WO2022048577A1 - Utilisation d'anticorps monoclonal dirigé contre emc10 humain dans la préparation de produits pour la prévention et/ou le traitement de maladies métaboliques - Google Patents

Utilisation d'anticorps monoclonal dirigé contre emc10 humain dans la préparation de produits pour la prévention et/ou le traitement de maladies métaboliques Download PDF

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WO2022048577A1
WO2022048577A1 PCT/CN2021/116096 CN2021116096W WO2022048577A1 WO 2022048577 A1 WO2022048577 A1 WO 2022048577A1 CN 2021116096 W CN2021116096 W CN 2021116096W WO 2022048577 A1 WO2022048577 A1 WO 2022048577A1
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emc10
preparation
monoclonal antibody
seq
animals
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PCT/CN2021/116096
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Chinese (zh)
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王宣春
李燕良
陈匡阳
景昱
胡仁明
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复旦大学附属华山医院
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Priority claimed from CN202010927627.7A external-priority patent/CN114149500B/zh
Priority claimed from CN202010927231.2A external-priority patent/CN114149498B/zh
Priority claimed from CN202010927577.2A external-priority patent/CN114149499B/zh
Application filed by 复旦大学附属华山医院 filed Critical 复旦大学附属华山医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the invention belongs to antibody drugs in the biomedical industry, and particularly relates to the application of anti-human EMC10 monoclonal antibodies in the preparation of products for preventing and/or treating metabolic diseases, specifically fatty liver, obesity and/or 2 type diabetes.
  • FLD fatty liver disease
  • CLD chronic liver disease
  • FLD can progress from simple hepatic steatosis to steatohepatitis and hepatic fibrosis.
  • ALD alcoholic fatty liver disease
  • NAFLD non-alcoholic fatty liver disease
  • NAFLD is the main cause of liver transplantation due to CLD in European countries in the past decade, and it is also the main cause of hepatocellular carcinoma in the United States and the United Kingdom, affecting the health of nearly 25% of the global population.
  • NAFLD non-alcoholic steatohepatitis
  • 6-30% have histologically confirmed progression to non-alcoholic steatohepatitis (NASH), and 40% of NASH patients have a tendency to progress to liver fibrosis.
  • NAFLD In addition to causing damage to the liver itself, NAFLD can cause or exacerbate insulin resistance. closely related. Patients with NAFLD have a 5-fold higher risk of developing T2DM than patients without NAFLD. Nearly 90% of patients with NAFLD have at least one clinical manifestation of MetS, and approximately 33% of patients are diagnosed with MetS. Studies have shown that at least 70% of T2DM patients with normal liver function coexist with NAFLD. In addition, NAFLD also increases the risk of coronary atherosclerosis, ischemic heart disease, and increases carotid intima-media thickness, so it is also considered to be a new independent risk factor for cardiovascular disease.
  • Fat is one of the main sources of energy for the body, and lipid metabolism disorder is a serious consequence of metabolic diseases and an important factor in its pathogenesis.
  • hormones or secreted proteins in the organism that regulate lipid metabolism such as classic insulin, thyroid hormone, adrenal glucocorticoid and growth hormone, which play an important role in regulating glucose and lipid metabolism and energy homeostasis.
  • adipokines secreted by fat such as: leptin, adiponectin, resistin, TNF, IL-6, PAI -1, MCP-1, etc.
  • liver factors secreted by the liver such as: Fetuin-A, FGF-21, sex hormone binding protein (SHBG), etc.
  • muscle factors secreted by muscles such as myostatin , irisin, IL-6, IL-15, etc.
  • cardiac factors secreted by the heart such as atrial natriuretic peptide (ANP)
  • these hormones or factors can be detected in serum or plasma, they are expected to be molecular markers for obesity diagnosis and potential targets for treatment.
  • Practice has proved that some of the above-mentioned hormones or cytokines can be used as molecular markers useful for evaluating obesity, such as leptin and adiponectin, but there are very few target drugs that can be used to treat obesity.
  • target drugs that can be used to treat obesity.
  • Diabetes is a multifactorial disease. So far, the pathogenesis of diabetes has not been fully elucidated. Therefore, it is of great scientific and practical significance to explore new pathogenic factors of diabetes and clarify its pathophysiological mechanism, and to develop new diabetes intervention and treatment methods. .
  • Glucose metabolism is the main source of energy supply in living organisms.
  • hormones or secreted proteins in the organism that regulate glucose metabolism such as classic insulin, glucagon, adrenal glucocorticoid and growth hormone, which play an important role in regulating glucose metabolism and energy homeostasis.
  • some non-classical endocrine tissues and organs have also been found to secrete some hormones or cytokines, and have been used for clinical treatment of diabetes, such as intestinal secretion of incretin (glucagon-like peptide 1, GLP-1), DPP-4 enzyme inhibitors and GLP-1 receptor agonists reduce blood sugar by increasing the level of GLP-1 in serum and enhancing the effect of GLP-1.
  • Leptin and Adiponectin secreted by adipose tissue can also regulate glucose metabolism, and there are currently clinical trials targeting these two targets to treat diabetes.
  • Neutralizing antibodies can effectively neutralize the activity and function of endocrine antigens in the body. By effectively inhibiting the biological activity of the target source, it can well verify the role of the target source in the occurrence and development of diseases.
  • the transformation to clinical application provides strong support.
  • the monoclonal therapeutic antibody of proprotein convertase subtilisinvertase/cosine type 9 (PCSK9) a novel lipid-lowering therapeutic target, has been revealed through relevant animal studies. important clinical value.
  • EMC10 was originally cloned from the cDNA library of human insulinoma tissue and was named as: INM02.
  • the nucleotide sequence of the INM02 (EMC10) gene and its encoded amino acid sequence are shown in the GenBank database (accession number: AY194293) (Wang XC ,Xu SY,Wu XY,Song HD,Mao YF,Fan HY,Yu F,Mou B,Gu YY,Xu LQ,Zhou XO,Chen Z,Chen JL,Hu RM.Gene expression profiling in human insulinoma tissue:genes involved in the insulin secretion pathway and cloning of novel full-length cDNAs.
  • EMC10 is a secreted protein that can be detected in human serum, and found that the expression of Emc10 (Inm02) gene in mouse pancreatic ⁇ cells is affected by glucose , suggesting that it may play an important role in glucose metabolism (Wang X, Gong W, Liu Y, Yang Z, Zhou W, Wang M, Yang Z, Wen J, Hu R. Molecular cloning of a novel secreted peptide, INM02, and regulation of its expression by glucose. J Endocrinol.
  • EMC10 was cloned from purified human hematopoietic stem cells.
  • Gluzman-Poltorak Z Miller JD, Wheeler CJ, Fan X, Basile LA.hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33.J Neurooncol.2011,102(2):197-211. ; Junes-Gill KS, Lawrence CE, Wheeler CJ, Cordner R, Gill TG, Mar V, Shiri L, Basile LA. Human Hematopoietic Signal peptide-containing Secret 1(hHSS1) modulates genes and paths in glioma:implications for the regulation of tumorigenicity and angiogenesis. BMC Cancer. 2014, 14:920.).
  • Diamantopoulou A Sun Z, Mukai J, Xu B, Fenelon K, Karayiorgou M, Gogos JA. Loss-of-function mutation in Mirta22/Emc10 rescues specific schizophrenia-related phenotypes in a mouse model of the 22q11.2 deletion.Proc Natl Acad Sci US A. 2017;114(30):E6127-E6136.). Recently, researchers from Germany found that in a mouse model of myocardial infarction, Emc10 deletion resulted in decreased angiogenesis in the infarct marginal zone and impaired left ventricular systolic and diastolic function.
  • Angiogenesis improves the impaired left ventricular function after myocardial infarction, suggesting that EMC10 is a growth factor with angiogenic function that promotes tissue repair after myocardial infarction (Reboll MR, Korf-Klingebiel M, Klede S, Polten F, Brinkmann E, Reimann I, HJ, Bobadilla M, Faix J, Kensah G, Gruh I, Klintschar M, Gaestel M, Niessen HW, Pich A, Bauersachs J, Gogos JA, Wang Y, Wollert KC.
  • EMC10 Endoplasmic Reticulum Membrane Protein Complex Subunit 10
  • EMC10 governs male fertility via maintaining sperm ion balance . J Mol Cell Biol. 2018 Dec 1;10(6):503-514.).
  • the purpose of the present invention is to prevent and/or treat insulin resistance-related metabolic diseases.
  • the metabolic disease may be fatty liver, obesity and/or type 2 diabetes.
  • the present invention first protects the application of the anti-human EMC10 monoclonal antibody or the biological material related to the anti-human EMC10 monoclonal antibody in the preparation of products for preventing and/or treating metabolic diseases; the metabolic diseases may be fatty liver, Obesity and/or type 2 diabetes.
  • the name of the anti-human EMC10 monoclonal antibody is 4C2, and the monoclonal antibody can specifically recognize the antigenic epitope whose amino acid sequence can be shown in SEQ ID No.5, namely VVGVSVVTHP.
  • the monoclonal antibody of described anti-human EMC10 contains the heavy chain variable region named VH and the light chain variable region named VL , and the VH and VL are both composed of determinant complementary regions and The framework region is composed; the determinant complementary regions of the VH and the VL are composed of CDR1, CDR2 and CDR3;
  • the amino acid sequence of the CDR1 of the VH is shown in positions 31-35 of SEQ ID No.1;
  • the amino acid sequence of the CDR2 of the VH is shown in positions 50-68 of SEQ ID No.1;
  • amino acid sequence of the CDR3 of the VH is shown in positions 101-103 of SEQ ID No.1;
  • the amino acid sequence of the CDR1 of the VL is shown in positions 24-39 of SEQ ID No.2;
  • the amino acid sequence of the CDR2 of the VL is shown in positions 55-61 of SEQ ID No.2;
  • the amino acid sequence of the CDR3 of the VL is shown in positions 94-102 of SEQ ID No.2.
  • the framework regions of VH and VL are derived from mice.
  • SEQ ID No.1 consists of 114 amino acid residues
  • SEQ ID No.2 consists of 113 amino acid residues.
  • the monoclonal antibody of described anti-human EMC10 can be any of the following:
  • the anti-human EMC10 monoclonal antibody may be a murine monoclonal antibody.
  • the biological material related to the monoclonal antibody against human EMC10 can be any one of B1) to B12):
  • B2 an expression cassette containing the nucleic acid molecule of B1);
  • B5 a recombinant microorganism containing the nucleic acid molecule of B1);
  • B9 a transgenic animal cell line containing the nucleic acid molecule of B1);
  • B11 a transgenic animal cell line containing the recombinant vector described in B3);
  • B12 a transgenic animal cell line containing the recombinant vector described in B4)
  • the metabolic disease may be fatty liver, obesity and/or type 2 diabetes.
  • the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.
  • the nucleic acid molecule of B1) can be a gene encoding the monoclonal antibody, and the gene can be the DNA molecule described in C1) or C2) below:
  • the coding sequence of the CDR1 of the VH is shown in the 91st-105th position of SEQ ID No.3, and the coding sequence of the CDR2 of the VH is shown in the 148th-204th position of SEQ ID No.3,
  • the coding sequence of the CDR3 of the VH is shown in the 301-309th position of SEQ ID No.3;
  • the coding sequence of the CDR1 of the VL is shown in the 70th-117th position of the SEQ ID No.4, the said
  • the coding sequence of CDR2 of VL is shown in position 163-183 of SEQ ID No.4, and the coding sequence of CDR3 of VL is shown in position 280-306 of SEQ ID No.4;
  • C2 A DNA molecule that is more than 90% identical to the DNA molecule defined in C1) and encodes the monoclonal antibody or antigen-binding portion thereof.
  • SEQ ID No.3 consists of 342 nucleotides
  • SEQ ID No.4 consists of 339 nucleotides.
  • identity refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
  • more than 90% identity may be DNA molecules that are at least 91%, 92%, 95%, 96%, 98% or 99% identical.
  • the expression cassette described in B2) refers to the DNA capable of expressing the monoclonal antibody or its antigen-binding portion in a host cell, and this DNA can not only include genes that start the monoclonal antibody or its antigen-binding portion.
  • the transcriptional promoter may also include a terminator that terminates the transcription of the monoclonal antibody or its antigen-binding portion of the gene.
  • the expression cassette may also include enhancer sequences.
  • a recombinant vector containing the monoclonal antibody gene expression cassette can be constructed by using an existing expression vector.
  • the recombinant vector can be a plasmid, cosmid, phage or viral vector.
  • the recombinant microorganism can be yeast, bacteria, algae or fungi.
  • the transgenic animal cell line may be non-reproductive material.
  • the present invention also protects the application of any of the above-mentioned anti-human EMC10 monoclonal antibodies in any of the following:
  • the present invention also protects the application of any of the above-mentioned biological materials related to the anti-human EMC10 monoclonal antibody in any of the following:
  • the present invention also contemplates a method of preventing and/or treating metabolic diseases.
  • the method comprises administering to the recipient animal any of the above-mentioned monoclonal antibodies against human EMC10;
  • the metabolic disease may be fatty liver, obesity and/or type 2 diabetes.
  • the present invention also protects the application of substances that inhibit the activity of the protein EMC10, reduce the gene expression of the protein EMC10 and/or reduce the content of the protein EMC10 in the preparation of products for preventing and/or treating metabolic diseases;
  • the metabolic disease may be fatty liver, obesity and/or type 2 diabetes.
  • the product may be a drug, vaccine, reagent or kit.
  • the animal may be a mammal, such as a mouse or a human.
  • the metabolic disease may be an insulin resistance-related metabolic disease.
  • the prevention and/or treatment of fatty liver in the present invention is mainly reflected in the aspects of reducing fatty infiltration of the liver, reducing serum insulin content, reducing serum triglyceride content, reducing serum free fatty acid content, reducing serum cholesterol content and improving insulin sensitivity, etc. .
  • the fatty liver of the present invention specifically refers to non-alcoholic fatty liver disease (NAFLD).
  • NAFLD non-alcoholic fatty liver disease
  • the prevention and/or treatment of diabetes in the present invention is mainly embodied in the aspects of lowering blood sugar, improving glucose tolerance, lowering serum insulin and improving insulin sensitivity.
  • the diabetes may specifically be type 2 diabetes.
  • the prevention and/or treatment of obesity described in the present invention is mainly reflected in weight loss, reduction in body fat content and visceral fat volume, activation of brown fat function and increase in thermogenesis, blood sugar, serum insulin, free fatty acids, triglycerides and Cholesterol lowering, etc.
  • the reduction of body fat content in the animal may be reduction of white fat, including volume and weight of visceral and subcutaneous adipocytes.
  • the reducing visceral fat volume in the animal can be reducing abdominal or epididymal adipocyte volume.
  • the activation of the brown fat of the animal to promote thermogenesis can reduce the accumulation of lipid droplets in the brown fat of the animal and promote the uptake and utilization of energy by the brown fat.
  • the anti-EMC10 monoclonal antibody provided by the present invention can significantly reduce the liver weight and fatty infiltration of the liver in mice, and can be used to prepare a product for preventing and/or treating fatty liver.
  • the anti-EMC10 monoclonal antibody provided by the invention can significantly reduce the blood sugar of the type 2 diabetes mouse model, improve the glucose tolerance and insulin sensitivity of the mouse, and can be used to prepare products for preventing and/or treating diabetes.
  • the anti-EMC10 monoclonal antibody provided by the present invention can significantly reduce the body weight of obese mice and can improve obesity-related metabolic disorders, and can be used to prepare products for preventing and/or treating obesity.
  • Fatty liver, obesity and type 2 diabetes are all metabolic diseases, thus it can be seen that the present invention can prevent and/or treat metabolic diseases, especially diabetes, obesity and fatty liver, and provide a new therapeutic target.
  • the invention has important application value.
  • Figure 1 shows the results of the identification of purified protein using the Dot blot method.
  • 1-9 represent protein weights of 30, 15, 7.5, 3.75, 1.875, 0.9375, 0.46875, 0.234375, and 0.1171875ng, respectively.
  • Figure 2 shows the results of the identification of purified protein using the Western blot method.
  • FIG. 3 shows the results of phosphorylation of CREB after mouse EMC10 protein and different mouse anti-human EMC10 monoclonal antibodies simultaneously intervene in HeLa cells; in the figure, Ctr means only EMC10 protein, no EMC10 antibody; 1, 2, 3 , 4 indicate 1F12, 4B12-1, 4B12-2, 4C2 antibodies, and EMC10 protein, respectively.
  • FIG. 4 shows the results of western blot detection of wild-type and different truncations of EMC10 protein with anti-Flag-tag antibody.
  • WT represents wild-type, namely EMC10 protein; ⁇ 28-105, ⁇ 66-145, ⁇ 106-183, ⁇ 146- 225, ⁇ 184-254, ⁇ 146-175, ⁇ 171-200, ⁇ 196-225, ⁇ 146-155, ⁇ 156-165, ⁇ 166-175 missing , 146-175, 171-200, 196-225, 146-155, 156-165, 166-175 amino acid EMC10 truncations.
  • Figure 5 shows the effect of anti-human EMC10 monoclonal antibody 4C2 on the body weight and fatty liver of diet-induced obese mice; wherein, A and B are the weight gain and weight gain of the control IgG group, 1F12 and 4C2 antibody groups, respectively C is the liver weight of the mice in the control IgG group, 1F12 and 4C2 antibody groups; D is the lipid infiltration in the liver tissue observed by HE staining; in the figure, *P ⁇ 0.05, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • Figure 6 shows the effect of anti-human EMC10 monoclonal antibody 4C2 on metabolic disorders associated with fatty liver in high-fat diet mice; wherein, AE are the insulin tolerance, serum insulin, Test results of triglycerides, non-esterified fatty acids and cholesterol; in the figure, *P ⁇ 0.05.
  • Figure 7 shows the weight gain of mice in the control IgG group, 1F12 and 4C2 antibody groups, in the figure, *P ⁇ 0.05.
  • Figure 8 shows the weight gain of mice in the control IgG group, 1F12 and 4C2 antibody groups, in the figure, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • Figure 9 is the fat and non-fat weight of mice in the control IgG group, 1F12 and 4C2 antibody groups measured by DEXA, in the figure, *P ⁇ 0.05.
  • Figure 10 shows that the mice in the control IgG group, 1F12 and 4C2 antibody groups were sacrificed after the experiment, and different tissues and organs ⁇ heart (Heart), liver (Liver), epididymal fat (eWAT), inguinal subcutaneous fat (iWAT), peritoneum) were weighed Weight of posterior fat (Retro), mesenteric fat (Mesen), brown fat (BAT), spleen (Spleen), kidney (Kidney), pancreas (Panc) ⁇ ; in the figure, *P ⁇ 0.05.
  • Figure 11 shows the HE staining of brown fat, subcutaneous fat and epididymal fat tissue sections after the mice of the control IgG group and the 4C2 antibody group were sacrificed after the experiment.
  • Figure 12 shows the blood glucose (6 hours fasting) and the levels of non-fasting insulin, non-esterified fatty acids, triglycerides, and cholesterol in mice on a high-fat diet with control IgG, 1F12 and 4C2 antibodies; in the figure, *P ⁇ 0.05 .
  • FIG 13 shows the energy metabolism status of high-fat diet mice intervened with control IgG and 4C2 antibody in metabolic cage study, in the figure, IgG: mouse control IgG, 4C2: mouse anti-human EMC10 monoclonal antibody 4C2.
  • Figure 14 shows the effect of anti-human EMC10 monoclonal antibody 4C2 on fasting blood glucose (A), glucose tolerance (B), serum insulin (C) and insulin tolerance (D) of mice fed a high-fat diet; in the figure, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Construction of recombinant plasmid pRAG2a-EMC10 Replace the small fragment between the restriction sites NheI and XhoI of the eukaryotic expression vector pRAG2a with the DNA fragment shown in positions 82-762 of SEQ ID NO.7 to obtain the recombinant plasmid pRAG2a-EMC10.
  • Washing impurities 5 column volumes of binding buffer wash impurities until no material flows out through the flow-through.
  • the purified protein obtained in step 3 was identified by the method of Dot blot.
  • the anti-EMC10 antibody used was a rabbit anti-human EMC10 polyclonal antibody (a polyclonal antibody obtained after immunizing New Zealand white rabbits with the EMC10 protein shown in SEQ ID NO. 6 of the sequence listing as the immunogen); the secondary antibody was goat anti-rabbit HRP antibody (Thermo Fisher, Catalog #65-6120).
  • the purified protein obtained in step 3 was identified by the method of Western blot.
  • the anti-EMC10 antibody used was a rabbit anti-human EMC10 polyclonal antibody (a polyclonal antibody obtained after immunizing New Zealand white rabbits with the EMC10 protein shown in SEQ ID NO: 6 of the sequence table as an immunogen); the secondary antibody was a goat anti-rabbit HRP antibody ( Thermo Fisher, Catalog #65-6120).
  • Blocking 250 ⁇ l/well of blocking solution, 37° C., 2 h.
  • step (2) Washing the plate: discard the liquid in the well, spin dry, and wash the plate 5 times, the method is the same as that of step (2).
  • Detection Draw the serum to be tested and dissolve it in the antibody diluent (0.1M PBS) to prepare working solutions with a dilution ratio of 1000, 3000, 9000, 27000 times, and add 100 ⁇ L of working solutions of different concentrations to each well. Be careful not to If there are air bubbles, add the sample to the bottom of the ELISA plate, try not to touch the wall of the well, and shake gently to mix. Cover the ELISA plate and incubate at 37°C for 2h.
  • the antibody diluent 0.1M PBS
  • step (2) Washing the plate: discard the liquid in the well, spin dry, and wash the plate 5 times, the method is the same as that of step (2).
  • step (2) Discard the liquid in the well, spin dry, and wash the plate 5 times, the method is the same as that of step (2).
  • DMEM was added to the mesh, and the mesh was rinsed to allow more spleen cells to be collected into the plate.
  • the cells were transferred to a 10 ml centrifuge tube, the spleen cells were washed twice with serum-free DMEM, centrifuged at 1000 rpm for 5 min, and the spleen cells were collected and counted.
  • the clone rate of hybridoma cells was more than 50%, there was a small amount of cell debris, and the cells grew well.
  • the screening test was started 10 days after fusion.
  • hybridoma cell lines capable of stably secreting anti-EMC10 monoclonal antibodies were obtained, numbered 8C11, 6B9, 1F12, 4B12-1, 1H11, 4C2, 4B12-2 and 8A3, respectively.
  • the western blotting method is as follows:
  • Monoclonal hybridoma cell line 4C2 secreting mouse anti-human EMC10 monoclonal antibody 4C2 (hereinafter referred to as 4C2 antibody) - mouse anti-human EMC10 monoclonal antibody hybridoma cell line 4C2 (hereinafter referred to as 4C2 hybridoma cells or 4C2 cells) has been On June 18, 2020, it was deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), and the preservation number is CGMCC No.19950.
  • CGMCC General Microbiology Center of China Microorganism Culture Collection Management Committee
  • the sequencing result showed that the coding gene sequence of the heavy chain variable region ( VH ) of the mouse anti-human EMC10 monoclonal antibody 4C2 was SEQ ID No.3 , the amino acid sequence of VH is SEQ ID No.1, wherein, the amino acid sequence of CDR1 of VH is shown in the 31-35th position of SEQ ID No.1, and the amino acid sequence of CDR2 of VH is as shown in SEQ ID No.1
  • the amino acid sequence of the CDR3 of VH is shown in the 101-103 position of SEQ ID No.1; the light chain variable region ( VL ) of the mouse anti-human EMC10 monoclonal antibody 4C2
  • the coding gene sequence is SEQ ID No.4, and the amino acid sequence of VL is SEQ ID No.2, wherein, the amino acid sequence of CDR1 of VL is shown in positions 24-39 of SEQ ID No.2, and the CDR2 of VL is shown in position 24-39.
  • the amino acid sequence of VL is shown in positions
  • the coding gene of the heavy chain fragment VH shown in SEQ ID No.3 is spliced with the coding gene of the heavy chain fragment constant region ( CH ) of mouse IgG1, and inserted into the eukaryotic expression vector pAH (HAS Bind, Wuhai, China) , the antibody heavy chain expression plasmid pAH-4C2 was obtained;
  • the coding gene of the light chain fragment ( VL ) shown in SEQ ID No.4 was spliced with the coding gene of the mouse CL-kappa fragment (constant region of the light chain fragment), and inserted into In the eukaryotic expression vector pAK (HAS Bind, Wuhai, China), the antibody light chain expression plasmid pAK-4C2 was obtained.
  • the forward and reverse sequencing primers were used for bidirectional testing, and then the sequence alignment analysis was performed; the antibody light chain expression plasmid pAK-4C2 was tested with forward sequencing primers, and then the sequences were compared.
  • SEQ ID No. 8 including the coding sequence of the secretion signal peptide
  • SEQ ID No. 9 positions 1-21 are the amino acids of the secretion signal peptide
  • the 22-459th position is the amino acid sequence of the mouse anti-human EMC10 monoclonal antibody 4C2 heavy chain); the nucleotide sequence of the light chain is shown in SEQ ID No. 10 (including the coding sequence of the secretion signal peptide), and the expression SEQ ID The protein represented by ID No. 11 (1-21st is the amino acid sequence of the secretion signal peptide, 22-240th is the amino acid sequence of the mouse anti-human EMC10 monoclonal antibody 4C2 light chain).
  • the above two eukaryotic expression plasmids (antibody heavy chain plasmid pAH-4C2 and antibody light chain plasmid pAK-4C2) were subjected to mid-suction (Plasmid Midiprepkit, AxyPrep, USA), and the plasmid quality was detected by agarose gel electrophoresis.
  • the antibody heavy chain plasmid pAH-4C2 and the antibody light chain plasmid pAK-4C2 were co-transfected into 40 mL of HEK293F cells. After the expression, the cell suspension culture supernatant was collected.
  • the expression supernatant and 4C2 antibody (the mouse anti-human EMC10 monoclonal antibody 4C2 secreted by the mouse anti-human EMC10 monoclonal antibody hybridoma cell line) were simultaneously subjected to a gradient dilution ELISA analysis.
  • the results are as follows As shown in Table 1 and Table 2: the ELISA value of the expression supernatant diluted about 30 times was equivalent to 11 ng/mL of the 4C2 antibody.
  • the amino acid sequence of the heavy chain variable region of the mouse anti-human EMC10 monoclonal antibody 4C2 secreted by the mouse anti-human EMC10 monoclonal antibody hybridoma cell line is shown in SEQ ID No. 1 (the coding sequence is shown in SEQ ID No. 1). 3), the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2 (the coding sequence is shown in SEQ ID No. 4).
  • the variable region of the heavy chain and the variable region of the light chain are both composed of a determinant complementary region and a framework region; the determinant complementary region of the heavy chain variable region is composed of CDR1 (positions 31-35 of SEQ ID No.1).
  • the coding sequence is shown in the 91st-105th position of SEQ ID No.3), CDR2 (the 50th-68th position of SEQ ID No.1 is shown, and the coding sequence is shown in the 148th-204th position of SEQ ID No.3 position) and CDR3 (shown at positions 101-103 of SEQ ID No. 1, and the coding sequence shown at positions 301-309 of SEQ ID No. 3);
  • the complementary region is represented by CDR1 (positions 24-39 of SEQ ID No. 2, and the coding sequence is shown in positions 70-117 of SEQ ID No. 4), CDR2 (positions 55-61 of SEQ ID No. 2) shown, the coding sequence is shown in positions 163-183 of SEQ ID No. 4) and CDR3 (positions 94-102 of SEQ ID No. 2 are shown, and the coding sequence is shown in positions 280-306 of SEQ ID No. 4) bits shown) composition.
  • the mouse anti-human EMC10 monoclonal antibody 4C2 in the following experiments is the mouse anti-human EMC10 monoclonal antibody 4C2 secreted by the mouse anti-human EMC10 monoclonal antibody hybridoma cell line.
  • the EMC10 protein (containing 28-254 amino acids) with the signal peptide removed was divided into 5 different truncations, which were deleted 28-105 ( ⁇ 28-105), 66-145 ( ⁇ 66-145), 106- EMC10 truncations of amino acids 183 ( ⁇ 106-183), 146-225 ( ⁇ 146-225) and 184-254 amino acids ( ⁇ 184-254) (shown in A in Figure 4) were co-immunoprecipitated (IP) with the 4C2 antibody as described above.
  • PCR was used to amplify the gene sequences of EMC10 wild-type (WT) and different truncations (as shown in Figure 4) with a Flag tag at the C-terminus, and construct them into an untagged pLEX-MCS vector (Thermo Scientific) .
  • Example 2 Application of mouse anti-human EMC10 monoclonal antibody 4C2 in treating fatty liver in mice and improving its accompanying metabolic disorder
  • the mouse anti-human EMC10 monoclonal antibody was used to intervene in obese mice to observe whether fatty liver and its accompanying metabolic disorder could be improved.
  • mice 1. The application of mouse anti-human EMC10 monoclonal antibody 4C2 in the treatment of fatty liver in mice
  • mice on a high-fat diet (60% of dietary calories come from fat) with a body weight of about 35 grams for 7 weeks were randomly divided into three groups, namely the control IgG group, the control 1F12 group and the 4C2 antibody group, with 8-10 mice in each group.
  • Each mouse in the 4C2 antibody group was given mouse anti-human EMC10 monoclonal antibody 4C2 at a dose of 3 mg/kg body weight
  • each mouse in the control 1F12 group was given mouse anti-human EMC10 monoclonal antibody 1F12 at a dose of 3 mg/kg body weight
  • Mouse IgG was administered to each mouse in the control IgG group at a dose of 3 mg/kg body weight.
  • the control IgG group, 1F12 group and 4C2 antibody group were injected twice a week for a total of two weeks.
  • the body weights of mice in different groups were measured.
  • the results of changes in body weight and weight gain of mice in different groups are shown in Figure 5A and As shown in B, the results show that the weight of mice in the control IgG group (indicated by "IgG” in the figure) and the 1F12 group (indicated by "1F12” in the figure) continued to increase, while the 4C2 antibody group (indicated by "4C2” in the figure)
  • the body weight of the mice decreased significantly, and the body weight decreased to 4 grams within 2 weeks, and its body weight was negative, and there was a significant statistical difference compared with the control IgG group and the 1F12 group.
  • mice were sacrificed, and the liver weights of the three groups of mice in the control IgG group, the 1F12 group and the 4C2 antibody group were weighed. Compared with the control IgG group (indicated by "IgG” in the figure) or compared with the 1F12 group (indicated by "1F12” in the figure), the liver weight of mice in the 4C2 antibody group (indicated by "4C2" in the figure) was significantly reduced.
  • the liver tissue of the mice in the control IgG group and the 4C2 antibody group was sectioned by HE staining. The results are shown in D in Figure 5. The results showed that the livers of the mice in the control IgG group (represented by "IgG” in the figure) had a large number of livers. The fatty infiltration was consistent with the appearance of fatty liver, while the fatty infiltration of the liver of the mice in the 4C2 antibody group (represented by "4C2" in the figure) was not obvious.
  • mouse anti-human EMC10 monoclonal antibody 4C2 in improving metabolic disorders associated with fatty liver
  • mice with a body weight of about 35 grams on a high-fat diet for 7 weeks were randomly divided into three groups, namely the control IgG group, the control 1F12 group and the 4C2 antibody group, with 8-10 mice in each group.
  • Each mouse in the 4C2 antibody group was given mouse anti-human EMC10 monoclonal antibody 4C2 at a dose of 3 mg/kg body weight;
  • each mouse in the control 1F12 group was given mouse anti-human EMC10 monoclonal antibody at a dose of 3 mg/kg body weight 1F12;
  • Each mouse in the control IgG group was administered mouse IgG at a dose of 3 mg/kg body weight.
  • mice in the control IgG group, the 1F12 group and the 4C2 antibody group were injected twice a week for a total of two weeks.
  • IPITT intraperitoneal insulin tolerance test
  • the mice in the control IgG group, 1F12 group and 4C2 antibody group were given intraperitoneal injection of insulin at a dose of 1 mU/g body weight per mouse, monitoring 0, 30, 60 and 90, respectively.
  • the results are shown in BE in Figure 6.
  • the results show that: the 4C2 treatment group (indicated by “4C2” in the figure) ) mice compared with the control IgG group (indicated by “IgG” in the figure) and the 1F12 group (indicated by "1F12” in the figure), serum triglycerides and non-esterified fatty acids were significantly reduced (C and D), serum insulin and cholesterol were also significantly reduced (B and E in Figure 6).
  • mice anti-human EMC10 monoclonal antibody 4C2 can significantly reduce the body weight of obese mice, and significantly improve fatty liver and its accompanying metabolic disorders, which provides a new therapeutic target for the treatment of fatty liver and other metabolic diseases. .
  • mice with a body weight of about 35 grams on a high-fat diet (60% of dietary calories come from fat) for 7 weeks were randomly divided into three groups, namely the control IgG group, the control 1F12 group and the 4C2 antibody group, with 8-10 mice in each group.
  • Each mouse in the 4C2 antibody group was given mouse anti-human EMC10 monoclonal antibody 4C2 at a dose of 3 mg/kg body weight;
  • each mouse in the control 1F12 group was given mouse anti-human EMC10 monoclonal antibody 1F12 at a dose of 3 mg/kg body weight;
  • Mouse IgG was administered to each mouse in the control IgG group at a dose of 3 mg/kg body weight.
  • mice in the control IgG group, 1F12 group and 4C2 antibody group were injected twice a week for a total of two weeks.
  • the results of body weight and weight gain of mice in different groups are shown in Figure 7 and Figure 8
  • the results show that the mice in the control IgG group (indicated by "IgG” in the figure) and the 1F12 group (indicated by "1F12” in the figure) continued to gain weight, while the 4C2 antibody group (indicated by "4C2” in the figure)
  • the body weight of the mice decreased significantly, and the body weight decreased to 4 grams within 2 weeks, and its body weight was negative.
  • Compared with the control IgG group and the 1F12 group there was a significant statistical difference (*P ⁇ 0.05, ***P ⁇ 0.001, ****P ⁇ 0.0001).
  • Dual energy X-ray absorptiometry was used to measure the fat weight and non-fat weight of mice in the control IgG group, 1F12 group and 4C2 antibody group two weeks after injection.
  • the results are shown in Figure 9.
  • the results show that: the same as the control IgG group Compared with the 1F12 group (indicated by "IgG” in the figure) and the 1F12 group (indicated by "1F12” in the figure), the fat weight of the mice in the 4C2 antibody group (indicated by "4C2” in the figure) was significantly reduced (*P ⁇ 0.05) , and the non-fat weight did not differ among the control IgG group, 1F12 group and 4C2 antibody group; different tissues and organs (heart, liver, epididymal fat, inguinal subcutaneous fat, retroperitoneal fat, mesenteric fat, brown fat, spleen, kidney, pancreas) weight, the results are shown in Figure 10, the results show that: the same as the control
  • mice were sacrificed 2 weeks after the antibody injection, and brown fat, subcutaneous fat and epididymal adipose tissue were taken.
  • the volume was significantly smaller than that of the control IgG group (indicated by "IgG” in the figure); the control IgG group (indicated by “IgG” in the figure) had a large number of lipid droplets accumulated in the brown fat, while the 4C2 antibody group (indicated by "IgG” in the figure).
  • Indicated as "4C2” mice had significantly reduced lipid droplets.
  • mice with a body weight of about 35 grams on a high-fat diet (60% of dietary calories come from fat) for 7 weeks were randomly divided into three groups, namely the control IgG group, the control 1F12 group and the 4C2 antibody group, with 8-10 mice in each group.
  • Each mouse in the 4C2 antibody group was given mouse anti-human EMC10 monoclonal antibody 4C2 at a dose of 3 mg/kg body weight;
  • each mouse in the control 1F12 group was given mouse anti-human EMC10 monoclonal antibody at a dose of 3 mg/kg body weight 1F12;
  • Each mouse in the control IgG group was administered mouse IgG at a dose of 3 mg/kg body weight.
  • the control IgG group, the 1F12 group and the 4C2 antibody group were injected twice a week for a total of two weeks.
  • the metabolism-related indexes in the serum were studied, and the blood glucose (6 hours fasting) and insulin, insulin, and insulin in the non-fasting state of the mice were detected.
  • Non-esterified fatty acids, triglycerides, and cholesterol as shown in Figure 12, the blood glucose, non-esterified fatty acids and triglycerides of mice in the 4C2 antibody group (represented by “4C2" in the figure) were significantly lower than those of the control Serum insulin and cholesterol were also significantly decreased in the IgG group (represented by "IgG” in the figure) and the 1F12 group (represented by "1F12” in the figure) (*P ⁇ 0.05).
  • mice with a body weight of about 35 grams on a high-fat diet (60% of dietary calories come from fat) for 7 weeks were randomly divided into two groups, namely the control IgG group and the 4C2 antibody group, with 8-10 mice in each group.
  • the Oxymax indirect calorimetry system (Oxymax, Columbus Instruments) was used to study the energy metabolism of mice. The mice were placed in metabolic cages for 3 days.
  • the first day was the adaptation period, the second and third days were the experimental period, and the second day
  • the antibody was injected once, and each mouse in the 4C2 antibody group was given mouse anti-human EMC10 monoclonal antibody 4C2 at a dose of 3 mg/kg body weight; each mouse in the control IgG group was given mouse IgG at a dose of 3 mg/kg body weight.
  • Mice had free access to food and water in the metabolic cage, and each day was divided into 12 hours of light/day (white area in Figure 13) and 12 hours of night (grey area in Figure 13).
  • Real-time monitoring of the mice's food intake, water intake, activity, oxygen consumption, carbon dioxide exhalation, heat production and other indicators in the metabolic cage is carried out through the built-in instrument of the system. Results As shown in Figure 13, the oxygen consumption, carbon dioxide exhalation and heat production of mice in the 4C2 antibody group (represented by "4C2" in the figure) were significantly higher than those in the control IgG group (P ⁇ 0.01).
  • the anti-EMC10 monoclonal antibody 4C2 can significantly reduce the body weight and body fat content of obese mice by increasing energy expenditure (calorie production) and can improve obesity-related glucose and lipid metabolism disorders, which provides a new perspective for obesity. therapeutic targets.
  • the anti-human EMC10 monoclonal antibody was used to intervene in type 2 diabetic mice to observe whether the glucose metabolism disorder of the mice could be improved.
  • mice with a body weight of about 35 grams on a high-fat diet (60% of dietary calories come from fat) for 7 weeks were randomly divided into three groups, namely the control IgG group, the control 1F12 group and the 4C2 antibody group, with 8-10 mice in each group.
  • Each mouse in the 4C2 antibody group was given mouse anti-human EMC10 monoclonal antibody 4C2 at a dose of 3 mg/kg body weight;
  • each mouse in the control 1F12 group was given mouse anti-human EMC10 monoclonal antibody 1F12 at a dose of 3 mg/kg body weight;
  • Mouse IgG was administered to each mouse in the control IgG group at a dose of 3 mg/kg body weight.
  • the control IgG group, 1F12 group and 4C2 antibody group were injected twice a week for a total of two weeks, and the fasting blood glucose and serum insulin of mice in different groups were detected.
  • the results are shown in A and C in Figure 14.
  • the results show that: 4C2
  • the fasting blood glucose of the mice in the antibody group represented by "4C2” in the figure
  • the 4C2 antibody group Compared with the control IgG group (indicated by "IgG” in the figure) and the 1F12 group (indicated by "1F12” in the figure) mice, the serum insulin content of the mice was also significantly reduced.
  • mice in the control IgG group, the 1F12 group and the 4C2 antibody group were given intraperitoneal injection of glucose at a dose of 2 g/kg body weight, and then used a blood glucose meter (Roche Excellence) The blood glucose of each group of mice was measured before glucose injection (0 minutes) and at 15, 30, 60 and 120 minutes after glucose injection. The results are shown in B in Figure 14.
  • IPITT Insulin tolerance test by intraperitoneal injection was used.
  • the control IgG group, 1F12 group and 4C2 antibody group were given intraperitoneal injection of insulin at a dose of 0.75mU/g body weight to each mouse.
  • the results showed that the percentage of blood glucose drop of mice in the 4C2 antibody group (indicated by "4C2" in the figure) was significantly greater than that in the control IgG group (indicated by "IgG” in the figure) and 1F12 group (indicated by "1F12” in the figure) , indicating that the insulin sensitivity of mice in the 4C2 antibody group was significantly increased.
  • mice anti-human EMC10 monoclonal antibody 4C2 can improve insulin resistance, increase insulin sensitivity and reduce blood sugar in type 2 diabetic mice, which provides a new drug target for the treatment of type 2 diabetes.
  • the anti-EMC10 monoclonal antibody provided by the present invention can significantly reduce liver weight and liver fat infiltration in mice, significantly reduce blood sugar in type 2 diabetes mouse model, improve glucose tolerance and insulin sensitivity of mice, and significantly reduce obesity and obesity.
  • the body weight of mice can also improve obesity-related metabolic disorders. Therefore, anti-EMC10 monoclonal antibodies can be used to prepare products for preventing and/or treating metabolic diseases (such as fatty liver, obesity, and type 2 diabetes).
  • the invention has important application value.

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Abstract

L'invention concerne l'utilisation d'un anticorps monoclonal dirigé contre l'EMC10 humain dans la préparation de produits pour la prévention et/ou le traitement de maladies métaboliques. Les maladies métaboliques sont le foie gras, l'obésité et/ou le diabète de type 2. L'anticorps monoclonal contre EMC10 selon l'invention peut réduire de manière significative le poids du foie de souris et l'infiltration de graisse du foie, réduire de manière significative le glucose sanguin de modèles de souris diabétiques de type 2, améliorer la tolérance au glucose et la sensibilité à l'insuline de souris, réduire de manière significative le poids de souris obèses et améliorer les troubles métaboliques associés à l'obésité. Par conséquent, l'anticorps monoclonal dirigé contre EMC10 peut être utilisé dans la préparation de produits pour la prévention et/ou le traitement du foie gras, de l'obésité et/ou du diabète de type 2. La présente invention concerne une nouvelle marque de cible de traitement pour la prévention ou le traitement du diabète, de l'obésité et du foie gras, et a des valeurs d'application importantes.
PCT/CN2021/116096 2020-09-07 2021-09-02 Utilisation d'anticorps monoclonal dirigé contre emc10 humain dans la préparation de produits pour la prévention et/ou le traitement de maladies métaboliques WO2022048577A1 (fr)

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CN114149498A (zh) * 2020-09-07 2022-03-08 复旦大学附属华山医院 抗人emc10的单克隆抗体在防治2型糖尿病中的应用
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CN114149499B (zh) * 2020-09-07 2023-12-05 复旦大学附属华山医院 抗人emc10的单克隆抗体及其在治疗和/或预防肥胖症中的应用
CN114149498B (zh) * 2020-09-07 2023-12-05 复旦大学附属华山医院 抗人emc10的单克隆抗体在防治2型糖尿病中的应用

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