WO2022048577A1

WO2022048577A1 - Use of monoclonal antibody against human emc10 in preparation of products for preventing and/or treating metabolic diseases

Info

Publication number: WO2022048577A1
Application number: PCT/CN2021/116096
Authority: WO
Inventors: 王宣春; 李燕良; 陈匡阳; 景昱; 胡仁明
Original assignee: 复旦大学附属华山医院
Priority date: 2020-09-07
Filing date: 2021-09-02
Publication date: 2022-03-10

Abstract

Provided is a use of a monoclonal antibody against human EMC10 in the preparation of products for preventing and/or treating metabolic diseases. The metabolic diseases are fatty liver, obesity and/or type 2 diabetes. The provided monoclonal antibody against EMC10 can significantly reduce the liver weight of mice and fat infiltration of the liver, significantly reduce the blood glucose of type 2 diabetic mouse models, improve the glucose tolerance and insulin sensitivity of mice, significantly reduce the weight of obese mice, and improve metabolic disorders associated with obesity. Therefore, the monoclonal antibody against EMC10 can be used in the preparation of products for preventing and/or treating fatty liver, obesity and/or type 2 diabetes. The present invention provides a brand-new treatment target for the prevention or treatment of diabetes, obesity and fatty liver, and has important application values.

Description

Application of anti-human EMC10 monoclonal antibody in the preparation of products for preventing and/or treating metabolic diseases

technical field

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.

Background technique

Globally, with the control of viral hepatitis, fatty liver disease (FLD) has gradually become the main cause of the development of chronic liver disease (CLD). FLD can progress from simple hepatic steatosis to steatohepatitis and hepatic fibrosis. Clinically, FLD can be divided into alcoholic fatty liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). Among them, 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. Among patients with ultrasound-confirmed NAFLD, 6-30% have histologically confirmed progression to non-alcoholic steatohepatitis (NASH), and 40% of NASH patients have a tendency to progress to liver fibrosis.

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.

There is still a lack of effective drugs for the treatment of NAFLD. Strengthening exercise and diet control can effectively reduce liver fat content, reduce liver enzyme levels, and improve symptoms of metabolic syndrome. It is still the primary recommended method for the treatment of NAFLD, but for most patients Difficult to persist for a long time.

Affected by factors such as diet structure, lifestyle and environment, the incidence of obesity is increasing year by year. Obesity can greatly increase the morbidity and mortality of type 2 diabetes mellitus, cardiovascular disease and tumors, and bring a heavy burden to human health and economic development. Searching for the new etiology of obesity and clarifying its pathological mechanism is particularly urgent and extremely important for the development of new drugs to intervene and treat obesity, and to curb and reduce the harm of obesity to human beings.

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. There are many 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. In recent years, some non-classical endocrine tissues and organs have also been found to secrete some hormones or cytokines, including 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.; and cardiac factors secreted by the heart (such as atrial natriuretic peptide (ANP)); they also play an important role in regulating lipid metabolism, energy balance and the pathogenesis of obesity . Since 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. There is a need to continue the search for novel cytokines or secreted proteins that can be targeted for obesity treatment.

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. There are many 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. In recent years, 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. For example, 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. EndocrRelat Cancer. 2004, 11:295-303.). So far, several studies have revealed multiple biological functions of EMC10. Using the ELISA method established by Wang X et al., we report for the first time internationally that EMC10 is a secreted protein that can be detected in human serum, and found that the expression of Emc10 (Inm02) gene in mouse pancreatic β 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. 2009, 202: 355-364.). Later, another research group reported that EMC10 was cloned from purified human hematopoietic stem cells. The research group named it HSS1 and its other splice isomer named HSM1; ) can inhibit the proliferation, migration, and invasion of glioma cell lines, as well as the neovascularization of endothelial cells, so they believe that EMC10 is a potential target for the treatment of glioblastoma (Junes-Gill KS, Gallaher TK). , 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.). Another study found that in a mouse model of schizophrenia, elevated Mrita22 (the mouse homolog of human EMC10) could inhibit the development of neuronal dendrites and spinous processes, and reduced levels of Mrita22 could completely rescue the above Defects in dendrite and spinous process development in hippocampal vertebral neurons in a mouse model, suggesting that EMC10 plays an important role in the formation of dendrites and spinous processes in mouse neurons (Xu B, Hsu PK, Stark KL, Karayiorgou M, Gogos JA. Derepression of a neuronal inhibitor due to miRNA dysregulation in a schizophrenia-related microdeletion. Cell. 2013, 152(1-2):262-75.;

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) Is a Bone Marrow-Derived Angiogenic Growth Factor Promoting Tissue Repair After Myocardial Infarction. Circulation. 2017;136(19):1809-1823.). Zhou Y et al. found that EMC10 deletion led to infertility in male mice, and sperm lacking the Emc10 gene showed multiple defects, including morphological abnormalities, reduced sperm motility, impaired sperm capacitation, and loss of acrosome reaction. Molecular mechanism studies found that EMC10 deletion resulted in inactivation of sodium/potassium-ATPase and impaired HCO3 ^- induced activation of cAMP/PKA signaling pathway and decreased tyrosine phosphorylation of sperm capacitation-related proteins (Zhou Y, Wu F, Zhang M, Xiong Z, Yin Q, Ru Y, Shi H, Li J, Mao S, Li Y, Cao X, Hu R, Liew CW, Ding Q, Wang X, Zhang Y. EMC10 governs male fertility via maintaining sperm ion balance . J Mol Cell Biol. 2018 Dec 1;10(6):503-514.).

Despite these advances, whether there is a relationship between EMC10 and fatty liver, obesity and type 2 diabetes has not been reported.

Invention Disclosure

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. In the above application, 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.

In the above-mentioned application, 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;

The 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.

In the above application, the framework regions of _VH and _VL are derived from mice.

In the above application, the amino acid sequence of V _H can be shown in SEQ ID No.1; the amino acid sequence of _VL can be shown in SEQ ID No.2.

Among them, SEQ ID No.1 consists of 114 amino acid residues, and SEQ ID No.2 consists of 113 amino acid residues.

In the above-mentioned application, the monoclonal antibody of described anti-human EMC10 can be any of the following:

S1) a single-chain antibody obtained by connecting the V _H and the _VL ;

S2) a fusion antibody containing the single-chain antibody described in S1);

S3) a Fab containing said _VH and said _VL ;

S4) an intact antibody comprising said _VH and said _VL ;

S5) The monoclonal antibody secreted by the hybridoma cell line 4C2 whose deposit number is CGMCC No.19950.

In the above application, the anti-human EMC10 monoclonal antibody may be a murine monoclonal antibody.

In the above application, the biological material related to the monoclonal antibody against human EMC10 can be any one of B1) to B12):

B1) a nucleic acid molecule encoding the monoclonal antibody;

B2) an expression cassette containing the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule described in B1);

B4) a recombinant vector containing the expression cassette described in B2);

B5) a recombinant microorganism containing the nucleic acid molecule of B1);

B6) a recombinant microorganism containing the expression cassette described in B2);

B7) a recombinant microorganism containing the recombinant vector described in B3);

B8) a recombinant microorganism containing the recombinant vector described in B4);

B9) a transgenic animal cell line containing the nucleic acid molecule of B1);

B10) a transgenic animal cell line containing the expression cassette of B2);

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.

In the above application, 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.

In the above application, 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:

C1) 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.

Among them, SEQ ID No.3 consists of 342 nucleotides, and SEQ ID No.4 consists of 339 nucleotides.

In the above applications, "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.

In the above applications, more than 90% identity may be DNA molecules that are at least 91%, 92%, 95%, 96%, 98% or 99% identical.

In the above application, 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. Further, 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.

In the above application, the recombinant vector can be a plasmid, cosmid, phage or viral vector.

In the above application, the recombinant microorganism can be yeast, bacteria, algae or fungi.

In the above applications, the transgenic animal cell line may be non-reproductive material.

Any of the above-mentioned monoclonal antibodies against human EMC10 also belong to the protection scope of the present invention.

Any of the above-mentioned biological materials related to the anti-human EMC10 monoclonal antibody also belong to the protection scope of the present invention.

The present invention also protects the application of any of the above-mentioned anti-human EMC10 monoclonal antibodies in any of the following:

C1) use in the preparation of a product for reducing fatty infiltration of the liver of an animal;

C2) use in the preparation of products that reduce the serum triglyceride content of animals;

C3) application in the preparation of the product that reduces the serum free fatty acid content of animals;

C4) use in the preparation of products for reducing the serum cholesterol content of animals;

C5) application in the preparation of products for reducing the blood sugar of animals;

C6) application in the preparation of products for improving the glucose tolerance of animals;

C7) use in the preparation of products for improving the insulin sensitivity of animals;

C8) use in the preparation of a product for reducing the body weight of an animal;

C9) use in the preparation of a product for reducing the body fat content of an animal;

C10) use in the preparation of a product for reducing the volume of visceral fat in animals;

The use of C11) in the preparation of a product that activates brown fat in animals to promote thermogenesis.

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.

In the above, the product may be a drug, vaccine, reagent or kit.

In the above, the animal may be a mammal, such as a mouse or a human.

In the above, 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).

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.

In the above applications, 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.

Preservation Instructions

Reference Biomaterials Co., Ltd.: 4C2

Proposed nomenclature: mouse anti-human EMC10 monoclonal antibody hybridoma cell line

Preservation institution: General Microbiology Center of China Microorganism Culture Collection Management Committee

Abbreviation of depositary institution: CGMCC

Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: June 18, 2020

Deposit number: CGMCC No.19950

Description of drawings

Figure 1 shows the results of the identification of purified protein using the Dot blot method. In the figure, 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.

Figure 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.

Figure 4 shows the results of western blot detection of wild-type and different truncations of EMC10 protein with anti-Flag-tag antibody. In the figure, WT represents wild-type, namely EMC10 protein; Δ28-105, Δ66-145, Δ106-183, Δ146- 225, Δ184-254, Δ146-175, Δ171-200, Δ196-225, Δ146-155, Δ156-165, Δ166-175 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 .

Figure 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.

Best way to implement your invention

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Acquisition of mouse anti-human EMC10 monoclonal antibody 4C2

1. Preparation of mouse anti-human EMC10 monoclonal antibody

Eight strains of mouse anti-human EMC10 monoclonal antibodies were prepared. Specific steps are as follows:

1. Construction of EMC10 eukaryotic expression recombinant plasmid

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.

2. Transfection and expression

(1) Culture >1×10 ⁸ HEK 293F cells for later use.

(2) Dilute 100 μg of recombinant plasmid pRAG2a-EMC10 to 1 ml with diluent (Opti-MEM), and mix gently.

(3) Dilute 200 μl of Lipofectamine ^TM 2000 liposome to a final volume of 1 ml with diluent (Opti-MEM). Mix gently and leave at room temperature for 5 min.

(4) Add the diluted plasmid to the diluted Lipofectamine ^TM 2000 liposomes to make the final volume of the mixture 2 ml, and mix gently.

(5) Incubate at room temperature for 30 min.

(6) Transfer 1×10 ⁸ HEK 293F cells into a 500 ml shake flask, and add fresh, pre-warmed Expression Medium to bring the final volume to 98 ml.

(7) Add 2 ml of the incubated DNA-Lipofectamine ^™ 2000 mixture.

(8) In an incubator with 8% CO ₂ concentration, culture at 37° C. and 125 rpm for 4-5 days.

(9) 4°C, collect the supernatant. The supernatant contains the mature EMC10 protein with the signal peptide removed (as shown in positions 28-254 of SEQ ID NO. 6 of the Sequence Listing).

3. Purified protein and identification by SDS-PAGE

(1) Take the supernatant obtained in (9) of step 2, add binding buffer (8M urea, 20mM sodium phosphate, 500mM NaCl, pH 7.8), then filter with a 0.45 μm filter to collect the filtrate.

(2) Equilibrium: 5 column volumes of binding buffer equilibrate the nickel column.

(3) Loading: The filtrate obtained in step (1) is loaded.

(4) Washing impurities: 5 column volumes of binding buffer wash impurities until no material flows out through the flow-through.

(5) Elution: 5 column volumes of elution buffer (8M urea, 20mM _NaH2PO4 , 500mM NaCl, pH _4.0 ) were eluted, and the eluted product was collected.

(6) SDS-PAGE detection.

Only a band of about 36KD was displayed, indicating that the electrophoresis pure target protein was obtained.

4. Identification of Dot blot and Western blot

(1) 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 results are shown in Figure 1. It can be seen from the figure that the sample protein diluted to about 0.47ng has a positive result (No. 7) after 2000-fold dilution, and no positive result (No. 8) is about 0.23ng.

(2) 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).

The results are shown in Figure 2. As can be seen from the figure, 10ng sample was loaded, and there was a positive band around 36KD.

The results of Dot blot and Western blot identification indicated that the eukaryotic expression of EMC10 protein was successful.

5. Animal immunity

Select 6 female BALB/c mice aged 6-8 weeks, mix the purified protein obtained in step 3 with Freund's complete adjuvant in a volume ratio of 1:1 for the first immunization, subcutaneously inject 100 μg, and boost the immunization every 2-3 weeks. 100 μg of the mixture was injected subcutaneously. After four immunizations, blood was collected for detection, and the titer of antiserum against EMC10 protein was determined by indirect ELISA method (the titer was expressed by the maximum dilution ratio of the serum with the OD value of the sample well/the OD value of the negative well ≥ 2.1), and the titer was greater than 1:10000 , select 1-2 mice for cell fusion arrangement.

The steps of determining the titer of antiserum against EMC10 protein by the above-mentioned indirect ELISA method are as follows:

(1) Plate coating: draw the self-prepared standard EMC10 (expressed and purified in step 3 of Example 1) and dissolve it in 0.1M PBS buffer to prepare a coating solution with a concentration of 1 μg/ml, 100 μl/well , 4 ℃ coating overnight.

(2) Wash the plate: discard the liquid in the well, spin dry, wash the plate twice, soak for 1-2 minutes each time, about 200 μL/well, spin dry and pat the liquid in the well dry on absorbent paper.

(3) Blocking: 250 μl/well of blocking solution, 37° C., 2 h.

(4) 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).

(5) 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 μL 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.

(6) 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).

(7) Add 100 μL of rabbit anti-mouse-HRP to each well, add the membrane, and incubate at 37°C for 1 hour.

(8) Discard the liquid in the well, spin dry, and wash the plate 5 times, the method is the same as that of step (2).

(9) Color development: After 1:1 (volume ratio) mixing of substrate color A and B solution, add 100 μL to each well, cover the ELISA plate, and incubate at 37°C for 15 minutes in the dark.

(10) Add 50 μL of stop solution to each well to stop the reaction, at which time the blue turns to yellow. The order of addition of the stop solution should be the same as the order of addition of the substrate solution.

(11) Immediately measure the optical density (OD value) of each well with a microplate reader at 450/630nm dual wavelength, and read. Turn on the power of the microplate reader in advance, preheat the instrument, and set the detection program.

(12) Judgment of results: positive when the OD value of the sample well/negative well (ie, the blank control well) OD value ≥ 2.1. The results showed that the sample wells with a dilution ratio of serum anti-EMC10 antibody greater than 10,000 were positive, indicating that the antibody titer was greater than 1:10,000.

6. Cell fusion

(1) Preparation of myeloma cells: One week before fusion, SP2/0 cells were expanded and cultured in DMEM medium containing 10% FBS. By the time of confluence, the cells were filled with about 6 flasks of T25 cell culture flasks. On the day of confluence, SP2/0 cells were collected into a 50 ml centrifuge tube, and centrifuged at 1000 rpm for 5 min. Discard the supernatant, then add 20ml of DMEM basal medium, blow off the cells and count.

(2) Preparation of splenocytes: Mice with serum ELISA titers of more than 1:10000 after four immunizations were finally immunized 3 days before fusion, and the volume ratio of EMC10 protein purified in step 3 to Freund's complete adjuvant was 1 intraperitoneally. : 1 Mixture 100μg. Mice to be fused were euthanized by cervical dislocation on the day of fusion. Soak with 75% alcohol for 5min. Take the spleen aseptically and put the spleen into a petri dish containing 10 ml of DMEM basal culture. Take the sieve and place it in another plate, transfer the spleen to the sieve, and grind the spleen internally with a syringe. 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.

(3) Cell fusion: mix myeloma cells and spleen cells so that the ratio of myeloma cells to spleen cells is 1:20. The cells were placed in a 50 ml centrifuge tube, diluted with DMEM basal medium, and centrifuged at 1000 rpm for 5 min. Discard the supernatant. Shake the centrifuge tube to homogenize the cells. Slowly add 0.8 ml of 50% PEG, react for 90 seconds, then add 20-30 ml of DMEM medium to stop the PEG. The fused cells were placed in a 37°C water bath for 10 minutes. Centrifuge at 1000rpm for 5min, discard the supernatant and add HAT DMEM medium. Confluent cells were plated in 96-well plates at 100 μl per well. The cell culture plate was then placed in a _CO2 incubator.

4 days after fusion, 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.

7. Fusion screening and subcloning

(1) Fusion screening: One day before detection, 5 μg/ml antigen (EMC10 protein purified in step 3) was coated on ELISA plate with PBS overnight. The next day, 100 μl/well of cell supernatant was drawn for ELISA detection. According to the ELISA results, positive wells were judged (sample well OD value/negative well (blank control well) OD value ≥ 2.1, it was judged as positive well). Use a single-channel pipette to check the positive wells detected in the whole plate, and perform a second confirmation test to further confirm the positive wells. Subcloned cells from positive wells were determined.

(2) Subcloning: pipetting the cells in the positive wells, counting, adding 4ml DMEM medium to the centrifuge tube, taking 100μl of cell suspension into the centrifuge tube, blowing evenly and leaving 1ml, adding DMEM to 4ml, blowing evenly, leaving 100 μl (about 2 drops) at the bottom of the tube. Add DMEM to 5ml in the centrifuge tube, and after mixing, add dropwise to the first three rows of the 96-well plate, leaving about 1.8-2ml at the bottom of the dropper in each well, add DMEM to 5ml, blow evenly and add dropwise to the bottom of the 96-well plate Three rows D, E and F, one drop per well, about 1.5-1.8ml left at the bottom of the tube, add DMEM to about 2.8-3ml, blow well and add dropwise to rows G and H of the 96-well plate, one drop per well, 7 - Observe under the microscope after 10 days, detect the wells with clonal growth, mark the wells with monoclonal, pick the positive monoclonal cells as much as possible for subcloning again, after the detection is 100% positive, pick out the monoclonal wells to expand the culture Fixed strains.

Finally, eight 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.

8. Preparation and purification of ascites

(1) Preparation of ascites: 0.5 ml of liquid paraffin was intraperitoneally injected into each mouse, and hybridoma cells were injected intraperitoneally into the pretreated mice within 30 days after 7 days. Hybridoma cells were injected in an amount of 1×10 ⁶ cells per mouse. 7 to 10 days, carefully withdraw as much liquid as possible from the abdominal cavity with a syringe needle, and perform titer determination by indirect ELISA method (the titer is expressed by the maximum dilution factor of the serum with the OD value of the sample hole/the OD value of the negative hole ≥ 2.1, Negative wells are blank controls). Mice were sacrificed by cervical dislocation after the last collection.

(2) Purification: Centrifuge the collected ascites to get the supernatant, prepare the protein A agarose medium and pack it into a column, dilute the ascites 10 times with PBS and slowly load the sample. After the sample is loaded, wash with phosphate buffer to UV The detector reaches the minimum value, and the glycine elution buffer is eluted to obtain the desired purified antibody, which is immediately dialyzed in PBS at 4 °C overnight, and the purity, concentration and titer are determined every other day (titer is determined by the OD value of the sample hole / negative The maximum dilution factor of the serum with OD value ≥ 2.1 in the well is indicated, and the negative well is the blank control).

HeLa cells were simultaneously intervened with 1ug/ml of mouse EMC10 protein and 1ug/ml of different mouse anti-human EMC10 monoclonal antibodies (1F12, 4B12-1, 4B12-2 and 4C2), and the phosphorylation level of CREB was detected by western blotting (Previous studies have shown that EMC10 can inhibit the phosphorylation of CREB), the results are shown in Figure 3, it can be seen that EMC10 protein reduces the phosphorylation of CREB, and 4B12-1 and 4C2 antibodies can restore the originally decreased phosphorylation of CREB to before the intervention of EMC10 protein , indicating that 4B12-1 and 4C2 antibodies can block the effect of EMC10 protein, while 1F12 and 4B12-2 cannot change the decrease in CREB phosphorylation caused by EMC10 protein, indicating that these two antibodies cannot block the effect of EMC10 protein. Results: Screening Two mouse anti-human EMC10 monoclonal antibodies that can block the biological effect of mouse EMC10 protein were developed: 4B12-1 and 4C2, and 1F12 could not block the biological effect of mouse EMC10 protein as a control antibody.

The western blotting method is as follows:

1 ug/ml of mouse EMC10 protein and 1 ug/ml of different mouse anti-human EMC10 monoclonal antibodies (1F12, 4B12-1, 4B12-2 and 4C2) were added to the culture medium to intervene in HeLa cells for 6 hours. The protein was separated by electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide gel, transferred to a polyvinylidene fluoride (PVDF) membrane, and then treated with rabbit anti-p-CREB monoclonal antibody (CST, Cat. No.: 9198, 1:1000 dilution), rabbit anti-CREB1 monoclonal antibody (ABclonal, Cat. No. A10826, 1:1000 dilution) and rabbit anti-α-Tubulin polyclonal antibody (CST, Cat. No. 2144, 1:2000 dilution) were incubated, two Anti-goat anti-rabbit antibody (Sigma) conjugated with horseradish peroxidase (dilution 1:10000) was used, and finally the band of interest was visualized by ECL Plus (Amersham) chemiluminescence.

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.

2. Sequence of mouse anti-human EMC10 monoclonal antibody 4C2

1. Extraction of total RNA from 4C2 hybridoma cells

Trizol Reagent (Thermofisher, USA) was used to extract the total RNA of 4C2 cell samples, and the concentration of the above total RNA samples was determined by Nanodrop, and a total of 15 μg total RNA was obtained (concentration: 511.3ng/μL, volume: 30 μL, A260/A280=2.01), 500ng was taken for agarose gel electrophoresis analysis. The results showed that the 28S and 18S bands in the total RNA samples were clearly visible, and the brightness of the 28S band was greater than that of 18S, indicating that the integrity of the two RNAs was good.

2. Mouse antibody fragment amplification and sequence analysis

Design specific primers in the constant regions of the antibody heavy chain (mouse IgG1 subtype) and light chain (kappa), respectively. The primer sequences are as follows: MouseIgG1 CHouter (5'-3'): ACAATCCCTGGGCACAAT, MouseCL-Kappaoter (5'-3') ): ACACTCATTCCTGTTGAAGCTCTTGAC. The antibody heavy and light chain fragments were amplified separately using 5'RACE. The amplified fragment was inserted into the cloning vector pUC57 (Addgene, USA) and sequenced. 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 55-61 of SEQ ID No.2, and the amino acid sequence of CDR3 of _VL is shown in positions 94-102 of SEQ ID No.2.

3. Construction of eukaryotic expression vector

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. For the antibody heavy chain expression plasmid pAH-4C2, 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. Analysis, the nucleotide sequence of the obtained heavy chain is shown in SEQ ID No. 8 (including the coding sequence of the secretion signal peptide), and the protein shown in SEQ ID No. 9 (positions 1-21 are the amino acids of the secretion signal peptide) are expressed. Sequence, 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).

4. Eukaryotic expression and detection of antibodies

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. In order to evaluate and confirm the activity of the expressed antibody, 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.

Table 1 Expression supernatant gradient dilution ELISA detection

Table 2 4C2 antibody concentration gradient dilution ELISA detection

Therefore, it was confirmed that 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). shown, 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.

3. Epitope sequence of mouse anti-human EMC10 monoclonal antibody 4C2

The EMC10 protein (containing 28-254 amino acids) with the signal peptide removed was divided into 5 different truncations, which were deleted 28-105 (Δ28-105), 66-145 (Δ66-145), 106- EMC10 truncations of amino acids 183 (Δ106-183), 146-225 (Δ146-225) and 184-254 amino acids (Δ184-254) (shown in A in Figure 4) were co-immunoprecipitated (IP) with the 4C2 antibody as described above. Different truncations were detected by western blotting with anti-Flag antibody, and it was found that the 4C2 antibody could not IP the EMC10 truncation (Δ146-225) missing amino acids 146-225 (shown in A in Figure 4), It shows that the epitope targeted by the 4C2 antibody is in the region of amino acids 146-225 of EMC10 protein; then three different truncations were constructed for amino acids 146-225, which were deleted 146-175 (Δ146-175) , 171-200 (Δ171-200) and 196-225 amino acids (Δ196-225) EMC10 truncations (shown in B in Figure 4), the same experiment as above was performed, and the epitope was again narrowed down to 146-175 The region of the amino acid (shown in B in Figure 4); repeat the above research, and finally determine that the epitope targeted by the 4C2 antibody is in the region 156-165 (shown in C in Figure 4), the corresponding EMC10 protein amino acid in this region The sequence is: VVGVSVVTHP.

EMC10 epitope acquisition experiment:

(1) 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) .

(2) Prepare 293T cells in a 10 cm dish, change serum-free DMEM medium (Gibco), and transfect wild-type and different truncated EMC10 plasmids into 293T cells. After 5 hours, replace with 10% fetal Bovine serum (Gibco) in DMEM medium, and the medium was changed after 1 day.

(3) After another day, the cells were collected with 400ul EBC buffer (50mM Tris-HCl pH=7.5, 120mM NaCl, 0.5% NP-40), lysed, centrifuged at 12000rpm at 4°C for 10min, and the supernatant was collected.

(4) Add 2ug of anti-human EMC10 monoclonal antibody 4C2 to the supernatant (the specific preparation method is as in step 1), and incubate at 4°C for 4h.

(5) Add protein A/G agarose (Santa Cruz Biotechnology), incubate for 1 h at 4°C, and wash the beads with PBS.

(6) Add 1X loading (Beyotime) containing SDS, boil samples at 100°C for 5 min, denature, and detect wild-type (ie EMC10 protein) and EMC10 proteins of different truncations by western blot with anti-Flag-tag antibody (Cell Signaling Technology).

Example 2. Application of mouse anti-human EMC10 monoclonal antibody 4C2 in treating fatty liver in mice and improving its accompanying metabolic disorder

In this example, 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.

1. The application of mouse anti-human EMC10 monoclonal antibody 4C2 in the treatment of fatty liver in mice

The 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. During the process, 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.

After the experiment (2 weeks after antibody treatment), the 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.

2. Application of 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. The control IgG group, the 1F12 group and the 4C2 antibody group were injected twice a week for a total of two weeks. Using the intraperitoneal insulin tolerance test (IPITT), 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. Minute blood sugar, taking the blood sugar before injection (0 minutes) as 100%, detecting the percentage of blood sugar drop at other time points, and testing the insulin tolerance of the three groups of mice, the results are shown in Figure 6, A, the results show that: compared with The blood glucose of the control IgG group (indicated by "IgG" in the figure), 1F12 group (indicated by "1F12" in the figure), and 4C2 treatment group (indicated by "4C2" in the figure) at 15, 30 and 15 after insulin injection 90 minutes were significantly reduced, indicating that 4C2 antibody can significantly improve the insulin resistance associated with fatty liver. After the study, the mice were sacrificed to separate serum, and the serum insulin, triglycerides, non-esterified fatty acids and cholesterol were detected. 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).

The above results show that the mouse 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. .

Example 3. Application of mouse anti-human EMC10 monoclonal antibody 4C2 in weight loss and improvement of obesity-related metabolic disorders

In this example, by intervening obese mice with an anti-human EMC10 monoclonal antibody, it was observed whether the body weight of the mice could be reduced and the obesity-related metabolic disorders could be improved.

1. Effects on high-fat diet-induced obesity in mice

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. During the process, the body weights of mice in different groups were measured. 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 (DEXA) 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 IgG group (indicated by "IgG" in the figure) and 1F12 group (indicated by "1F12" in the figure) Compared with the mice in the 4C2 antibody group (represented by "4C2" in the figure), the weights of subcutaneous fat and liver were significantly reduced (*P<0.05), and the weights of epididymal, mesenteric and retroperitoneal fat were also significantly reduced, while other tissues and organs ( The weight of heart, brown fat, spleen, kidney, pancreas) did not differ among the three groups of control IgG, 1F12 group and 4C2 antibody group.

The 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.

2. Effects on diet-induced metabolic disorders in mice

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).

3. Effects on energy metabolism in mice

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 above results indicate that 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.

Example 4. Application of anti-human EMC10 monoclonal antibody 4C2 in improving glucose metabolism disorder

In this example, 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) was significantly lower than that of the mice in the control IgG group (represented by "IgG" in the figure) and the mice in the 1F12 group (represented by "1F12" 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.

Using the intraperitoneal glucose tolerance test (IPGTT), each mouse in the control IgG group, the 1F12 group and the 4C2 antibody group was 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. The results show that: the 4C2 antibody group (represented by "4C2" in the figure) ) mouse blood sugar at 0, 15, 30 and 60 minutes was significantly lower than that of mice in the control IgG group (represented by "IgG" in the figure) and 1F12 group (represented by "1F12" in the figure), indicating that 4C2 antibody treatment can significantly Improves glucose tolerance in type 2 diabetic mice.

Insulin tolerance test (IPITT) 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 blood glucose before insulin injection (0 minutes) and 15, 30, 60 and 90 minutes after insulin injection, taking the blood glucose before injection (0 minutes) as 100%, and detecting the percentage of blood glucose drop at other time points, the results are shown in Figure 14 D As shown in the figure, 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.

The above results show that the mouse 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 present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

Industrial application

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.

Claims

The application of an anti-human EMC10 monoclonal antibody or a biological material related to the anti-human EMC10 monoclonal antibody in the preparation of a product for preventing and/or treating metabolic diseases, characterized in that the monoclonal antibody can specifically recognize amino acids The antigenic epitope whose sequence is shown in SEQ ID No.5;

The biological material is any one of B1) to B12):

B1) a nucleic acid molecule encoding the monoclonal antibody;

B2) an expression cassette containing the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule described in B1);

B4) a recombinant vector containing the expression cassette described in B2);

B5) a recombinant microorganism containing the nucleic acid molecule of B1);

B6) a recombinant microorganism containing the expression cassette described in B2);

B7) a recombinant microorganism containing the recombinant vector described in B3);

B8) a recombinant microorganism containing the recombinant vector described in B4);

B9) a transgenic animal cell line containing the nucleic acid molecule of B1);

B10) a transgenic animal cell line containing the expression cassette of B2);

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 is fatty liver, obesity and/or type 2 diabetes.
The application according to claim 1, wherein the monoclonal antibody against human EMC10 contains a heavy chain variable region named VH and a light chain variable region named VL , and the VH and VL is composed of determinant complementary region and framework region; said VH and said determinant complementary region of 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;

The 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 application according to claim 2, wherein the framework regions of the VH and VL are all derived from mice.
The application according to claim 2, characterized in that: the amino acid sequence of the V H can be shown as SEQ ID No.1; the amino acid sequence of the VL can be shown as SEQ ID No.2.
The application according to claim 2, wherein the monoclonal antibody is any of the following:

S1) a single-chain antibody obtained by connecting the V H and the VL ;

S2) a fusion antibody containing the single-chain antibody described in S1);

S3) a Fab containing said VH and said VL ;

S4) an intact antibody comprising said VH and said VL ;

S5) The monoclonal antibody secreted by the hybridoma cell line 4C2 whose deposit number is CGMCC No.19950.
The application according to claim 1, wherein: B1) the nucleic acid molecule is a gene encoding the monoclonal antibody, and the gene is the DNA molecule described in C1) or C2) below:

C1) 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.
The application according to claim 1, wherein the product is a medicine, a vaccine, a reagent or a test kit.
The monoclonal antibody against human EMC10 according to any one of claims 1-7.
The biological material related to the monoclonal antibody against human EMC10 according to any one of claims 1-7.
The application of the monoclonal antibody against human EMC10 described in any one of claims 1-7 in any of the following:

C1) use in the preparation of a product for reducing fatty infiltration of the liver of an animal;

C2) use in the preparation of products that reduce the serum triglyceride content of animals;

C3) application in the preparation of the product that reduces the serum free fatty acid content of animals;

C4) use in the preparation of products for reducing the serum cholesterol content of animals;

C5) application in the preparation of products for reducing the blood sugar of animals;

C6) application in the preparation of products for improving the glucose tolerance of animals;

C7) application in the preparation of products for improving the insulin sensitivity of animals;

C8) use in the preparation of a product for reducing the body weight of an animal;

C9) use in the preparation of a product for reducing the body fat content of an animal;

C10) use in the preparation of a product for reducing the volume of visceral fat in animals;

The use of C11) in the preparation of a product that activates brown fat in animals to promote thermogenesis.
The application of the biological material related to the monoclonal antibody against human EMC10 described in any one of claims 1-7 in any of the following:

C1) use in the preparation of a product for reducing fatty infiltration of the liver of an animal;

C2) use in the preparation of products that reduce the serum triglyceride content of animals;

C3) application in the preparation of the product that reduces the serum free fatty acid content of animals;

C4) use in the preparation of products for reducing the serum cholesterol content of animals;

C5) application in the preparation of products for reducing the blood sugar of animals;

C6) application in the preparation of products for improving the glucose tolerance of animals;

C7) use in the preparation of products for improving the insulin sensitivity of animals;

C8) use in the preparation of a product for reducing the body weight of an animal;

C9) use in the preparation of a product for reducing the body fat content of an animal;

C10) use in the preparation of a product for reducing the volume of visceral fat in animals;

The use of C11) in the preparation of a product that activates brown fat in animals to promote thermogenesis.
The application according to claim 10 or 11, wherein the product is a medicine, a vaccine, a reagent or a kit.
A method for preventing and/or treating metabolic diseases, characterized in that: the method comprises administering to a recipient animal the monoclonal antibody against human EMC10 according to any one of claims 1-7;

The metabolic disease is fatty liver, obesity and/or type 2 diabetes.
Application of substances that inhibit the activity of protein EMC10, reduce the expression of the gene of said protein EMC10 and/or reduce the content of said protein EMC10 in the preparation of products for preventing and/or treating metabolic diseases;

The metabolic disease is fatty liver, obesity and/or type 2 diabetes.