desired biological activity is retained.
  • sTREM2 polypeptides may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like.
  • Covalent derivatives of sTREM2 may be prepared by linking the chemical moieties to functional groups on sTREM2 amino acid side chains or at the N-terminus or C-terminus of a sTREM2 polypeptide or the extracellular domain thereof.
  • sTREM2 within the scope of this disclosure include covalent or aggregative conjugates of sTREM2 or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N- terminal or C-terminal fusions.
  • the conjugate may comprise a signal or leader polypeptide sequence.
  • the disclosure also contemplates sTREM2 polypeptide fusions that can be used to facilitate purification of sTREM2.
  • Such peptides include, for example, poly-His or the FLAG peptide which can be used for purification by a specific monoclonal antibody, thus enabling rapid assay and facile purification of expressed recombinant protein.
  • the disclosure further includes sTREM2 polypeptides with or without associated native-pattern glycosylation.
  • sTREM2 expressed in yeast or mammalian expression systems may be similar to or significantly different from a native sTREM2 polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system.
  • Expression of sTREM2 polypeptides in bacterial expression systems, such as E. coli, provides non- glycosylated molecules.
  • Glycosylation sites in the sTREM2 extracellular domain can be modified to preclude glycosylation while allowing expression of a homogeneous, reduced carbohydrate analog using yeast or mammalian expression systems.
  • N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr.
  • Appropriate modifications to the nucleotide sequence encoding this triplet will result in substitutions, additions or deletions that prevent attachment of carbohydrate residues at the Asn side chain.
  • Attorney Docket No.00015-428WO1 Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Pat. No. 5,071,972 and EP 276,846.
  • sTREM2 polypeptides in the form of oligomers, such as dimers, trimers, or higher oligomers. Oligomers may be formed by disulfide bonds between cysteine residues on different sTREM2 polypeptides, or by non- covalent interactions between sTREM2 polypeptide chains, for example. In other embodiments, oligomers comprise from two to four sTREM2 polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the sTREM2 polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization.
  • Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of sTREM2 polypeptides attached thereto, as described in more detail below.
  • the sTREM2 polypeptide oligomers are soluble. [0058] Preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al.
  • an sTREM2 dimer is created by fusing sTREM2 to an Fc region polypeptide derived from an antibody.
  • the term "Fc polypeptide” includes native and mutein forms, as well as truncated Fc polypeptides containing the hinge region that promotes dimerization.
  • the Fc polypeptide typically is fused to a soluble sTREM2 (e.g., comprising only the extracellular domain).
  • a gene fusion encoding the sTREM2/Fc fusion protein is inserted into an appropriate expression vector.
  • the Fc polypeptide is fused to the N-terminus of a soluble sTREM2 polypeptide.
  • the sTREM2/Fc fusion proteins are allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc polypeptides, yielding divalent sTREM2.
  • sTREM2 may be substituted for the variable Attorney Docket No.00015-428WO1 portion of an antibody heavy or light chain.
  • Fc polypeptide is the native Fc region polypeptide derived from a human IgG1, which is described in PCT application WO 93/10151, hereby incorporated by reference.
  • Another useful Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035.
  • oligomeric sTREM2 may comprise two or more soluble sTREM2 polypeptides joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Fusion proteins comprising multiple sTREM2 polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
  • Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found.
  • Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different proteins.
  • leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
  • the zipper domain (also referred to herein as an oligomerizing, or oligomer-forming, domain) comprises a repetitive heptan repeat, often with four or five leucine residues interspersed with other amino acids.
  • Examples of zipper domains are those found in the yeast transcription factor GCN4 and a heat- stable DNA-binding protein found in rat liver (C/EBP; Landschulz et al., Science 243:1681, 1989).
  • the fusogenic proteins of several Attorney Docket No.00015-428WO1 different viruses including paramyxovirus, coronavirus, measles virus and many retroviruses, also possess zipper domains (Buckland and Wild, Nature 338:547,1989; Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and Human Retrovirtises 6:703, 1990).
  • the zipper domains in these fusogenic viral proteins are near the transmembrane region of the proteins; it has been suggested that the zipper domains could contribute to the oligomeric structure of the fusogenic proteins. Zipper domains fold as short, parallel coiled coils.
  • the disclosure provides recombinant expression vectors for expression of sTREM2, and host cells transformed with the expression vectors.
  • vector can refer to a nucleic acid construct deigned for transfer between different hosts, including but not limited to a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc. Any suitable expression system may be employed.
  • the vectors include a DNA encoding a sTREM2 polypeptide, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene.
  • suitable transcriptional or translational regulatory nucleotide sequences such as those derived from a mammalian, microbial, viral, or insect gene.
  • regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination.
  • Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the sTREM2 DNA sequence.
  • a promoter nucleotide sequence is operably linked to an sTREM2 DNA sequence if the promoter nucleotide sequence controls the transcription of the sTREM2 DNA sequence.
  • An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.
  • a sequence encoding an appropriate signal peptide can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide secretory leader
  • a signal peptide that is functional in the intended host cells promotes extracellular secretion of the sTREM2 polypeptide.
  • sTREM2 polypeptide is cleaved from the sTREM2 polypeptide upon secretion of sTREM2 from the cell.
  • Suitable host cells for expression of sTREM2 polypeptides include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-free translation systems could also be employed to produce sTREM2 polypeptides using RNAs derived from DNA constructs disclosed herein.
  • Prokaryotes include gram negative or gram positive organisms, for example, E. coli or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • a sTREM2 polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant STREM2 polypeptide.
  • Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes.
  • a phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement.
  • useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.
  • An appropriate promoter and a STREM2 DNA sequence are inserted into the pBR322 vector.
  • Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include beta-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res.
  • a particularly useful prokaryotic host cell expression system employs a phage lambda P promoter and a cI857ts thermolabile repressor sequence.
  • Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the lambda P promoter include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).
  • sTREM2 alternatively may be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveroinyces, may also be employed.
  • yeast vectors will often contain an origin of replication sequence from a 2 ⁇ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem.
  • glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho-glucose isomerase, and glucokinase.
  • yeast alpha-factor leader sequence may be employed to direct secretion of the sTREM2 polypeptide.
  • the alpha-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984.
  • Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art.
  • a leader sequence may be modified near its 3' end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
  • Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.
  • suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CVI/EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991). [0074] CHO cells are useful as host cells.
  • DX-B 11 is the cell line designated DX-B 11, which is deficient in dihydrofolate reductase (DHFR), as described in Urlaub and Chasin (Proc. Natl. Acad. Sci USA 77:4216-4220, 1980), hereby incorporated by reference.
  • DX-B 11 cells may be transformed with expression vectors that encode DHFR, which serves as a selectable marker (Kauffman et al., Meth. in Enzymology, 185:487- 511, 1990).
  • the host cells are CHO cells that can be grown in suspension culture, and that are adapted to grow in media that does not contain serum.
  • the cells may be further adapted to grow in media lacking insulin-like growth factor (IGF-1) and/or transferrin.
  • IGF-1 insulin-like growth factor
  • transferrin insulin-like growth factor
  • the host cells may be adapted to grow in media that does not contain any exogenous growth factors that are animal proteins.
  • Such CHO cell lines may be generated by any suitable procedure. One such procedure is conducted generally as follows.
  • DX-B 11 cells are adapted to growth in serum free medium by a gradual reduction of serum supplementation in the media, and replacement of serum with low levels of the growth factors transferrin and insulin-like growth factor (IGF-1), in an enriched cell growth media.
  • Cells adapted to serum-free medium then are weaned off transferrin and insulin-like growth factor-1.
  • the resulting CHO cells maintain vigorous growth and high viability, as well as a DHFR-deficient phenotype, in serum-free, essentially protein-free, media.
  • Transformed host cells provided herein include, but are not limited to, host cells in which heterologous DNA, including a STREM2-encoding sequence, is inserted into the cell's genomic DNA.
  • Transcriptional and translational control sequences for mammalian host cell expression vectors may be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.
  • SV40 Simian Virus 40
  • DNA sequences derived from the SV40 viral genome may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell.
  • Viral early and late Attorney Docket No.00015-428WO1 promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
  • Expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983), for example.
  • a useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986).
  • a high expression vector, PMLSV N1/N4 described by Cosman et al., Nature 312:768, 1984 has been deposited as ATCC 39890. Additional mammalian expression vectors are described in EP-A-0367566, and in WO 91/18982.
  • the vector may be derived from a retrovirus.
  • viral vector is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • plasmid vectors can be prepared from commercially available vectors.
  • viral vectors can be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art.
  • the viral vector is a lentiviral vector.
  • viral vectors examples include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
  • Infectious tobacco mosaic virus (TMV)-based vectors can be used to manufacturer proteins (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104).
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med.
  • a vector construct can refer to the polynucleotide comprising the retroviral genome or part thereof, and a gene of interest. Further details as to modern methods of vectors for use in gene transfer can be found in, for example, Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art.
  • AAV adeno-associated virus
  • Non-limiting exemplary serotypes useful for the purposes disclosed herein include any of the 11 serotypes, e.g., AAV2 and AAV8.
  • the term “lentivirus” as used herein refers to a member of the class of viruses associated with this name and belonging to the genus lentivirus, family Retroviridae. While some lentiviruses are known to cause diseases, other lentivirus are known to be suitable for gene delivery. See, e.g., Tolich et al. (2013) Biochemistry, Genetics and Molecular Biology: “Gene Therapy – Tools and Potential Applications,” ISBN 978-953-51-1014-9, DOI: 10.5772/52534.
  • a method for producing a heterologous recombinant protein involves culturing mammalian host cells transformed with an expression vector of the disclosure under conditions that promote expression and secretion of the heterologous protein, and recovering the protein from the culture medium.
  • a particularly useful signal peptide for expression of STREM2 polypeptides is a signal peptide derived from a growth hormone gene.
  • Attorney Docket No.00015-428WO1 [0084]
  • the disclosure provides purified sTREM2 polypeptides, which may be produced by recombinant expression systems as described above or purified from naturally occurring cells. The desired degree of purity may depend on the intended use of the polypeptide. A relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example.
  • One process for producing the sTREM2 polypeptide comprises culturing a host cell transformed with an expression vector comprising a polynucleotide sequence that encodes sTREM2 under conditions such that sTREM2 is expressed.
  • the sTREM2 polypeptide is then recovered from the culture (from the culture medium or cell extracts).
  • procedures for purifying the recombinant sTREM2 will vary according to such factors as the type of host cells employed and whether or not the sTREM2 is secreted into the culture medium.
  • the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a purification matrix such as a gel filtration medium.
  • an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
  • a cation exchange step can be employed.
  • Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • One or more reversed-phase high performance liquid chromatography (RP- LIPLC) steps employing hydrophobic RP-HPLC media (e.g., silica gel having pendant methyl or other aliphatic groups) can be employed to further purify sTREM2.
  • RP- LIPLC reversed-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • the disclosure provides methods of treating a liver disease selected from ALD, NAFL and NASH comprising administering an sTREM2 polypeptide or pharmaceutical composition comprising Attorney Docket No.00015-428WO1 sTREM2 to a subject having the liver disease.
  • the pharmaceutical composition comprising sTREM2 or sTREM2 polypeptide is administered in a therapeutically effective amount such that the amount reduces markers of liver diseases to a normal control level.
  • the level of sTREM2 is administered to obtain a plasma level of about 50-400 ng/ml.
  • the level of sTREM2 should be administered to reach a level greater than the level of sTREM2 in a NASH subject to treat NASH or greater than a level of sTREM2 in a subject that has ALD (e.g., greater than about 50 ng/ml).
  • the disclosure provides a method to treat a liver disease or disorder selected from the group consisting of ALD, NAFL or NASH comprising administering a vector that can transform a cell in vivo or ex vivo (e.g., a hepatic cell), wherein the vector comprising an sTREM2 coding sequence, such that sTREM2 is expressed in the cell.
  • the vector is a AAV vector.
  • the vector comprises a polynucleotide sequence encoding a secretable sTREM2 polypeptide (e.g., an sTREM2 polypeptide operably linked to a signal sequence).
  • a secretable sTREM2 polypeptide e.g., an sTREM2 polypeptide operably linked to a signal sequence.
  • TREM2 is upregulated in NASH associated liver M ⁇ , it is not clear at what stage during the disease progression TREM2 expression gets triggered and what transcriptional factors regulate its expression.
  • TREM2 expression is induced at the onset of liver damage (Fig. 2A).
  • sn ATACseq transcription factors are identified that Attorney Docket No.00015-428WO1 regulate TREM2 expression during NASH development. Since TREM2 is NASH protective, identification of these transcription factors and ways to activate them pave the way for future drug development.
  • sTREM2 Upon ligand binding TREM2 is cleaved by ADAM10/17 metalloproteinases producing a soluble form (sTREM2) that is released into circulation (Fig. 1A, B). Serum sTREM2 levels increase during NASH progression in both mouse models and human patients. Although sTREM2 can be a potential non-invasive biomarker for NASH, the signaling properties of sTREM2 remains obscure.
  • Membrane bound TREM2 can transmit a protective intracellular signal via DAP12, however, it is not known whether sTREM2 plays any functional role in NASH and HCC pathogenesis.
  • the data presented herein (e.g., Fig. 7) provide evidence that sTREM2 protects the liver from NASH/fibrosis development and therefore, can be developed as a therapeutic molecule.
  • the current dogma in the TREM2 field is that since TREM2 intracellular signaling is protective, the cleavage of the extracellular ligand-binding domain of TREM2 terminates the protective intra-cellular signaling. Therefore, there are ongoing efforts to develop anti-TREM2 antibodies that prevent TREM2 shedding to prolong TREM2 intracellular signaling.
  • TREM2 cleavage provides a distinct trans-signaling axis of TREM2 that also exerts a protective function.
  • Studies on Alzheimer's disease also indicate potential signaling abilities of cleaved TREM2.
  • the experiments presented herein show that introduction of sTREM2 in mice that already have advanced NASH and fibrosis (Fig. 7) significantly improved NASH pathology demonstrating the therapeutic benefits of sTREM2.
  • sTREM2 also induced fibrotic scar resorption in advanced NASH livers.
  • Translational studies show that sTREM2 can be used to treat NASH and fibrosis and prevent subsequent progression to HCC.
  • the disclosure provides strong preclinical data that will guide the development of sTREM2 as a novel and unconventional therapeutic approach for NASH/HCC.
  • the disclosure contemplate administration of soluble/extracellular domains of TREM2 polypeptides as a therapeutic for the treatment of liver diseases and disorders.
  • the disclosure contemplates delivery of a vector encoding an sTREM2 domain such that the vector integrates into a liver cell or other suitable cell type and produces sTREM2.
  • the term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.
  • active ingredient refers to a compound or biological agent, which is administered, alone or in combination with one or more pharmaceutically acceptable excipients or carriers, to a subject for treating, preventing, or ameliorating one or more symptoms of a disorder.
  • agent as used herein generally refers to a biological agent used in the methods and compositions of the disclosure.
  • an agent can refer to a sTREM2 polypeptide, polynucleotide or a vector encoding an sTREM2 polypeptide or containing a polynucleotide encoding an sTREM2 polypeptide.
  • X and Y represent numbers. Unless otherwise specified, this notation is intended to include not only the numbers represented by X and Y themselves, but all the numbered positions that are bounded by X and y. For example, “from 1 to 4” or “1-4” would, unless otherwise specified, be equivalent to 1, 2, 3, and 4.
  • the term “combination therapy’ means the administration of two or more therapeutic agents to treat a therapeutic disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules or administration steps for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner. In either case, the Attorney Docket No.00015-428WO1 treatment regimen will provide beneficial effects of the drug combination in treating the disorders described herein.
  • disorder as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disease”, “syndrome”, and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms.
  • the disorder is alcoholic liver disease (ALD).
  • the disorder is non-alcoholic fatty liver (NAFL).
  • NAFL non-alcoholic fatty liver
  • NASH non-alcoholic steatohepatitis
  • the disorder is liver fibrosis and/or inflammation.
  • Homology or “identity” or “similarity” can refer to sequence similarity between two peptides/polypeptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated” or “non- homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the disclosure.
  • Homology refer to a % identity of a sequence to a reference sequence.
  • any particular sequence can be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described herein (which can correspond with a particular nucleic acid sequence described herein)
  • such particular polypeptide sequence can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711).
  • the parameters can be set such that the percentage of identity is calculated over the full length of the reference sequence and that gaps in homology of up to 5% of the total reference sequence are allowed.
  • identity between a reference sequence query sequence, i.e., a sequence of the disclosure
  • subject sequence also referred to as a global sequence alignment
  • FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity can be corrected by calculating the number of residues of the query sequence that are lateral to the N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue is matched/aligned can be determined by results of the FASTDB sequence alignment. This percentage can be then subtracted from the percent identity, calculated by the FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, Attorney Docket No.00015-428WO1 only query residue positions outside the farthest N- and C-terminal residues of the subject sequence are considered for this manual correction.
  • a 90 residue subject sequence can be aligned with a 100 residue query sequence to determine percent identity.
  • the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity can be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence.
  • Hybridization can refer to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex can comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about 10x SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
  • Examples of moderate hybridization Attorney Docket No.00015-428WO1 conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
  • Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, 0.1x SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
  • "Inhibit” as used herein refers to the ability to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, alter, eliminate, stop, or reverse the progression or severity of the activity of a particular agent (e.g., inhibiting advancement of a disorder or symptom thereof) or disease.
  • pharmaceutically acceptable carrier refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or Solid filler, diluent, excipient, solvent, or encapsulating material.
  • pharmaceutically-acceptable material such as a liquid or Solid filler, diluent, excipient, solvent, or encapsulating material.
  • Each component must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, Remington.
  • the subunits can be linked by peptide bonds. In another embodiment, the subunit can be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide can contain at least two amino acids and no limitation is placed on the maximum number of amino acids which can comprise a protein's or peptide's sequence.
  • amino acid can refer to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • fusion protein can refer to a protein comprised of domains from more than one naturally occurring or recombinantly produced protein, where generally each domain serves a different function.
  • linker can refer to a protein fragment that is used to link these domains together - optionally to preserve the conformation of the fused protein domains and/or prevent unfavorable interactions between the fused protein domains which can compromise their respective functions.
  • exemplary sTREM2 polypeptide sequences e.g., human and mouse
  • polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three dimensional structure and can perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or Attorney Docket No.00015-428WO1 after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also can refer to both double and single stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double stranded form and each of two complementary single stranded forms known or predicted to make up the double stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • the polynucleotide can comprise one or more other nucleotide bases.
  • the term "polynucleotide sequence” or “polypeptide sequence” is the alphabetical representation of a polynucleotide molecule or polypeptide molecule, respectively.
  • This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • the terms “prevent”, “preventing”, and “prevention” refer to a method of delaying or precluding the onset of a disorder, and/or its attendant symptoms, barring a subject from acquiring a disorder or reducing a subject's risk of acquiring a disorder.
  • subject refers to an animal, including, but not limited to, a primate (e.g., human, monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, and the like), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, and the like.
  • a primate e.g., human, monkey, chimpanzee, gorilla, and the like
  • rodents e.g., rats, mice, gerbils, hamsters, ferrets, and the like
  • lagomorphs e.g., pig, miniature pig
  • swine e.g., pig, miniature pig
  • equine canine
  • feline feline
  • the term “substantially” as used herein refers to a majority of, or mostly, as in at least about 51%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.
  • Attorney Docket No.00015-428WO1 [00115]
  • the term “therapeutically acceptable” refers to those compounds/polypeptides or biological agents (or salts, prodrugs, tautomers, Zwitterionic forms, etc.) which are suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, immunogenicity, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.
  • terapéuticaally effective amount refers to the amount of a compound, polypeptide or biological agent that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder being treated.
  • therapeutically effective amount also refers to the amount of a compound, polypeptide or biological agent that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, Veterinarian, medical doctor, or clinician.
  • the terms “treat”, “treating”, and “treatment” are meant to include alleviating or abrogating a disorder or one or more of the symptoms associated with a disorder; or alleviating or eradicating the cause(s) of the disorder itself.
  • compositions of the disclosure may be formulated as a modified release dosage form, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms.
  • These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see, Remington. The Science and Practice of Pharmacy, supra; Modified-Release Drug Delivery Technology, Rathbone et al., Eds. Drugs and the Pharmaceutical Science, Marcel Dekker, Inc.: New York, N.Y., 2002; Vol. 126).
  • compositions include those suitable for oral, parenteral (including Subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, Sublingual and intraocular) administration.
  • parenteral including Subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary
  • intraperitoneal transmucosal
  • transdermal rectal
  • topical including dermal, buccal, Sublingual and intraocular
  • the composition is administered to the hepatic artery or hepatic portal vein.
  • the most suitable route for administration depends on a variety of factors, including interpatient variation or disorder type, and therefore the disclosure is not limited to just one form of administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • these methods include the step of bringing into association a compound of the disclosure or a pharmaceutically salt, prodrug, or solvate thereof (“active ingredient”) with the carrier which constitutes one or more accessory ingredients.
  • active ingredient a compound of the disclosure or a pharmaceutically salt, prodrug, or solvate thereof
  • the carrier which constitutes one or more accessory ingredients.
  • the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • Formulations of the compounds, sTREM2 polypeptides, or biological agents (e.g., vectors) disclosed herein suitable for administration may be presented as discrete units such as capsules, cachets or tablets, vials comprising polypeptides or vectors, each containing a predetermined amount of the active ingredient; as a powder or granules (including enterically formulations); as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • compositions which can be used orally include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with binders, inert diluents, or lubricating, surface active or dispersing agents. Moulded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so Attorney Docket No.00015-428WO1 as to provide slow or controlled release of the active ingredient therein. All formulations for oral administration should be in dosages suitable for such administration.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or 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.
  • the polypeptides or vectors of the disclosure may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use.
  • sterile liquid carrier for example, saline or sterile pyrogen-free water
  • Formulations for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compounds which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic Attorney Docket No.00015-428WO1 with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or 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 compounds to allow for the preparation of highly concentrated solutions.
  • the polypeptides may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the polypeptides may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example as an emulsion in an acceptable oil
  • ion exchange resins for example, as a sparingly soluble salt.
  • compositions may be delivered from an insufflator, nebulizer pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the compositions according to the disclosure may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form, in for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.
  • Typical unit dosage formulations are those containing an effective dose, as herein below recited, or an appropriate fraction thereof, of the active ingredient.
  • Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of one or more agents which is effective at such dosage or as a multiple of the same, for instance, units containing 1 mg to 1000 mg of the polypeptide disclosed herein, usually around 100 mg to 500 mg of the compound.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • the polypeptides, vectors and/or compositions of the disclosure can be administered in various modes, e.g. orally or by injection. The precise amount to be administered to a patient will be the responsibility of the attendant physician.
  • the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, sex, diets, time of administration, route of administration, rate of excretion, drug combination, the precise disorder being treated, and the severity of the disorder being treated. Also, the route of administration may vary depending on the disorder and its severity. Generally, the amount of sTREM2 to be administered should provide an sTREM2 level in the blood greater than that of the disease/disorder to be treated (e.g., ALD, NAFL or NASH).
  • the administration of the agents may be administered chronically, that is, for an extended period of time, including throughout the duration of the patient’s life in order to ameliorate or otherwise control or limit the symptoms of the patient’s disorder.
  • the administration of the agents may be given continuously or temporarily suspended for a certain length of time (i.e., a "drug holiday'). Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary.
  • compositions and methods of the disclosure can also be used to treat NAFLD and NASH as well as liver fibrotic diseases.
  • NAFLD Non-alcoholic fatty liver disease
  • NAFLD also sometimes referred to as NAFL
  • NAFLD steatosis
  • steatosis fat in the liver
  • metabolic syndrome including obesity, diabetes and hypertriglyceridemia
  • NAFLD is linked to insulin resistance, it causes liver disease in adults and children and may ultimately lead to cirrhosis (Skelly et al., J Hepatol 2001; 35: 195-9; Chitturi et al., Hepatology 2002;35(2):373-9).
  • NAFLD nonalcoholic fatty liver or NAFL
  • NASH non-alcoholic steatohepatitis
  • Angulo et al. J Gastroenterol Hepatol 2002;17 Suppl:S186-90
  • NASH is characterized by the histologic presence of steatosis, cytological ballooning, scattered inflammation and pericellular fibrosis (Contos et al., Adv Anat Pathol 2002;9:37-51).
  • Hepatic fibrosis resulting from NASH may progress to cirrhosis of the liver or liver failure, and in some instances may lead to hepatocellular carcinoma.
  • the degree of insulin resistance correlates with the severity of NAFLD, being more pronounced in patients with NASH than with simple fatty liver (Sanyal et al., Gastroenterology 2001;120(5):1183-92).
  • insulin- mediated suppression of lipolysis occurs and levels of circulating fatty acids increase.
  • Two factors associated with NASH include insulin resistance and increased delivery of free fatty acids to the liver. Insulin blocks mitochondrial fatty acid oxidation. The increased generation of free fatty acids for hepatic re- esterification and oxidation results in accumulation of intrahepatic fat and increases the liver’s vulnerability to secondary insults.
  • triglyceride During periods of increased calorie intake, triglyceride will accumulate and act as a reserve energy source. When dietary calories are insufficient, stored triglycerides (in adipose) undergo lipolysis and fatty acids are released into the circulation and are taken up by the liver. Oxidation of fatty acids will yield energy for utilization.
  • Treatment of NASH currently revolves around the reduction of the two main pathogenetic factors, namely, fat accumulation within the liver and excessive accumulation of free radicals causing oxidative stress. Fat accumulation is diminished by reducing fat intake as well as increasing caloric expenditure. One therapeutic approach is sustained and steady weight loss.
  • ROS can be generated in the liver through several pathways including mitochondria, peroxisomes, cytochrome P450, NADPH oxidase and lipooxygenase (Sanyal et al., Nat Clin Pract Gastroenterol Hepatol, 2005;2(1):46-53). Insulin resistance and hyperinsulinism has been shown to increase hepatic oxidative stress and lipid peroxidation through increased hepatic CYP2EI activity (Robertson et al., Am J Physiol Gastrointest Liver Physiol, 2001 281(5):G1135-9; Leclercq et al., J Clin Invest 2000, 105(8):1067- 75).
  • the medical conditions most commonly associated with NAFLD are obesity, Type II diabetes and dyslipidemia. These conditions can be induced by feeding mice and rats with high fat or sucrose diets. Rats fed with a >70% fat-rich diet for 3 weeks developed pan-lobular steatosis, patchy inflammation, enhanced oxidative stress, and increased plasma insulin concentrations suggesting insulin resistance.
  • Mouse models of NASH can be used to study various therapies.
  • Mouse models are created through specific diets (methionine choline deficient, MCD) or intragastric overfeeding. These mice develop serologic and histologic features of NASH.
  • NASH mice are useful in screening and measuring the effects vectors or peptide compositions on NASH related disease and disorders. For example, the effect of treatment can be measured by separating the NASH mice into a control group where animals will continue to receive MCD diet only and three other treatment groups where mice will receive MCD diet as well as anti-oxidant therapy.
  • NASH is a disease subset falling under the umbrella of NAFLD and is characterized by various biomarkers and histological examination.
  • Type 1 NASH has been characterized as including two types: Type 1 and Type 2, having some distinct biomarker and histological characteristics, while certain others that overlap between the two types. These two types, Type 1 and Type 2 NASH are typically identified in juvenile patients.
  • Type 1 NASH is characterized by steatosis, lobular inflammation, ballooning degeneration and perisinusoidal fibrosis.
  • Type 2 NASH is characterized by steatosis, portal inflammation, and portal fibrosis.
  • Weg et al. (Hepatology, 42(3):641-649, 2005; incorporated herein by reference) described various criteria and biomarkers used to differentiate NASH Type 1 from NASH Type 2. In particular, Berger et al.
  • NASH Type 1 had higher AST, ALT and triglyceride levels compared to patients with NASH Type 2.
  • the strongest factor demonstrating a difference in the two types of NASH are best found upon histological examination.
  • Type 1 NASH demonstrates a prevalent lobular inflammation in the liver in Attorney Docket No.00015-428WO1 contrast with a prevalent portal inflammation in Type 2 NASH.
  • one of the key differentiating factors that can be used in the methods disclosed herein is identifying, by histological examination, the presence of Type 1 vs. Type 2 NASH.
  • the diagnosis of steatosis is typically made when lipid deposition is visible in more than 5% of hepatocytes.
  • NASH NAFLD Activity Score
  • the NAFLD Activity Score was developed to provide a numerical score for patients who most likely have NASH. Accordingly, the NAS is the sum of the separate scores for steatosis (0-3), hepatocellular ballooning (0-2) and lobular inflammation (0-3), with the majority of patients with NASH having a NAS score of ⁇ 5 (Kleiner DE, Brunt EM, Van Natta M et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41(6), 1313-1321 (2005)).
  • cytokeratin 18 is a useful indicator of inflammation in NASH, due to cytokeratin 18’s release from hepatocytes undergoing apoptosis.
  • Normal cytokeratin 18 levels are typically characterized as being less than 200 units per liter.
  • subjects with liver disease, including NALFD and NASH have a statistically significant elevation in cytokeratin 18 (e.g., above 200 U/L; 200-300 U/L).
  • cytokeratin 18 levels can be used as a marker to determine whether a treatment is being effective.
  • a reduction in cytokeratin 18 levels of greater than 10% is indicative that the therapy is having a beneficial effect.
  • Other markers include commonly used liver function tests including measuring one or more of, for example, serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (GGT).
  • ALT serum alanine aminotransferase
  • ALP alkaline phosphatase
  • AST aspartate aminotransferase
  • GTT gamma-glutamyl transpeptidase
  • Such diagnostics can utilize antibodies against sTREM2 or using other common assay Attorney Docket No.00015-428WO1 techniques.
  • the method contemplates obtaining a biological sample (e.g., blood, plasma or tissue biopsy) and measuring the amount of sTREM2 in the sample compared to a normal control (i.e., a control subject not having any symptoms of liver disease). If the test patient/subject has a value of sTREM2 increased compared to the normal control, then test subject can be identified as having NALFD or NASH.
  • the effectiveness of a method or composition of the disclosure can be assessed by measuring fatty acid content and metabolism in the liver.
  • ALT alanine aminotransferase
  • ALT levels have been shown to be indicative of liver function. For example, normal ALT levels are about 7 to 55 units (e.g., 10-40 units) per liter has been shown to correlate with normal liver function. This value is somewhat varied in children and adolescents. Thus, in some instances ALT levels less than 25 units per liter are “normal” in children and adolescents. Increased levels of ALT have been shown to correlate with liver disease and disorders.
  • NAFLD and NASH subjects typically show ALT levels of between 60 to 150 (e.g., 60-145, 70- 140, 80-135, 90-130, 105-125, 110-120, or any number between any two values thereof).
  • ALT levels above 25 units per liter can be indicative of NASH or NAFLD.
  • ALT may be measured alone, but preferably, the determination should be made in combination with one or more other markers of liver function or dysfunction. For example, a subject having ALT levels above about 80 is indicative of liver disease or dysfunction.
  • AST levels have been shown to be indicative of liver function. For example, AST levels between about 8 to 48 units (e.g., 10-40 units) per liter has been shown to correlate with normal liver function. Increased levels of AST have been shown to correlate with liver disease and disorders. For example, NAFLD and NASH subjects typically show AST levels of between 40 to 100 (e.g., 45-95, 55-90, 65-85, 70-80, or any number between any two values thereof). In determining if a subject has NAFLD or NASH or is susceptible to treatment using a composition of the disclosure, AST may be measured alone, but preferably the determination should be made in combination with one or more other markers of liver function or dysfunction.
  • ALP levels have been shown to be indicative of liver function.
  • ALP levels between about 45 to 115 units (e.g., 50-110 units) per liter has been shown to correlate with normal liver function.
  • Increased levels of ALP have been shown to correlate with liver disease and disorders.
  • NAFLD and NASH subjects typically show ALP levels of between 150 to 250 (e.g., 155-245, 160-240, 165-235, 170-230, 175-225, 180-220, 185- 215, 190-210, 195-200, or any number between any two values thereof).
  • ALP may be measured alone, but preferably the determination should be made in combination with one or more other markers of liver function or dysfunction.
  • a subject having ALP levels above about 150 is indicative of liver disease or dysfunction.
  • GGT levels have been shown to be indicative of liver function.
  • GGT levels between about 9 to 48 units (e.g., 10-40 units) per liter has been shown to correlate with normal liver function.
  • Increased levels of GGT have been shown to correlate with liver disease and disorders.
  • NAFLD and Attorney Docket No.00015-428WO1 NASH subjects typically show GGT levels of between 50 to 100 (e.g., 55-95, 60-90, 65-85, 70-80, or any number between any two values thereof).
  • GGT may be measured alone, but preferably the determination should be made in combination with one or more other markers of liver function or dysfunction.
  • a subject having GGT levels above about 50 is indicative of liver disease or dysfunction.
  • Triglycerides levels have been shown to be indicative of liver function.
  • triglyceride levels less than about 150 mg/dL has been shown to correlate with normal liver function.
  • Increased levels of triglycerides have been shown to correlate with liver disease and disorders.
  • NAFLD and NASH subjects typically show triglyceride levels of between 150 to 200 (e.g., 155-195, 160-190, 165-185, 170-180, or any number between any two values thereof).
  • triglycerides may be measured alone, but preferably the determination should be made in combination with one or more other markers of liver function or dysfunction.
  • a subject having triglyceride levels above about 150 mg/dl is indicative of liver disease or dysfunction.
  • High triglyceride levels are known to be a leading cause of various forms of inflammation.
  • Triglycerides are the form in which fat moves through the bloodstream.
  • Triglycerides can be metabolized by various organs, including the liver, to form phospholipids (LDLs and HDLs), cholesterol and oxidized forms thereof.
  • Oxidized phospholipids (OxPL) including OxLDL are known inflammatory mediators and strongly correlated with cardiovascular diseases. For example, Bieghs et al.
  • Adiponectin circulates as trimer (low molecular weight Attorney Docket No.00015-428WO1 adiponectin), hexamer (medium molecular weight adiponectin) and higher order multimer (high molecular weight adiponectin) in serum and isoform-specific effects have been demonstrated.
  • Epidemiological studies revealed that low adiponectin levels are associated with NASH.
  • adiponectin is believed to have a hepatoprotective effect due to protective effects against oxidative damage.
  • Normal levels of adiponectin vary by age and sex. For example, females have a higher baseline adiponectin level compared to males.
  • a normal weight female typically has an adiponectin level of between about 8.5 and 11 ⁇ g/ml and males typically have an adiponectin level of between about 6 and 8 ⁇ g/ml.
  • subject with fatty liver disease, NASH and/or obesity have adiponectin levels that are about 50-90% of normal levels (e.g., decreased by 10-50% from normal, or any value there between) (see, e.g., Merl et al. Int. J. Obes (Lond), 29(8), 998-1001, 2005).
  • the resistin protein is increased in NASH subjects compared to normal subjects.
  • Human resistin is a cysteine-rich, 108-amino-acid peptide hormone with a molecular weight of 12.5 kDa. In adult humans, resistin is expressed in bone marrow. Moreover, in adipocytes of subjects having a low or healthy BMI, resistin mRNA is almost undetectable. Consistent with this resistin concentrations in serum, and women may have higher resistin concentrations than men. Resistin mRNA expression in human peripheral mononuclear cells is increased by proinflammatory cytokines. Serum resistin is significantly elevated in both NASH and simple steatotic subjects. Hepatic resistin is significantly increased in NASH patients in both mRNA and protein levels than those in simple steatosis and normal control subjects.
  • the disclosure provides a method of treating a subject suffering from ALD or fatty liver disease, such as NASH, comprising administering a therapeutically effective amount of an sTREM2 polypeptide or a vector that produces sTREM2.
  • the fatty liver disease is selected from the group consisting of non-alcoholic fatty acid liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fatty liver disease resulting from hepatitis, fatty liver disease resulting from obesity, fatty liver disease resulting from diabetes, fatty liver disease resulting from insulin resistance, fatty liver disease resulting from hypertriglyceridemia, Abetalipoproteinemia, glycogen storage diseases, Weber-Christian disease, Wolman’s disease, acute fatty liver of pregnancy, alcoholic liver disease and lipodystrophy.
  • NAFLD non-alcoholic fatty acid liver disease
  • NASH non-alcoholic steatohepatitis
  • fatty liver disease resulting from obesity fatty liver disease resulting from diabetes
  • fatty liver disease resulting from insulin resistance fatty liver disease resulting from hypertriglyceridemia
  • Abetalipoproteinemia glycogen storage diseases
  • Weber-Christian disease Wolman’s disease
  • acute fatty liver of pregnancy alcoholic liver disease and lip
  • the administration results in a decrease in NAFLD Activity Score of two or more points, no worsening or an improvement of fibrosis, reduction in serum aminotransferases and gammaglutamyl transpeptidase (GGT); reduction in MRI-determined hepatic fat fraction; changes to markers of oxidation and anti-oxidant status; changes in fasting insulin and glucose; an increase in circulating adiponectin levels; a decrease in circulating resistin levels; a decrease in triglyceride levels; a decrease in oxidized phospholipids; changes in weight, height, body mass index (BMI) and waist circumference; changes in the Pediatric Quality of Life score; changes to any symptoms that patient may have experienced; proportion with a change from a histological diagnosis of definite NASH or indeterminate for NASH to not NASH at end of treatment; individual histological characteristics at end of treatment compared to baseline such as steatosis (fatty liver), lobular inflammation, portal chronic inflammation, ballooning,
  • GTT gammaglutamy
  • the methods and composition of the disclosure can also include administering a second agent in combination with a compound of the disclosure to treat a disease or disorder.
  • a second agent in combination with a compound of the disclosure to treat a disease or disorder.
  • the subject can be treated with a combination of active agents for treating fatty liver disorders, such as NAFLD and NASH.
  • the Attorney Docket No.00015-428WO1 combination includes an sTREM2 polypeptide or vector of the disclosure and one or more of metformin, statins, anti-oxidants, and/or antibodies against oxidized phospholipids. Such a combination can have unexpected synergy due to a multifaceted approach to modulating inflammation and inflammatory mediators.
  • the Foz/Foz model thoroughly recapitulates the human NASH pathology and associated co-morbidities. Moreover, this model progresses to cirrhosis and HCC which very few NASH models have achieved to date. Furthermore, these findings will be translated from the mouse to human using primary cells isolated from transplant rejected human livers that were declined for transplantation. Primary human liver cell derived 3-D spheroids (NASH-in-a-dish) has been established in the lab that are formed by mixing multiple cell types will provide key insights into cell-cell cross talk in NASH. [00159] Presence or absence of TREM2 in NASH associated M ⁇ reveals distinct changes in functional pathways. TREM2 is one of the most highly differentially expressed genes upregulated in both Foz and human NASH livers.
  • TREM2 upregulation a time course analyses of TREM2 expression in WT and Foz/Foz livers was performed (Fig. 2A).
  • TREM2 transcript levels in Foz+WD livers increased beginning at 8w post WD feeding and continued to increase as NASH and fibrosis progressed (Fig. 2A).
  • Attorney Docket No.00015-428WO1 Furthermore, TREM2 expression is downregulated during NASH regression when Foz+WD 12w mice switch to normal chow (Fig. 2A).
  • Analyses of human patient samples confirm that TREM2 (both mRNA and protein) is upregulated in most NASH livers (Fig. 2B-D).
  • TREM2 M ⁇ in fibrotic NASH livers were reported as “scar associated M ⁇ (SAMac)”. TREM2 M ⁇ expand in NASH (vs healthy) (Fig. 2C), and accumulate next to collagen deposits in F3 fibrotic NASH, in patients with lower fibrosis scores (F1 NASH), the TREM2 M ⁇ were scattered in the liver parenchyma (Fig. 2C). Most TREM2 expressing cells co-express the M ⁇ marker Cd11b (Fig. 2D), indicating that M ⁇ are the major cellular source of TREM2 upregulation. This finding is further validated with single cell (sc) RNAseq (Fig.
  • NPC non-parenchymal cells
  • pH sensitive fluorescently labeled pH sensitive fluorescently labeled
  • TREM2 is protective. The mechanistic role of TREM2 in NASH, fibrosis and HCC development has been elusive so far.
  • mice lacking TREM2 have increased NASH and fibrosis in two models of NASH, demonstrating that the findings are generalizable (Fig. 4).
  • Foz::Trem2 mice were generated and subjected to WD. Deletion of TREM2 made Foz mice develop more NASH and fibrosis when fed WD for 8-12w (Fig. 4A, B).
  • Trem2 was analyzed by itself (without the Alms1 mutation found in Foz mice) to determine if they are prone to NASH. Since WD alone is not sufficient to induce NASH/fibrosis in non-Foz mice, Fructose was included in the drinking water.
  • Trem2 mice When fed with WD+30% Fructose in drinking water, Trem2 mice had more severe NASH and fibrosis by 16w (Fig. 4C). Strikingly, about 60% of Trem2 mice spontaneously progressed to HCC by 32w (Fig. 4D). Taken together, these studies indicate that TREM2 plays a mechanistic ‘protective’ role in NASH, fibrosis and HCC development. [00164] TREM2 dampens NLRP3 Inflammasome activation. NOD-like receptor protein 3 (NLRP3) inflammasome activation plays a pivotal role in NASH pathology.
  • NLRP3 NOD-like receptor protein 3
  • NLRP3 inflammasome is a critical component of the innate immune system that mediates caspase-1 activation and the secretion of inflammatory cytokines IL-1 ⁇ /IL-18.
  • TREM2 was observed to lead to pronounced activation of caspase-1 in NASH livers (Fig. 5A).
  • BMDM bone marrow derived M ⁇
  • sTREM2 could be a potential biomarker for NASH, the signaling effects of sTREM2 in NASH is unknown.
  • sTREM2 protects microglial cells from cell death and its activation aids in clearing up beta-amyloid plaques.
  • prevention of TREM2 cleavage improves Alzheimer’s pathology.
  • AAV8 vectors exhibit strong liver tropism and stably express ectopic proteins in hepatocytes for at least 12 months.
  • the concept is to convert the hepatocytes into micro- factories to produce and release systemic sTREM2 (Fig. 7C). Indeed, when introduced into Trem2 mice, successful expression of sTREM2 and release into the serum was accomplished in a dose dependent manner (Fig. 7C).
  • sTREM2 expression was confirmed by serum ELISA (Fig. 7E).

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Abstract

La divulgation concerne des vecteurs et des agents recombinants comprenant des fragments solubles de sTREM2 destinés à être utilisés dans le traitement de la NAFLD et de la NASH ainsi que d'autres maladies et troubles hépatiques.
PCT/US2024/034618 2023-06-19 2024-06-19 Fragments de trem2 pour le traitement de la nafld, de la nash et de maladies et de troubles hépatiques Pending WO2024263639A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8236294B2 (en) * 2003-12-24 2012-08-07 The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Gene therapy using transposon-based vectors
WO2017147509A1 (fr) * 2016-02-25 2017-08-31 Marco Colonna Compositions comprenant de la trem2 et leurs procédés d'utilisation
WO2022133461A1 (fr) * 2020-12-18 2022-06-23 Baylor College Of Medicine Administration de variants abeta pour l'inhibition de l'agrégation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8236294B2 (en) * 2003-12-24 2012-08-07 The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Gene therapy using transposon-based vectors
WO2017147509A1 (fr) * 2016-02-25 2017-08-31 Marco Colonna Compositions comprenant de la trem2 et leurs procédés d'utilisation
WO2022133461A1 (fr) * 2020-12-18 2022-06-23 Baylor College Of Medicine Administration de variants abeta pour l'inhibition de l'agrégation

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