ANGPTL3-resistant humanized monoclonal antibody stably transfected strain and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an ANGPTL3 (anti-ANGPTL3) humanized monoclonal antibody stably transformed strain and application thereof. According to the application, an anti-ANGPTL3 humanized monoclonal antibody expression vector containing different nucleotide sequences is introduced into a host cell through electrotransfection, and a recombinant cell line with the highest yield is preliminarily screened out. The recombinant cell line is further screened for multiple times through main cloning and subcloning, and finally an ANGPTL3-resistant humanized monoclonal antibody cell strain with the advantages of high expression quantity, stability and controllability is screened out. The invention further discloses an ANGPTL3 humanized monoclonal antibody based on the cell, and the monoclonal antibody can be used for preparing a reagent for detecting ANGPTL3 and can also be used for preparing a medicine for preventing and / or treating kidney diseases or metabolism-related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an anti-ANGPTL3 humanized monoclonal antibody stable strain and application thereof. BACKGROUND

[0002] Angiopoietin-like protein-3 (ANGPTL3) was first proposed by Conklin et al. in 1999. It belongs to the angiotensin-like protein family, which is named for its structural similarity to angiotensin. The ANGPTL gene family encodes a class of secreted proteins, ANGPTL1-8, which have similar structures and a series of studies have confirmed that some members play an important role in lipid metabolism (Mattijssen F, Kersten S, Regulation of triglyceride metabolism by Angiopoietin-like proteins, Biochim Biophys Acta, 2012, 1821(5): 782-789.). ANGPTL3 is a sequence with a unique signal peptide molecular structure. More studies have confirmed that it is mainly expressed in hepatocytes and is a key regulator of lipoprotein metabolism, inhibiting the activity of lipoprotein lipase and endothelial lipase (KERSTEN S. ANGPTL3 as therapeutie target [J]. Curr Opin li-pidal, 2021. 32(6): 335-341.), significantly increasing the expression level of serum very low density lipoprotein, ultimately leading to a significant decrease in the decomposition of triglycerides (TG) and total cholesterol (TC), and a significant increase in blood lipid levels, thereby causing insulin resistance and vascular disorders. ANGPTL3 is involved in the development of various metabolic-related diseases and has been recognized by researchers as a key factor in regulating the physiological metabolism of blood lipids in the body (KERSTEN S. Angiopoietin-like 3 in lipoprotein metabolism [J]. Nat Rev Enlocrinol, 2017, 13(12): 731; GRAHAM MJ, LEE RG, BRANIT TA, et al. Cardiovascular and metabolic effects of ANGPTL3 antisense oligonucleotides [J]. N Engl J Med, 2017, 377(3): 222; XU YX, REDON V, YU H, et al. Role of angiopoietin-like 3 (ANGPTL3) in regulating plasma level of low-density lipoprotein cholesterol [J]. Atherosclerosis, 2018, 58(268): 196.).ANGPTL3 abnormal high expression can increase plasma lipid levels, further induce the formation of foam cells and the proliferation and migration of vascular smooth muscle cells, thereby causing atherosclerosis (SUN T, ZHAN W, WEI L, et al. Circulating ANGPTL3 and ANGPTL4 levels predict coronary artery atherosclerosis severity [J]. Lipids Health Dis. 2021. 20(1): 154-162.).

[0003] So far, three types of ANGPTL3 inhibitor drugs are in development, namely monoclonal antibodies, antisense oligonucleotides (ASO) and CRISPR / Cas9 gene editing, which can promote the hydrolysis of VLDL. Evinacumab is a full human IgG4 subtype monoclonal antibody targeting ANGPTL3 developed by Regeneron Pharmaceuticals, which can reduce the dangerous high cholesterol levels of patients with familial hypercholesterolemia to normal levels when used in combination with other lipid-lowering therapies. It was approved for marketing by the FDA on February 11, 2021, and is used to treat children and adults with familial homozygous hypercholesterolemia aged 12 and above. Currently, LY3475766, a monoclonal antibody targeting ANGPTL3 / 8 developed by Lilly, is in the phase I clinical trial stage, which can significantly reduce the TG and residual cholesterol content in the plasma of patients with dyslipidemia. In addition, there are some drugs that develop innovative therapies targeting ANGPTL3, such as Vupanorsen, an antisense oligonucleotide targeting ANGPTL3 developed by Pfizer, which can reduce the level of TG-rich lipoprotein (TRL) by inhibiting the synthesis of ANGPTL3 and increasing the activity of lipoprotein lipase (LPL).

[0004] With the development of biological drugs, various recombinant protein expression platforms are also developing, among which CHO cells are one of the most commonly used hosts for expressing recombinant proteins (especially fusion proteins and genetically engineered antibodies) (Haimanti Dorai, Subinay Ganguly. Mammalian cell-produced therapeutic proteins: heterogeneity derived from protein degradation [J]. Curr opin Biotechnol, 2014, 30: 198-204; Wuest DM, Harcum SW, Lee KH. Genomics in mammalian cell culture bioprocessing [J]. Biotechnol Adv, 2012, 30(3) 629-638.), and the efficient expression of recombinant protein genes in host cells is the most critical step, which is gene transfection. The commonly used transfection methods at present mainly include electroporation, liposome transfection, microinjection method, calcium phosphate coprecipitation method, etc. (Wang Yuli, Wei Jilou, Cheng Honglei, et al. Research progress of foreign gene transfection technology [J]. Modern Biomedicine Progress, 2014, 14(7): 1382-138). The expression of the anti-ANGPTL3 humanized monoclonal antibody in the past relies on the transient transfection technology, which needs to extract and transfect plasmid every time, and the process is complicated and the expression amount is unstable, which is not conducive to large-scale industrial production. Compared with other transfection technologies, the electroporation method has the advantages of simple operation, good repeatability and high transfection efficiency, and the electroporation is almost suitable for all cells, but different electroporation conditions will lead to different transfection efficiency of foreign genes, even under the optimal conditions, the same cells are transfected, and different foreign genes have different transfection efficiency (Deng Xiaofen, Yang Xiaojia, Yi Tianhong, et al. Optimization of electroporation conditions for fusion protein gene and antibody gene in CHO-S cells [J]. Biotechnology Bulletin, 2019, 35(04): 223-228.). SUMMARY

[0005] The present application introduces the anti-ANGPTL3 humanized monoclonal antibody expression vector containing different nucleotide sequences into the host cells by electroporation, and preliminarily screens a recombinant cell line with the highest yield. And further through the master clone and subclone, the recombinant cell line is screened for many times, and finally a strain of anti-ANGPTL3 humanized monoclonal antibody cell strain with high expression amount and stable controllable advantage is screened out. Accordingly, the present application is completed.

[0006] In a first aspect, the present application provides a stable cell strain which can stably express anti-ANGPTL3 humanized monoclonal antibody, and the cell is named as anti-ANGPTL3 humanized monoclonal antibody stable cell strain (FD36-A44), and the preservation number is CGMCC No.46049, and the biological preservation is provided to China General Microbiological Culture Collection Center (CGMCC).

[0007] In a second aspect, the present application provides a method for screening a cell which can stably express anti-ANGPTL3 humanized monoclonal antibody, and the screening method comprises the following steps:

[0008] S1. Different nucleotide sequences are designed according to the amino acid sequence of anti-ANGPTL3 humanized monoclonal antibody, gene synthesis is carried out, and primers are designed for amplification, the amplified gene fragments are connected to vectors to obtain recombinant plasmids;

[0009] S2. The recombinant plasmids obtained in S1 are mixed with host cells and then transfected, and the transfected cells are cultured in A culture medium, diluted into B culture medium after two days, and recombinant cell strains are obtained;

[0010] S3. The recombinant cells obtained in S2 are grown to a certain abundance, and the yield is detected by ELISA method to preliminarily screen the recombinant cell strains which can highly express anti-ANGPTL3 humanized monoclonal antibody, and the culture is continued for subsequent screening;

[0011] S4. The recombinant cell strains screened in S3 are further screened by using a culture medium without glutamine component and MSX, and finally the cell strain which can highly express and stably control anti-ANGPTL3 humanized monoclonal antibody is screened.

[0012] Further, in step S1, the amino acid sequence of the anti-ANGPTL3 humanized monoclonal antibody is shown as SEQ ID NO: 1-2; and the nucleotide sequence of the anti-ANGPTL3 humanized monoclonal antibody is shown as SEQ ID NO: 3-24.

[0013] Preferably, the nucleotide sequence of the anti-ANGPTL3 humanized monoclonal antibody is shown as SEQ ID NO: 3-4.

[0014] Further, in step S1, the cloning method is IN-FUSION technology.

[0015] Further, in step S2, the host cell is a eukaryotic cell.

[0016] Preferably, the host cell is CHO-K1Q.

[0017] Further, in step S2, the A medium is CHO CD02+ glutamine medium, and the B medium is CHO CD02+ methionine sulfoximine medium.

[0018] Further, in step S3, the screening method comprises five steps of Gln-free culture screening, MSX pressure screening, batch culture screening, shake flask flow process screening, and secondary subcloning screening.

[0019] In a third aspect, the present application provides an ANGPTL3 humanized monoclonal antibody, which is secreted by the stable cell strain of the first aspect.

[0020] In a fourth aspect, the present application provides an antibody conjugate, which comprises the monoclonal antibody of the third aspect.

[0021] In a fifth aspect, the present application provides use of the monoclonal antibody of the third aspect in the preparation of a reagent for detecting ANGPTL3.

[0022] In a sixth aspect, the present application provides a kit for detecting ANGPTL3, which comprises the monoclonal antibody of the third aspect.

[0023] Further, the kit can be one or more of an ELISA detection kit, a colloidal gold detection kit, an immunohistochemical kit, an immunofluorescence kit, and / or an in situ hybridization staining kit.

[0024] Still further, the kit diagnosis method comprises one or more of a direct method, an indirect method, a double-antibody sandwich method, and / or a competition method.

[0025] In a seventh aspect, the present application provides use of the monoclonal antibody of the third aspect or the antibody conjugate of the fourth aspect in the preparation of a medicament for preventing and / or treating kidney diseases or metabolic-related diseases.

[0026] Further, the kidney diseases include, but are not limited to, various kidney diseases accompanied by proteinuria and podocyte injury, such as minimal change disease, focal segmental glomerulosclerosis, diabetic nephropathy, IgA nephropathy, Henoch-Schonlein purpura nephritis, and lupus nephritis.

[0027] Further, the metabolic-related diseases include, but are not limited to, diabetes, and dyslipidemia diseases such as hypercholesterolemia and hypertriglyceridemia.

[0028] The high expression and stable controllable cell strain in the application is named: anti-ANGPTL3 humanized monoclonal antibody stable cell strain (FD36-A44), the preservation number is: CGMCC No. 46049, and the biological preservation has been provided to the China General Microbiological Culture Collection Center (CGMCC) on September 23, 2024, and the preservation address is No. 1, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology.

[0029] Beneficial effects

[0030] The application first uses a plurality of anti-ANGPTL3 humanized monoclonal antibody nucleotide sequences to construct a plasmid to transfect CHO-K1Q cells, preliminarily culture a recombinant anti-ANGPTL3 humanized monoclonal antibody stable cell line of different nucleotide sequences, and screen a recombinant cell line with the highest yield (recombinant FD36 strain). And further screening the recombinant cell line through master cloning and subcloning, finally screening an anti-ANGPTL3 humanized monoclonal antibody cell strain with high expression and stable controllable advantages, and preserving it. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Stable cell line screening process.

[0032] Figure 2 Minipool well plate evaluation of expression detection results.

[0033] Figure 3 Minipool shake flask evaluation of expression detection results.

[0034] Figure 4 Minipool shake flask fed-batch evaluation of expression detection results.

[0035] Figure 5 Master clone fed-batch culture purification yield.

[0036] Figure 6 Master clone fed-batch culture viable cell density and cell viability.

[0037] Figure 7 Subclone well plate evaluation of expression detection results.

[0038] Figure 8 Subclone shake flask evaluation of expression detection results.

[0039] Figure 9 Subclone fed-batch evaluation of expression detection results.

[0040] Figure 10 Subclone and its corresponding master clone purification yield.

[0041] Figure 11To subclone the cell density and cell viability of the fed-batch culture.

[0042] Figure 12 To subclone and its corresponding main clone protein product SDS-PAGE results. DETAILED DESCRIPTION

[0043] The specific embodiments of the present application are further described below. It is to be understood that the description of these embodiments is intended to help understand the present application and does not constitute a limitation of the present application. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0044] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0045] TERMS

[0046] Transient transfection: refers to the introduction of a constructed plasmid into mammalian cells by a certain way, the exogenous gene on the plasmid is not integrated into the cell's own genome, and the whole process of rapid transfection to obtain protein is called transient transfection expression.

[0047] Electroporation transfection method: (electrotransformation) by applying electric current to change the structure of cell membrane, making the cell membrane produce transient, reversible nanoscale water-filled cavities, prompting the exogenous molecules to enter the cells. The operation of electroporation transfection method is simple and fast, only needs to prepare cell suspension to carry out electric shock, the whole transfection process is less than 1s, in theory, through optimizing the electric shock conditions, better transfection effect can be obtained in various types of cells, and a large number of cells can be batch transfected. It is worth noting that the solution system of electroporation transfection needs to contain as little inorganic salt as possible, because the presence of salt may cause electric arc discharge, affect cell viability and even cause cell death.

[0048] Seamless cloning: (IN-FUSION) is a new technology that is significantly different from traditional enzyme digestion and ligation method, which is a gene cloning technology relying on homologous sequences, with the characteristics of simplicity, rapidness and high efficiency. Through the action of recombinase, any DNA fragment containing the terminal overlapping region of the vector can be recombined to the linearized vector, which is not limited by the restriction enzyme site, and the background of vector self-ligation is very low.

[0049] Heavy chain: The heavy chain class defines the class of the antibody. There are five types of Ig heavy chain in mammals, denoted by a Greek letter: alpha, delta, epsilon, gamma, and mu. These heavy chains occur in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. The size and composition of heavy chains vary, with alpha and gamma having about 450 amino acids, and mu and epsilon having about 550 amino acids. Each heavy chain has two regions, the constant region and the variable region.

[0050] Light chain: There are only two types of light chain in mammals, lambda and kappa. The light chain has two consecutive domains, a constant domain and a variable domain. The length of the light chain is about 211-217 amino acids. The two light chains contained in each antibody are usually identical.

[0051] Example 1 Construction of Anti-ANGPTL3 Humanized Monoclonal Antibody Expression Vector

[0052] After converting the amino acid sequence of the anti-ANGPTL3 humanized monoclonal antibody (FD36) (the amino acid sequence of FD36 is shown in Table 1) into the corresponding nucleotide sequence (the nucleotide sequences of FD36, FD36-a to FD36-j are shown in Table 2), the gene fragment and the vector were amplified by PCR using the designed upstream and downstream primers (universal upstream primer, primer sequence shown in Table 3). The DNA was linearized and the cohesive end was added, and the gene fragment and the vector were ligated by IN-FUSION (seamless cloning) technology to construct pKS001-FD36, pKS001-FD36-a, pKS001-FD36-b, pKS001-FD36-c, pKS001-FD36-d, pKS001-FD36-e, pKS001-FD36-f, pKS001-FD36-g, pKS001-FD36-h, pKS001-FD36-i, and pKS001-FD36-j plasmids. After the constructed plasmids were verified by sequencing to be error-free, they were amplified and prepared in large quantities for subsequent steps.

[0053] Table 1 Amino acid sequence of anti-ANGPTL3 humanized monoclonal antibody (FD36)

[0054]

[0055] Table 2 Nucleotide sequence of anti-ANGPTL3 humanized monoclonal antibody

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] Table 3 Primer Sequences

[0073]

[0074] Example 2 Anti-ANGPTL3 Humanized Monoclonal Antibody Expression Vector Introduction into Host Cells

[0075] Recovery CHO-K1Q working cell bank cells were used for the construction of engineered cell strains. The cells were cultured with CHO CD02 medium supplemented with 4 mM L-glutamine. The cells were inoculated and cultured for 3 days before being passaged again or directly transfected. The plasmids pKS001-FD36, pKS001-FD36-a, pKS001-FD36-b, pKS001-FD36-c, pKS001-FD36-d, pKS001-FD36-e, pKS001-FD36-f, pKS001-FD36-g, pKS001-FD36-h, pKS001-FD36-i, and pKS001-FD36-j prepared in Example 1 were mixed uniformly with CHO-K1Q cells respectively before being electroporated, and the cells were incubated in CHO CD02 + 4 mM L-glutamine medium after transfection. Two days later, the cells were diluted into CHO CD02 medium supplemented with L-methionine sulfoximine for incubation. The recombinant cells (recombinant FD36, FD36-a, FD36-b, FD36-c, FD36-d, FD36-e, FD36-f, FD36-g, FD36-h, FD36-i, and FD36-j strains) were grown to a certain abundance (more than 3e6 / mL) before being screened for yield using an ELISA method. It was found that the recombinant FD36 strain had the highest yield. The recombinant FD36 strain was further cultured for subsequent screening.

[0076] Example 3 Screening of Anti-ANGPTL3 Humanized Monoclonal Antibody Stable Cell Lines

[0077] 3.1 Experimental Methods

[0078] The recombinant FD36 strain was screened using glutamine-free medium CHO CD02 and MSX. Glutamine is an amino acid that plays an important role in cell growth and reproduction in cell culture. It not only participates in cellular energy metabolism, but also participates in protein synthesis and nucleic acid metabolism. In the absence of or insufficient supply of glutamine, glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamic acid and ammonium ions to supply cell metabolism and protein synthesis. MSX is a competitive inhibitor of GS activity. After MSX binds to the glutamate site of GS, it is phosphorylated by ATP, thereby irreversibly inhibiting GS activity. The vector has a GS gene and an FD36 gene connected to it. A small amount of vector is integrated into the host cell genome after transfection. Cells that do not integrate the FD36 gene and cells that express low levels of glutamine synthetase will not survive. Only cells that express high levels of glutamine synthetase genes can survive, thereby screening for cell strains that express high levels of FD36 genes.

[0079] During the whole screening process, the recombinant FD36 stable cell line went through five screening steps, including serum-free, animal component-free, glutamine-free medium CHO CD02 screening, MSX pressure screening, batch culture screening, shake flask perfusion process screening, and secondary subcloning screening (see Figure 1 ).

[0080] 3.2 Experimental results

[0081] 3.2.1 Minipool clone evaluation and screening

[0082] Well plate evaluation: After dilution and culture of the transfected cells, the minipool clones formed were grown to a certain abundance (more than 3e6 / mL) and then screened for high expression of yield by ELISA. A total of 353 clones were screened, of which 235 clones had a yield of 0-11 mg / L, 118 clones had a yield of 3.95-55.83 mg / L, and the latter were used for scale-up evaluation (Table 4). Figure 2

[0083] Table 4 Well plate evaluation results

[0084]

[0085]

[0086] Shake flask evaluation: Of the above 118 clones, 3 clones were discarded due to poor growth, and the remaining 115 clones were further cultured in shake flasks. Of these, 76 clones had a yield of 0-50 mg / L, 19 clones had a yield of 50-82 mg / L, and 20 clones had a yield of 82-296 mg / L. The top 20 clones with the highest yield were used for fed-batch culture screening (Table 5). Figure 3

[0087] Table 5 Shake flask evaluation results

[0088]

[0089]

[0090] Shake flask perfusion evaluation: After shake flask perfusion culture of the above 20 clones, the expression amount was detected by ELISA and ranked, and the top 3 clones FD36-34, FD36-36, and FD36-37 with the highest yield were selected as the main clones for further evaluation (Table 6). Figure 4

[0091] Table 6 Shake flask perfusion evaluation

[0092] ID Titer (mg / L) ID Titer (mg / L) ID Titer (mg / L) FD36-04 289.4 FD36-80 662.3 FD36-70 802.6 FD36-15 390.5 FD36-06 274.2 FD36-71 165.9 FD36-18 856.8 FD36-07 210.7 FD36-76 658.8 FD36-34 1617.7 FD36-10 210.6 FD36-85 416.6 FD36-36 2484.1 FD36-14 403.8 FD36-88 433.6 FD36-37 1931.8 FD36-17 416.2 FD36-97 172.1 FD36-42 330.2 FD36-23 592.1 FD36-70 802.6

[0093] ​​​Main clone evaluation: The above 3 main clones were cultured to take the cell supernatant for protein purification, and the yield was evaluated by UV quantification. The results showed that the yield of FD36-34 was 801.17 mg / L, the yield of FD36-36 was 886.1 mg / L, and the yield of FD36-37 was 825.36 mg / L( Figure 5 ); and the viable cell density and cell viability of fed-batch culture were monitored, and the results showed that the 3 clones all showed high viable cell density and cell viability within 6-8 days of the protein expression regular cycle( Figure 6 ). After comprehensive consideration, the highest expression FD36-36 was finally selected as the main clone target strain for subsequent subclone screening.

[0094] 3.2.2 Subclone evaluation and screening

[0095] Well plate evaluation: the above FD36-36 main clone target strain was cultured by limiting dilution, and a total of 244 clones were screened, 152 clones had a yield of 0-39.8 mg / L, 92 clones had a yield of 14.5-92 mg / L, and the latter was used for expansion evaluation( Figure 7 and Table 7).

[0096] Table 7 Well plate evaluation results

[0097]

[0098]

[0099] Flask evaluation: of the above 92 clones, 44 clones had a yield of 267-500 mg / L, 30 clones had a yield of 500-644 mg / L, and 18 clones had a yield of 661-994 mg / L. Of the 18 clones with the highest yield, 3 clones were discarded due to poor growth, and the remaining 15 clones were used for fed-batch culture screening( Figure 8 and Table 8).

[0100] Table 8 Flask evaluation results

[0101]

[0102]

[0103] Fed-batch evaluation in shake flask: the above 15 clones were cultured by fed-batch in shake flask, the expression yield was detected by ELISA and sorted, and the 3 subclones FD36-A44, FD36-A90 and FD36-A06 with the highest yield and the corresponding main clone FD36-36 were selected for further evaluation( Figure 9 and Table 9).

[0104] Table 9 Fed-batch evaluation results in shake flask

[0105] ID Titer (mg / L) ID Titer (mg / L) ID Titer (mg / L) FD36-A44 3635.7 FD36-A15 2527.6 FD36-A40 1906 FD36-A90 3383.5 FD36-A80 2513.9 FD36-A68 1885.8 FD36-A06 3004.9 FD36-A10 2289.7 FD36-A13 1861.3 FD36-A26 2904.1 FD36-A76 2128.8 FD36-A84 1803.5 FD36-A18 2678.0 FD36-A07 2123.6 FD36-A74 1347.0 ​​​​​

[0106] Subclone evaluation: The above three subclones and their corresponding master clones were cultured, and the cell supernatant was taken for protein purification. The yield was evaluated quantitatively by UV. The results showed that the yield of master clone FD36-36 was 2405.55 mg / L, the yield of subclone FD36-A44 was 3177.90 mg / L, the yield of subclone FD36-A90 was 2949.43 mg / L, and the yield of subclone FD36-A06 was 2473.07 mg / L( Figure 10 );and the viable cell density and cell viability of the fed-batch culture were monitored. The results showed that the three subclones all showed high viable cell density and cell viability within 6-8 days of the regular protein expression cycle( Figure 11 );the above protein products showed good purity by SDS-PAGE with the expected band molecular weight( Figure 12 ). The highest yield of FD36-A44 in the above screening was selected and provided for biological preservation to the China General Microbiological Culture Collection Center (CGMCC) on September 23, 2024, with the name: Anti-ANGPTL3 humanized monoclonal antibody stable cell strain (FD36-A44), and the preservation number: CGMCC No. 46049.

Claims

1. A stable cell line that can stably express anti-ANGPTL3 humanized monoclonal antibody, the cell line being named: Anti-ANGPTL3 Humanized Monoclonal Antibody Stable Transformation Cell Line (FD36-A44), with accession number: CGMCC No. 46049, and has been provided for bio-extraction by the China General Microbiological Culture Collection Center (CGMCC).

2. A method for screening cells that stably express humanized monoclonal antibodies against ANGPTL3, the screening method comprising the following steps: S1. Based on the amino acid sequence of the anti-ANGPTL3 humanized monoclonal antibody, different nucleotide sequences were designed, gene synthesis was carried out, primers were designed to amplify the gene using the sequence as a template, and the amplified gene fragment was ligated into a vector to obtain a recombinant plasmid. S2. The recombinant plasmid obtained in S1 was mixed with the host cells and transfected. The transfected cells were cultured in medium A for two days and then diluted to medium B to obtain the recombinant cell line. After the recombinant cells obtained from S3 and S2 grew to a certain abundance, the yield was detected by ELISA to preliminarily screen recombinant cell lines that highly expressed anti-ANGPTL3 humanized monoclonal antibody, and they were further cultured for subsequent screening. S4. The recombinant cell lines selected in S3 were further screened using glutamine-free culture medium and MSX to finally screen out the cell lines that highly express and stably controllable anti-ANGPTL3 humanized monoclonal antibody as described in this invention.

3. The screening method as described in claim 2, wherein in step S1, the amino acid sequence of the anti-ANGPTL3 humanized monoclonal antibody is shown in SEQ ID NO: 1-2; and the nucleotide sequence of the anti-ANGPTL3 humanized monoclonal antibody is shown in SEQ ID NO: 3-24; In step S2, the host cell is CHO-K1Q, the A medium is CHO CD02 + glutamine medium, and the B medium is CHO CD02 + methionine sulfoxide imide medium. In step S3, the screening method includes five steps: Gln-free culture screening, MSX pressurized screening, batch culture screening, shake flask fed-batch process screening, and secondary subcloning screening.

4. An ANGPTL3 humanized monoclonal antibody, said monoclonal antibody being secreted by the stable transgenic cell line of claim 1.

5. An antibody conjugate comprising the monoclonal antibody of claim 4.

6. The use of the monoclonal antibody according to claim 4 in the preparation of reagents for detecting ANGPTL3.

7. A kit for detecting ANGPTL3, the kit comprising the monoclonal antibody of claim 4.

8. The kit according to claim 7, wherein the kit may be one or more of the following: ELISA detection kit, colloidal gold detection kit, immunohistochemistry kit, immunofluorescence kit, and / or in situ hybridization staining kit.

9. The use of the monoclonal antibody of claim 4 or the antibody-drug conjugate of claim 5 in the preparation of a medicament for the prevention and / or treatment of kidney disease or metabolic-related diseases.

10. The application as described in claim 9, wherein the kidney disease includes, but is not limited to, various kidney diseases accompanied by proteinuria and podocyte damage, such as minimal change disease, focal segmental glomerulosclerosis, diabetic nephropathy, IgA nephropathy, Henoch-Schönlein purpura nephropathy, and lupus nephropathy; and the metabolic-related diseases include, but are not limited to, diabetes mellitus and lipid metabolism disorders such as hypercholesterolemia and hypertriglyceridemia.

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