Anti-angptl3 humanized monoclonal antibody stable cell line and use thereof

Abstract

Provided are an anti-ANGPTL3 humanized monoclonal antibody stable cell line and a use thereof. An anti-ANGPTL3 humanized monoclonal antibody expression vector is introduced into a host cell by means of electrotransfection; and after multiple times of screening, a series of stable and controllable cell lines with high expression levels are finally selected. Further disclosed is an ANGPTL3 humanized monoclonal antibody on the basis of a hybridoma cell. The monoclonal antibody can be used in a reagent for detecting ANGPTL3, and can also be used in the preparation of a drug for preventing and / or treating kidney diseases or metabolic diseases.

Description

A stable anti-angptl3 humanized monoclonal antibody strain and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a stable anti-angptl3 humanized monoclonal antibody 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 metabalism [J]. Nat Rev Enlocrinol, 2017, 13(12): 731; GRAHAM MJ, LEE RG, BRANIT TA, et al. Cardiovascular and metabolie eleets of ANGPTl3 antisense oligonueleolides [J]. EngI J Med, 2017, 377(3): 222; XU YX, REDON V, YU H, et al. Role of angiopoietin-like 3 (ANGPTl3) in regulating plasa level of low-density lipoprotein cholesderol [J]. Atherasclensis, 2018, 58(268): 196.).ANGPTL3 abnormal high expression will increase the plasma lipid level, further induce the formation of foam cells and the proliferation and migration of vascular smooth muscle cells, leading to 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, which are 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 risk of high cholesterol levels in patients with familial hypercholesterolemia to normal levels when used in combination with other lipid-lowering therapies. It was approved for marketing by FDA on February 11, 2021, and is used to treat children and adults with familial homozygous hypercholesterolemia aged 12 years and older (Zhang Y, Li Y, Wang L, et al. ANGPTL3 and atherosclerosis [J]. China Biotechnology of Medicine, 2022, 17(02): 158-163.). Currently, LY3475766, a monoclonal antibody targeting ANGPTL3 / 8 developed by Lilly, is in the I phase of clinical trials, which can significantly reduce the TG and residual cholesterol content in the plasma of patients with dyslipidemia. Drugs targeting ANGPTL4 include monoclonal antibody REGN-1001 developed by Regeneron and 14D12 monoclonal antibody developed by Lexicon Pharmaceuticals, both of which are used to lower blood lipids. 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 inhibit the synthesis of ANGPTL3 and increase the activity of lipoprotein lipase (LPL), thereby reducing the level of TG-rich lipoprotein (TRL).

[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 exogenous gene transfection technology [J]. Modern Biomedicine Progress, 2014, 14(7): 1382-138). The expression of the anti-ANGPTL3 humanized monoclonal antibody relies on the transient transfection technology, which needs to extract and transfect the plasmid process complicatedly and the expression quantity 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 exogenous genes, even under the optimal conditions, the same cells are transfected, and different exogenous genes will 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.).

[0005] SUMMARY

[0006] The application introduces the anti-ANGPTL3 humanized monoclonal antibody expression vector into the host cells by electroporation, and finally screens a series of cell strains with high expression quantity and stable controllability, thereby completing the application.

[0007] In a first aspect, the present application provides a hybridoma cell 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).

[0008] 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:

[0009] S1. Designing a primer according to the sequence of anti-ANGPTL3 humanized monoclonal antibody, and amplifying an ANGPTL3 gene fragment from the genome as a template, and cloning the ANGPTL3 gene fragment into a vector to obtain a recombinant plasmid pKS001-FD36;

[0010] S2. Mixing the recombinant plasmid obtained in S1 with a host cell, and then transfecting, and culturing the transfected cell in A culture medium for two days, and then diluting into B culture medium;

[0011] S3. Screening the anti-ANGPTL3 humanized monoclonal antibody cell strain by using a glutamine-free culture medium and MSX, and finally screening the cell strain which can stably and controllably express anti-ANGPTL3 humanized monoclonal antibody.

[0012] Further, in step S1, the sequence of the anti-ANGPTL3 humanized monoclonal antibody is the amino acid sequence thereof.

[0013] Further, the heavy chain amino acid sequence of the anti-ANGPTL3 humanized monoclonal antibody is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2.

[0014] Further, in step S1, the primer is designed after converting the amino acid sequence of the anti-ANGPTL3 humanized monoclonal antibody into a corresponding nucleotide sequence.

[0015] Further, in step S1, the cloning method is IN-FUSION technology.

[0016] Further, in step S2, the host cell is a eukaryotic cell.

[0017] Preferably, the host cell is CHO-K1Q.

[0018] Further, in step S2, the A culture medium is CHO CD02+glutamine culture medium, and the B culture medium is CHO CD02+methionine sulfoximine culture medium.

[0019] Further, in step S3, the screening method comprises five steps of Gln-free culture screening, MSX pressure screening, batch culture screening, shake flask flow processing screening and secondary subcloning screening.

[0020] In a third aspect, the present application provides an ANGPTL3 humanized monoclonal antibody, which is secreted by the hybridoma cell of the first aspect.

[0021] In a fourth aspect, the present application provides an antibody conjugate comprising the monoclonal antibody of the third aspect.

[0022] 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.

[0023] In a sixth aspect, the present application provides a kit for detecting ANGPTL3, which comprises the monoclonal antibody of the third aspect.

[0024] 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.

[0025] 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.

[0026] 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 disease or metabolic-related disease.

[0027] Further, the kidney disease includes but is not limited to various types of 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.

[0028] Further, the metabolic-related disease includes but is not limited to diabetes, and dyslipidemia diseases such as hypercholesterolemia and hypertriglyceridemia.

[0029] The cell strain with high expression and stable controllability in the present application is named: anti-ANGPTL3 humanized monoclonal antibody stable cell strain (FD36-A44), and the preservation number is CGMCC No.46049. 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.3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard 3rd. Beneficial effects

[0030] The application prepares a series of cells expressing anti-ANGPTL3 humanized monoclonal antibodies, and further screens a strain of anti-ANGPTL3 humanized monoclonal antibody cells with high expression and stable controllable advantages. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a stable cell line screening process.

[0032] Figure 2 is a minipool well plate evaluation expression detection result.

[0033] Figure 3 is a minipool flask evaluation expression detection result.

[0034] Figure 4 is a minipool flask evaluation expression detection result.

[0035] Figure 5 is a main clone fed-batch culture purification yield.

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

[0037] Figure 7 is a subclone well plate evaluation expression detection result.

[0038] Figure 8 is a subclone flask evaluation expression detection result.

[0039] Figure 9 is a subclone evaluation expression detection result.

[0040] Figure 10 is a subclone and its corresponding main clone purification yield.

[0041] Figure 11 is a subclone fed-batch culture viable cell density and cell viability.

[0042] Figure 12 is a subclone and its corresponding main clone protein product SDS-PAGE result. DETAILED DESCRIPTION

[0043] The specific embodiments of the application are described below. It should be noted that the description of these embodiments is used to help understand the application, but does not constitute a limitation on the 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 EXPLANATION

[0046] Transient transfection: refers to the process of introducing a constructed plasmid into mammalian cells by a certain method, and the exogenous gene on the plasmid is not integrated into the genome of the cells themselves. The entire process of rapidly transfecting and expressing proteins is called transient transfection expression.

[0047] Electroporation transfection method: (electrotransformation) by applying an electric current to change the structure of the cell membrane, making the cell membrane produce a transient and reversible nanoscale water-filled cavity, 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, and the whole transfection process is less than 1s. In theory, by 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 methods. This technology is a gene cloning technology that relies on homologous sequences, and has 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 type of heavy chain defines the class of an antibody. There are five types of Ig heavy chains in mammals, denoted by Greek letters: α, δ, ε, γ, and μ. These heavy chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. The size and composition of heavy chains vary, with α and γ containing approximately 450 amino acids, and μ and ε containing approximately 550 amino acids. Each heavy chain has two regions, a constant region and a variable region.

[0050] Light chain: there are only two types of light chains in mammals, λ and κ. Light chains have 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 the same.

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

[0052] The amino acid sequence of the anti-ANGPTL3 humanized monoclonal antibody (FD36) (the heavy chain amino acid sequence of FD36 is shown as SEQ ID NO: 1, and the light chain amino acid sequence of FD36 is shown as SEQ ID NO: 2) is converted into the corresponding nucleotide sequence after gene synthesis, and the upstream and downstream primer pairs are designed for PCR amplification of the gene fragment and the vector. The DNA is linearized and the cohesive end is added, the gene fragment and the vector are connected by IN-FUSION (seamless cloning) technology, the pKS001-FD36 plasmid is constructed, and the constructed plasmid is verified by sequencing to be correct, then it is amplified and prepared in large quantities for subsequent steps.

[0053] Example 2 Introduction of anti-ANGPTL3 humanized monoclonal antibody expression vector into host cells

[0054] Recovery CHO-K1Q working cell bank cells are used for the construction of engineered cell strains. The cells are cultured with CHO CD02 medium supplemented with 4 mM L-glutamine. The cells are inoculated, and after 3 days of culture, they are passaged again or directly transfected. The pKS001-FD36 plasmid is mixed uniformly with CHO-K1Q cells before electroporation, and after transfection, the cells are incubated in CHO CD02 + 4 mM L-glutamine medium, and after two days, they are diluted into CHO CD02 medium supplemented with L-methionine sulfoximine for selection.

[0055] Example 3 Screening of anti-ANGPTL3 humanized monoclonal antibody stable cell lines

[0056] 3.1 Experimental method

[0057] CHO CD02 and MSX are used to screen the recombinant FD36 strain. 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 and ammonium ion to supply cell metabolism and protein synthesis. MSX is a competitive inhibitor of GS activity. MSX is phosphorylated by ATP after binding to the glutamate site of GS, 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 gene can survive, thereby screening for cell strains that express high levels of FD36 gene.

[0058] During the whole screening process, the recombinant FD36 stable cell line underwent five screening steps, including serum-free, animal component-free, stable cell line screening in serum-free, animal component-free, glutamine-free medium CHO CD02, MSX pressure screening, batch culture screening, shake flask perfusion process screening, and secondary subclone screening (see FIG. 1).

[0059] 3.2 Experimental results

[0060] 3.2.1 minipool clone evaluation and screening

[0061] Well plate evaluation: After transfection, the cells were diluted and cultured, and the minipool clones formed were detected for yield screening to screen high expression cell lines by ELISA method after the minipool clones grew to a certain abundance. 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 expanded evaluation (FIG. 2).

[0062] Shake flask evaluation: Of the above 118 clones, 3 clones were discarded due to poor growth, and the remaining 115 clones were continuously cultured in shake flasks. The yield of 76 clones was 0-50 mg / L, the yield of 19 clones was 50-82 mg / L, the yield of 20 clones was 82-296 mg / L, and the 20 clones with the highest yield were used for fed-batch culture screening (FIG. 3).

[0063] Shake flask perfusion evaluation: After the above 20 clones were cultured by shake flask perfusion, the expression amount was detected by ELISA and sorted, and the three clones with the highest yield, FD36-34, FD36-36 and FD36-37, were selected as master clones for further evaluation (FIG. 4).

[0064] Master clone evaluation: After the above three master clones were cultured, the cell supernatant was taken 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 (FIG. 5); and the viable cell density and cell viability of fed-batch culture were monitored, and the results showed that the three clones all showed high viable cell density and cell viability within 6-8 days of the conventional protein expression cycle (FIG. 6). After comprehensive consideration, the highest expression FD36-36 was finally selected as the master clone target strain for subsequent subclone screening.

[0065] 3.2.2 Subclone evaluation and screening

[0066] Well plate evaluation: After the above FD36-36 master clone target strain was cultured by limited dilution, a total of 244 clones were screened, of which 152 clones had a yield of 0-39.8 mg / L, and 92 clones had a yield of 14.5-92 mg / L. The latter was used for expanded evaluation (FIG. 7).

[0067] Shake flask evaluation: Among the above 92 clones, 44 clones had a yield of 267-500 mg / L, 30 clones had a yield of 500-644 mg / L, 18 clones had a yield of 661-994 mg / L, among 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);

[0068] Fed-batch shake flask evaluation: After fed-batch shake flask culture of the above 15 clones, the expression level was detected by ELISA and ranked, and the three subclones FD36-A44, FD36-A90 and FD36-A06 with the highest yield and their corresponding master clone FD36-36 were further evaluated (Figure 9);

[0069] Subclone evaluation: After culture of the above three subclones and their corresponding master clone, the cell supernatant was taken for protein purification, and the yield was quantitatively evaluated 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 fed-batch culture were monitored, and the results showed that the three subclones all showed high viable cell density and cell viability within 6-8 days of the conventional cycle of protein expression (Figure 11); the protein products were subjected to SDS-PAGE, which showed that the band molecular weight was consistent with the expectation and the purity was good (Figure 12).

Claims

1. A hybridoma cell, which can stably express anti-ANGPTL3 humanized monoclonal antibody, and is named as anti-ANGPTL3 humanized monoclonal antibody stable cell strain (FD36-A44), has a preservation number of CGMCC No.46049, and has been provided to China General Microbiological Culture Collection Center (CGMCC). 2.A method for screening a cell stably expressing anti-ANGPTL3 humanized monoclonal antibody, comprising the following steps: S1. designing primers according to the sequence of anti-ANGPTL3 humanized monoclonal antibody, and amplifying an ANGPTL3 gene fragment from the genome as a template, and cloning the ANGPTL3 gene fragment into a vector to obtain a recombinant plasmid pKS001-FD36; S2. mixing the recombinant plasmid obtained in S1 with host cells, and then transfecting, culturing the transfected cells in A medium, and diluting to B medium after two days; S3. screening the cell strain expressing anti-ANGPTL3 humanized monoclonal antibody by using a glutamine-free medium and MSX, and finally screening the cell strain of the application which can stably and controllably express anti-ANGPTL3 humanized monoclonal antibody. 3.The screening method of claim 2, in step S1, the sequence of the anti-ANGPTL3 humanized monoclonal antibody is the amino acid sequence thereof; the heavy chain amino acid sequence of the anti-ANGPTL3 humanized monoclonal antibody is shown as SEQ ID NO: 1, and the light chain amino acid sequence is shown as SEQ ID NO: 2; and the primers are designed by converting the amino acid sequence of the anti-ANGPTL3 humanized monoclonal antibody into a corresponding nucleotide sequence; in step S2, the host cells are CHO-K1Q, the A medium is CHO CD02+ glutamine medium, and the B medium is CHO CD02+ methionine sulfoximine medium; in step S3, the screening method comprises five steps of glutamine-free culture screening, MSX pressure screening, batch culture screening, shake flask flow process screening and secondary subcloning screening. 4.Angiopoietin-like 3 (ANGPTL3) humanized monoclonal antibody, which is secreted by the hybridoma cell of claim 1. 5.An antibody conjugate comprising the monoclonal antibody of claim 3. 6.Use of the monoclonal antibody of claim 3 in the preparation of a reagent for detecting ANGPTL3. 7.A kit for detecting ANGPTL3, comprising the monoclonal antibody of claim 3. 8.The kit of claim 7, which 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. 9.Use of the monoclonal antibody of claim 3 or the antibody conjugate of claim 4 in the preparation of a drug for preventing and / or treating kidney disease or metabolic-related disease.

10. The use according to claim 9, wherein the kidney diseases include, but are not limited to, various types of kidney diseases accompanied with proteinuria and podocyte injury, such as minimal change disease, focal segmental glomerulosclerosis, diabetic nephropathy, IgA nephropathy, Henoch-Schonlein purpura nephritis and lupus nephritis; and the metabolic related diseases include, but are not limited to, diabetes, and dyslipidemia such as hypercholesterolemia and hypertriglyceridemia.

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