CN117603361A - Fusion protein containing ANGPTL3 monoclonal antibody - Google Patents

Abstract

This application discloses a fusion protein ANGPTL3 monoclonal antibody-B containing an ANGPTL3 monoclonal antibody. The fusion protein ANGPTL3 monoclonal antibody-B is composed of the fusion of ANGPTL3 monoclonal antibody and sequence B; the fusion protein ANGPTL3 monoclonal antibody-B disclosed in this application is used for Treatment of diseases or conditions mediated by it, such as diabetes and its complications such as: diabetic nephropathy, diabetic retinopathy, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular disease (cerebral arteriosclerosis, silent stroke), Diabetic neuropathy, kidney-related diseases such as membranous nephropathy, IgA nephropathy, purpura kidney, lupus kidney, etc., and tumor-related diseases such as hepatocellular carcinoma, renal cell carcinoma, ovarian cancer, cervical cancer, oral cancer, esophageal cancer, etc.

Description

Fusion protein containing ANGPTL3 monoclonal antibody

Technical field

The present invention relates to the fields of medical immunology and molecular biology. Specifically, it relates to a fusion protein of a monoclonal antibody containing angiopoietin-like protein 3 (ANGPTL3) and its medical use.

Background technique

Angiopoietin-like protein 3 (ANGPTL3) is a secreted glycoprotein whose structure includes an amino-terminal coiled-coil domain (CCD), a secretory signal peptide, a short linker peptide, and a carboxyl-terminal globular fibrinogen-like domain (FLD). . The coiled-coil region at the amino terminus of ANGPTL3 increases plasma triglyceride (TG) levels by inhibiting the activity of lipoprotein esterase LPL, and the carboxyl terminus FLD (207-460aa) binds to the integrin αvβ3 receptor to promote angiogenesis (Camenisch G , Pisabarro MT, Sherman D, et al.ANGPTL3 stimulates endothelial cell adhesion and migration via integrinαvβ3and induces blood vessel formation in vivo.J BiolChem, 2002, 277(19):17281-17290), the amino-terminal helix-like domain is involved in regulating lipids Metabolism (Ono M, Shimizugawa T, Shimamura M, et al. Protein region important for regulation oflipid metabolism inangiopoietin-like 3(ANGPTL3):ANGPTL3 is cleaved andactivated in vivo[J].J Biol Chem, 2003, 278(43): 41804-41809), while affecting podocyte injury (Liu J, Gao X, Zhai Y, et al. A novel role of angiopoietin-like-3 associated with podocyte injury[J]. Pediatr Res, 2015, 77(6):732- 739.), insulin resistance (Robciuc MR, Maranghi M, Lahikainen A, et al. Angptl3 deficiency is associated with increased insulin sensitivity, lipoprotein lipase activity, and decreased serum freefatty acids [J]. Arterioscler Thromb Vasc Biol, 2013, 33(7 ):1706-1713.), atherosclerosis (Dewey FE, Gusarova V, Dunbar RL, et al. Genetic and pharmacologicinactivation of ANGPTL3 and cardiovascular disease [J]. N Engl JMed, 2017, 377(3): 211- 221.) and tumor occurrence and metastasis (Koyama T, Ogawara K, Kasamatsu A, et al. ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer[J]. Cancer Med, 2015, 4(5): 759-769), obesity, diabetes, familial hypobetalipoproteinemia, etc. also play an important role. In addition, studies have shown that ANGPTL3 is related to the occurrence and development of other metabolic diseases such as kidney disease (Liu J, Gao X, Zhai Y, et al. A novel role of angiopoietin-like-3 associated with podocyte injury. Pediatr Res, 2015, 77 (6):732-739), ANGPTL3 is only slightly expressed in normal-functioning kidneys, but its expression is significantly increased in a variety of kidney diseases with proteinuria as the main manifestation (Zhang Qiang, Yin Weidong, Tang Chaoke; ANGPTL3 is associated with metabolic disorders Progress in disease research[J] Chemistry of Life, 2018, 38(1):30-34).

Inflammatory factors are a type of highly efficient small molecule soluble proteins secreted by immune cells or tissue cells that regulate each other between cells. Common types of inflammatory factors include interleukin (IL) and interferon (IFN). ) and tumor necrosis factor (TNF), etc., which can regulate the development and differentiation of immune cells, participate in innate immunity and adaptive immune responses, and immune regulation, and play an important role in inflammatory responses (EKTanYX.Chao, A.WestLL .ChanWPoeweJ.JankovicParkinson diseaseand the immune system-associations,mechanisms and therapeuticsJ].NatRevNeurol202016(6):303-318).

Diabetes is a systemic disease that can cause a variety of complications, among which diabetic nephropathy is one of the complications of diabetes. Diabetic nephropathy is a kidney disease secondary to diabetes. In the past ten years, the incidence of end-stage renal disease caused by diabetic nephropathy has ranked first in my country (Zhao Yanxiang. Analysis of changes in serum TNF-a and IL-6 in patients with early diabetic nephropathy and their correlation with urinary albumin/creatinine [U]. Chinese Medical Innovation, 2020, 17(4):121-124.) Shao Changjuan, Shen Meng, Sun Lili. Level changes and significance of serum IL-6, TNF-a, and high-sensitivity C-reactive protein in patients with diabetic nephropathy[J] . Chinese Health Engineering, 2020,19(3):444-446.). In recent years, many studies have pointed out that serum inflammatory factors play an important role in the pathogenesis of diabetic nephropathy. Because pro-inflammatory cytokines act on the body, they induce the production of pro-inflammatory cytokines and promote the inflammatory factor response, thereby promoting the development of diabetic nephropathy. (Effects of hemodialysis combined with hemoperfusion on serum inflammatory factor levels in Cao Juan patients with end-stage diabetic nephropathy [J]. Clinical Research, 2020, 28(3):76-77.)

Podocyte injury is a common pathological change in nephrotic syndrome (NS). As an important component of the glomerular filtration barrier, damage to podocytes can cause proteinuria, which is the main driving factor in the development of NS. When proteinuria reaches a specific threshold, hypoalbuminemia, edema and other symptoms will appear. (Macé C, Chugh SS. Nephrotic syndrome: components, connections, and angiopoietin-like-4-related therapeutics[J]. J AmSoc Nephrol, 2014, 25(11): 2393-2398), Proteinuria is a common clinical symptom of kidney disease Performance: Chronic kidney disease can cause renal fibrosis when it develops to a certain stage. Renal fibrosis can be induced by a variety of factors, including severe trauma and infection to the kidney, obstruction of renal blood circulation, or immune response. After renal tissue is damaged by various factors, a large number of collagen fibers are deposited in the interstitium and scars are formed, leading to changes in renal structure and function, thereby causing renal fibrosis. The mechanism of renal fibrosis is not completely clear so far.

At present, the pathological diagnosis of NS mainly relies on renal biopsy, but its invasiveness limits its widespread use. As the basic drugs for treating NS, hormones and immunosuppressants also have many toxic and side effects. Although a mouse monoclonal antibody of ANGPTL3-FLD was prepared in an anti-ANGPTL3 monoclonal antibody and its use in preparing drugs for the treatment of nephrotic syndrome (Patent No.: 201911177503.5), it effectively reduced podocyte damage in vivo and in vitro. It has the therapeutic effect of reducing proteinuria, but it does not play a role in preventing renal fibrosis, a key problem in the treatment of diabetic nephropathy.

There is currently a need to develop a therapeutic method and agent that can achieve multi-dimensional, multi-temporal and spatial pharmacological effects of protecting podocytes, reducing urinary protein, alleviating renal inflammation, and inhibiting renal fibrosis. The present invention can meet this need.

Contents of the invention

The present application discloses a fusion protein ANGPTL3 monoclonal antibody-B containing an ANGPTL3 monoclonal antibody. The fusion protein ANGPTL3 monoclonal antibody-B is composed of the fusion of ANGPTL3 monoclonal antibody and sequence B; the B is selected from cytokines.

Furthermore, the cytokines may be selected from interleukin (IL), interferon (IFN), tumor necrosis factor (tumornecrosis factor, TNF), etc. In some embodiments, the cytokine is further selected from: IL-22, IL-1RA, IL-4, IL-10, IL-13, TGF-β and/or EPO, etc. Human IL-22 (SEQ ID No: 5) and/or murine IL-22 (SEQ ID No: 6) are further preferred. In some embodiments, ANGPTL3 monoclonal antibody and sequence B can be connected through a linker. The linker It can be selected from (GGGGS)2, (GGGGS)3, (GGGGS)4, (EAAAK)2, and/or (EAAAK)3, etc.

The present application also discloses a pharmaceutical composition comprising the disclosed fusion protein ANGPTL3 monoclonal antibody-B and a pharmaceutically acceptable carrier.

This application also discloses isolated nucleotides encoding the fusion protein ANGPTL3 monoclonal antibody-B disclosed herein. The present invention also covers vectors containing the disclosed nucleotides, and vectors containing the vectors or encoding the fusion protein ANGPTL3 monoclonal antibodies of the present invention. The host cell of the polynucleotide of -B.

The present application also discloses the use of the fusion protein ANGPTL3 monoclonal antibody-B in the preparation of drugs for treating diseases or conditions mediated by the fusion protein ANGPTL3 monoclonal antibody-B. Pharmaceutical compositions.

Description of drawings

Figure 1. SDS-PAGE detection of molecular weight of fusion protein

Figure 2. SEC-HPLC detection of fusion protein purity

Figure 3. SPR detection of affinity of fusion protein

Figure 4. Thermal stability analysis detects the melting point of fusion protein

Figure 5. In vitro fluorescence experiment to verify the in vitro activity of the fusion protein ANGPTL3 monoclonal antibody end

Figure 6. Western Blot to verify the in vitro activity of the IL-22 end of the fusion protein

Figure 7. Fusion protein intervention reduces urinary protein in diabetic nephropathy mouse model

Figure 8. Fusion protein intervention protects renal function in diabetic nephropathy mouse model.

Figure 9. Fusion protein intervention reduces blood sugar and blood lipids in diabetic nephropathy mouse model

Figure 10. Fusion protein intervention reduces podocyte damage in diabetic nephropathy mouse model

Figure 11. Fusion protein intervention inhibits renal fibrosis in diabetic nephropathy mouse model.

Figure 12. Fusion protein intervention reduces inflammatory response in diabetic nephropathy mouse model

Figure 13. Fusion protein protects the kidneys by inhibiting the NF/κB/NLRP3 signaling pathway in a mouse model of diabetic nephropathy.

Figure 14. Fusion protein intervention reduces urinary protein in mouse model of adriamycin nephropathy.

Figure 15. Fusion protein intervention protects renal function in the mouse model of doxorubicin nephropathy.

Figure 16. Fusion protein intervention improves hypoalbuminemia in the mouse model of doxorubicin nephropathy.

Figure 17. Fusion protein intervention improves hyperlipidemia in the mouse model of adriamycin nephropathy.

Figure 18. Fusion protein intervention reduces podocyte damage in the mouse model of adriamycin nephropathy.

Figure 19. Fusion protein intervention inhibits renal fibrosis in the mouse model of doxorubicin nephropathy.

Figure 20. Fusion protein protects the kidney by improving mitochondrial function in the mouse model of doxorubicin nephropathy.

Figure 21. Fusion protein protects the kidney by improving lysosomal autophagy in the mouse model of doxorubicin nephropathy.

Detailed ways

This application discloses the fusion protein ANGPTL3 monoclonal antibody-B. The fusion protein ANGPTL3 monoclonal antibody-B is connected to the ANGPTL3 monoclonal antibody and cytokines, and can protect podocytes, reduce urinary protein, reduce renal inflammation, and inhibit renal fibrosis.

Definitions and common techniques

Unless otherwise defined herein, technical and scientific terms used in connection with the present invention shall have the meaning commonly understood by one of ordinary skill in the art. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. of those.

As used herein, the term ANGPTL3 is an important member of the angiopoietin-like protein (ANGPTL) family. It is a secreted glycoprotein with a similar structure to angiopoietin and plays an important role in regulating angiogenesis. The structure of ANGPTL3 includes an amino-terminal coiled-coil domain, a secretion signal peptide, a short connecting peptide or a carboxyl-terminal globular fibrinogen-like domain (FLD). The term "fusion protein ANGPTL3 monoclonal antibody-B" refers to ANGPTL3 The protein or its protein fragments (amino-terminal coiled-coil domain, secretion signal peptide, short connecting peptide or carboxyl-terminal globular fibrinogen-like domain) are used as antigens to prepare monoclonal antibodies fused with other sequences. The ANGPTL3 monoclonal antibody can target the ANGPTL3 protein or its protein fragments such as the amino-terminal coiled-coil domain, secretory signal peptide, short connecting peptide or carboxyl-terminal globular fibrinogen-like domain FLD, and the ANGPTL3 monoclonal antibody is preferably targeted. in the FLD domain. Currently existing ANGPTL3 monoclonal antibodies such as SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3 and SEQ ID No: 4 are also suitable for use in the present invention.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids as well as polypeptides with modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, and the like.

Fusion protein ANGPTL3 monoclonal antibody-B can be obtained by fusing ANGPTL3 monoclonal antibody with heterologous sequences. As described herein, the heterologous sequence may be a polymer that contains an amino acid sequence or does not contain an amino acid sequence. The heterologous sequence can be fused to the ANGPTL3 mAb directly, chemically or by recombinant expression from a single polynucleotide, or it can be joined via a linker or linker molecule. A peptidyl linker or linker molecule can be one or more amino acid residues (or mers), such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 residues (or mers). Linkers or linker molecules can also be designed to have cleavage sites for DNA restriction endonucleases or proteases to allow isolation of the fused parts.

As the term "linker" is used herein, linkers may (but are not necessarily) employed when forming fusion proteins of the invention. Linkers can be composed of amino acids linked together by peptide bonds (i.e., peptidyl linkers). In some embodiments of the invention, the linker is composed of 1 to 20 amino acids connected by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. In some embodiments, suitable linkers include (GGGGS)2, (GGGGS)3, (GGGGS)4, (EAAAK)2, (EAAAK)3, etc.

The term "pharmaceutical composition" as used herein is prepared by mixing the fusion protein ANGPTL3 monoclonal antibody-B of the present invention with the desired purity and a pharmaceutically acceptable carrier in the form of a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to the recipient at the doses and concentrations employed and are formulated for oral ingestion in liquid, solid, aerosol, or other forms. In certain aspects, the pharmaceutical compositions provided in the present disclosure can be used in a method of treating a disease in a subject, wherein the method includes administering to the subject: a fusion protein and a vector comprising a polynucleotide encoding the fusion protein ; Modified host cells expressing the fusion protein; or pharmaceutical compositions thereof, wherein the disease is associated with the presence of an antigen bound by the fusion protein.

As used herein, the term "cytokine" refers to various cytokines involved in inflammatory responses. The cytokines described herein mainly include interleukin (IL), interferon (IFN) and tumor necrosis factor (tumornecrosis factor). , one or more of TNF). In one embodiment, the cytokine is further selected from: IL-22, IL-1RA, IL-4, IL-10, IL-13, TGF-β, EPO, etc.

The term "vector" as used herein may also be a nucleic acid molecule capable of transporting another nucleic acid. The vector may be, for example, a plasmid, a cosmid, a virus, a phage, an RNA vector or a linear or circular DNA or RNA molecule, which may include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules. In some embodiments, the vector is a "plasmid," that is, a circular double-stranded DNA loop to which other DNA segments can be ligated. In addition, certain vectors are capable of directing expression of the gene to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

The term "recombinant host cell" (or simply "host cell") as used herein means a cell into which exogenous nucleic acids and/or recombinant vectors are introduced. It will be understood that "recombinant host cell" and "host cell" refer not only to the specific subject cell, but also to the progeny of such cells. Because certain modifications may appear in progeny due to mutations or environmental influences, such progeny may in fact differ from the parent cell but still be included within the scope of the term "host cell" as used herein. Transformed host cells may include, but are not limited to, prokaryotic cells, eukaryotic cells, and cells of mammalian, plant, insect, fungal, or bacterial origin. In addition, for mammalian cells, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells, HEK293T cells, etc. can be used . However, mammalian cells are not limited thereto, and any cells known to those skilled in the art to be useful as mammalian host cells may be used.

The term "treatment," etc., as used herein, refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects attributable to said disease. "Treatment" as used herein encompasses any treatment of a disease in mammals, in particular humans, and includes: (a) preventing said disease in persons who may be susceptible to said disease but have not yet been diagnosed with said disease; occurs in a subject; (b) inhibits the disease, i.e. arrests its progression; and (c) alleviates the disease, i.e. causes regression of the disease.

The term "patient" is used interchangeably herein to include humans and non-human animals, such as mammals, such as mice, rats, guinea pigs, dogs, cats, rabbits, cattle, horses, sheep, goats and pigs. The term also includes birds, fish, reptiles, amphibians, and the like. It should be understood that a more specific patient is a human being. Likewise, more specific patients and subjects are non-human mammals, such as mice, rats and dogs.

The term "administration", which is used interchangeably herein, refers to an amount and manner of administration of a fusion protein of the present disclosure that is sufficient to ameliorate one or more symptoms of the disease being treated in a statistically significant manner. In one embodiment, the ANGPTL3 monoclonal antibody/IL-22 fusion protein is administered by injection at 5-100 mg/kg, 20-100 mg/kg, 30-100 mg/kg, 40-100 mg/kg, 50-100 mg/kg, 60-100mg/kg, 70-100mg/kg, 80-100mg/kg, 90-100mg/kg.

The term "monoclonal antibody" as used herein generally refers to a population of substantially homologous antibodies, and the individual antibodies comprised in the population may be identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and target directly a single antigenic site.

"Isolated nucleotide" refers to a nucleic acid (such as DNA) that does not contain a gene. In the present invention, the nucleotide refers to the nucleic acid molecule of the fusion protein ANGPTL3 monoclonal antibody-B. Thus, the term includes, for example, recombinant DNA incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryotic or eukaryotic organism; or as a separate molecule independent of other sequences (e.g., cDNA or by Genomic or cDNA fragments generated by PCR or restriction endonuclease digestion) are present. Furthermore, the term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that encodes a portion of a hybrid gene encoding additional polypeptide sequences.

As used herein, the term "variable light chain" (VL) refers to the variable binding region of an antibody light chain. The variable binding region consists of discrete, well-defined subregions called complementarity-determining regions (CDRs, also known as HVRs (hypervariable regions)) and framework regions (FRs). CDR refers to the amino acids within the variable region of an antibody that confer antigen specificity and/or binding affinity, separated by FR. There are three CDRs in each antibody light chain variable region (LCDR1, LCDR2, LCDR3) and three CDRs in each antibody heavy chain variable region (HCDR1, HCR2, HCDR3).

The term "CL" as used herein refers to an "immunoglobulin light chain constant region" or "light chain constant region", ie, a constant region derived from an antibody light chain.

The term "expression" as used herein refers to the process of producing a polypeptide based on the coding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

As used herein, the term "condition or disease" includes diabetes and its complications such as: diabetic nephropathy, diabetic retinopathy, diabetic foot, cardiovascular complications of diabetes, diabetic cerebrovascular disease (cerebral arteriosclerosis, silent stroke), Diabetic neuropathy, kidney-related diseases such as membranous nephropathy, IgA nephropathy, purpura kidney, lupus kidney, etc., and tumor-related diseases such as hepatocellular carcinoma, renal cell carcinoma, ovarian cancer, cervical cancer, oral cancer, esophageal cancer, etc.

As used herein, the term "transient transfection" refers to the introduction of a constructed plasmid into mammalian cells in some way. The foreign genes on the plasmid are not integrated into the cell's own genome. This entire rapid transfection The process of obtaining proteins is called transient transfection expression.

Example 1 Fusion protein expression and purification

1.1 Construct two plasmids using pTT5 as vector

1.1.1 Insert the monoclonal antibody heavy chain variable and constant region sequences (VH+CH1-CH3), linker sequence linker (GGGGS) into the HindIII and BamHI restriction sites _{. 3.} Murine IL-22 sequence (mIL-22 );

1.1.2 Insert the monoclonal antibody light chain variable region and constant region sequences (VL+CL) into the EcoRI and BamHI restriction sites, as shown in Figure 1. After the constructed plasmid is sequenced correctly by Sanger, proceed to the next step.

1.2 Transform competent cells with DH5α amplification plasmid

Mix the plasmid and competent cells DH5α and put them in an ice bath. After heat shock in a 42°C water bath, quickly transfer to ice and let stand. Then add LB liquid culture medium and resuscitate on a shaking table at 37°C. Take a small amount of the recovered liquid and spread it. Place on the LB solid medium plate, invert the culture at 37°C overnight, select single clones and expand them into shake flasks, and then use the Tiangen Max Extraction Kit to extract the plasmid for later use.

1.3 The amplified two plasmids were transiently transfected into ExpiCHO-S cells to express the protein.

Cultivation of ExpiCHO-S cells in a constant temperature incubator with 5% CO2 to a density of 3-6×10 ⁶ cells/mL, adjusted the cell concentration to 3.5×10 ⁶ cells/mL, and then cultured under the same culture conditions for 18 -20h until the cell density is 7-9×10 ⁶ cells/mL and the viability rate is greater than 95%, use pre-warmed culture medium to dilute the cells to 6×10 ⁶ cells/mL, and use Thermo Fisher ExpiFectamine ^TM CHO for transfection Kit, mix the plasmid and transfection reagent and let it stand for 1 minute, then slowly add it to the cells, shake the flask while adding (to avoid excessive local concentration), mix thoroughly and continue to culture under the previous conditions, and in the first Feeding medium was added on the first and fifth days, and the cell density was measured during the period. After 12 days of culture, the supernatant was collected by centrifugation for subsequent protein purification.

1.4 Protein purification and ultrafiltration concentration

Use a Protein A affinity chromatography column to purify proteins, prepare Buffer A (1×TBS solution) and Buffer B (0.1M glycine solution), turn on the purifier, and balance 1 mL of Protein A affinity chromatography column with Buffer A for 10 column volumes. Then, inject the above cell culture supernatant into the sample. After loading, rinse with Buffer A for at least 10 column volumes, and then elute with Buffer B for 5 column volumes. Collect the eluate and use Tris-HCl buffer to Adjust the eluent to neutral. Ultrafiltrate the eluent using a 50KD ultrafiltration tube, add PBS solution to make up to 15ml, and centrifuge three times at 3500rpm and 10min. Collect the final 600-1000ul of liquid in the ultrafiltration tube into a clean 1.5ml EP tube and store at -80°C.

Example 2 Fusion protein characterization method

2.1SDS-PAGE detects the molecular weight of fusion protein

Configure a 10% Tris-glycine gel, load 20ul of the sample under reducing and non-reducing conditions for electrophoresis, set the voltage to 80V for concentration and 120V for separation. After electrophoresis, take out the gel and stain it with Coomassie Brilliant Blue for 10 minutes, and then use Rinse overnight with a destaining solution composed of 30% methanol and 10% acetic acid, take images with a Biorad gel imager, and determine the molecular weight based on the band position.

2.2 SEC-HPLC to detect the purity of fusion protein

Using Agilent 1260Infinity II SFC system, 100 μg of purified protein was diluted with PBS buffer and then

Inject the TOSOH TSKgel G3000WXL chromatographic column (7.8mm×30cm, 5μm) at a flow rate of 1.0ml/min, and detect it under 37°C and 280nm UV irradiation for 30 minutes. The purity of the fusion protein can be judged by understanding the degree of aggregation and degradation.

2.3SPR detection of affinity of fusion protein

The Biacore T200 system (GE Healthcare, USA) was used for SPR analysis, and the human IgG capture antibody was pre-fixed on the CM5 chip (GE Healthcare, USA). By adjusting the capture time, the fusion protein was captured on the chip in about 60RU, and then The antigen hANGPTL3 (S17-E460) or the antigen mANGPTL3 (S17-T455) flows through the chip, binds for 120 seconds, and dissociates for 600 seconds. The affinity is calculated through the binding and dissociation parameters generated by the system.

2.4 Thermal stability analysis to detect the melting point of fusion protein

Using Nano Temper PR.48 instrument (Nano Temper Scientific, Germany), the fusion protein was diluted with PBS buffer to 1.03 mg/ml, 0.5 mg/ml and 2.2 mg/ml in the temperature range of 20-95°C, and monitored by The changes in fluorescence intensity caused by slight changes in tryptophan residues are used to fit the original data into an S-shaped curve, and the inflection point is the melting point, thereby judging the thermal stability of the fusion protein.

2.5 In vitro fluorescence experiments verify the in vitro activity of the fusion protein ANGPTL3 monoclonal antibody end

Under high-glucose conditions, podocytes highly express ANGPTL3, which can bind to integrin ανβ3, resulting in PSI epitope exposure, which can be bound and displayed by the specific antibody AP5. In order to determine the activity of the fusion protein ANGPTL3 monoclonal antibody, glomerular podocytes (MPC5) were seeded in a 6-well plate at a density of 1×106/ml, a temperature of 37°C, and a concentration of 5% CO2 overnight. After the cells adhered, add The fusion protein was incubated at 126ng/ml for 1-2 hours, and then 30mM high glucose solution was added for injury intervention for 4-6 hours. Finally, the PSI domain-specific antibody AP5 was used to detect whether the fusion protein could compete with ANGPTL3 for binding to block the PSI epitope. exposed.

2.6 Western Blot to verify the in vitro activity of the IL-22 end of the fusion protein

IL-22 can induce the phosphorylation of STAT3 in mouse proximal tubular epithelial cells (mPTC). First, mPTC was seeded in a 6-well cell culture plate at a density of 1×106/ml, a temperature of 37°C, and a concentration of 5% CO2 overnight. Then, the cells were treated with 20mm fusion protein for 2h, 4h, and 6h. After collecting the cells, 50ul of Lyse with weak RIPA for 30 min, centrifuge at 4°C and 12000 rpm for 15 min, collect the lysate supernatant, and finally use rabbit anti-mouse phosphorylated STAT3 antibody (Biolegend) and HPR-labeled goat anti-rabbit antibody (Merck) for Western blot detection.

Example 3 Functional verification in animal experiments

3.1 Construction of diabetic nephropathy mouse model and dosing regimen

db/m and db/db mice (C57BKS/Lepr, male, 6-7 weeks old) produced by Changzhou Cavins Animal Experiment Co., Ltd. (Jiangsu, China) were selected and raised under specific pathogen-free (SPF) conditions, and the control group was The db/m mice were fed with ordinary feed, and the model group was db/db mice, which were fed with high-fat (fat content 40%) feed for 4-5 weeks. The DN mouse model was successfully established at about 4 weeks, and drug administration was started with intraperitoneal injection twice a week for 8 weeks. The normal control group and the model group were injected with the same dose of normal saline. Experimental groups: control group (7 animals), model group (7 animals), ANGPTL3 monoclonal antibody treatment group (7 animals, ANGPTL3 monoclonal antibody 20 mg/kg), IL-22Fc treatment group (7 animals, IL-22Fc 12 mg/kg) , ANGPTL3 monoclonal antibody/IL-22 fusion protein treatment group (7 animals, fusion protein 25.3 mg/kg), ANGPTL3 monoclonal antibody/IL-22Fc combination treatment group (7 animals, ANGPTL3 monoclonal antibody 20 mg/kg + IL-22Fc 12 mg/kg ), positive drug losartan treatment group (7 animals, losartan 20mg/kg/d, daily gavage).

3.2 Construction of doxorubicin nephropathy mouse model and dosage regimen

A total of 48 6-week-old male Balb/c mice were selected and raised under specific pathogen-free (SPF) conditions. During the experiment, they had free access to water and food. After 1 week of feeding (7 weeks of age), a one-time tail vein injection of 10.5 mg/kg of doxorubicin diluted to 2 mg/ml with physiological saline was used to create the model. Administration was started on the second day after modeling, with intraperitoneal injection twice a week for 12 weeks. The normal control group and the model group were injected with the same dose of normal saline. Experimental groups: control group (8 animals), model group (8 animals), ANGPTL3 monoclonal antibody treatment group (8 animals, ANGPTL3 monoclonal antibody 20 mg/kg), IL-22Fc treatment group (8 animals, IL-22Fc 12 mg/kg) , ANGPTL3 monoclonal antibody/IL-22 fusion protein treatment group (8 animals, fusion protein 25.3mg/kg), ANGPTL3 monoclonal antibody/IL-22Fc combination treatment group (8 animals, ANGPTL3 monoclonal antibody 20mg/kg+IL-22Fc 12mg/ kg).

3.3 Detect the levels of biological macromolecules or metabolites in blood and urine samples

Samples were collected at different time points after administration. Blood was collected through the inner canthus capillaries, and urine was collected through a 24-hour mouse metabolic cage and stored in a -80°C refrigerator. Subsequently, relevant detection kits (Nanjing Jiancheng, China) were used to detect serum creatinine, serum urea nitrogen, serum total cholesterol, serum triglycerides, blood sugar, urine creatinine and 24-hour urine protein levels; ELISA kits (Nanjing Jiancheng, China) were used for detection. Urinary microalbumin, serum TNF-α, IL-6 and IL-1β, etc.

3.4 Observe the morphological changes of kidney tissue with the help of light microscope or electron microscope

After the mice were sacrificed, the kidneys were removed and cut longitudinally, fixed in 4% paraformaldehyde solution overnight, embedded in paraffin, cut into 4 μm thick sections, stained with H&E, PAS, and Masson, respectively, and examined under 200× and 400× microscopes. The pathological damage and fibrosis degree of the renal tissue were observed, and Image J software was used to quantify and analyze the percentage of positive areas in the entire image. In order to observe changes in the ultrastructure of glomeruli, kidney tissue of appropriate size was fixed overnight at 4°C, and then observed under an electron microscope at 60kv. Kidney tissue from 3-5 mice in each group was taken to observe the ultrastructure of podocytes. , changes in glomerular basement membrane thickness, mitochondria and lysosomes.

Example 4: Experimental results

1. SDS-PAGE detects the molecular weight of fusion protein

SDS-PAGE showed that the molecular weight of the ANGPTL3 monoclonal antibody-IL22 fusion protein under reducing conditions was 72.39KD for the heavy chain and 25.94KD for the light chain, which was consistent with expectations and showed its high purity (Figure 1)

2. SEC-HPLC to detect the purity of fusion protein

SEC-HPLC showed that the main peak of the protein was located at 5.583 minutes, with only a weak peak around 8 minutes, indicating that the ANGPTL3 monoclonal antibody-IL22 fusion protein did not undergo obvious aggregation and dissociation, further confirming its good purity. (See Figure 2)

3.SPR detection of affinity of fusion protein

SPR analysis showed that the KD value of the ANGPTL3 monoclonal antibody-IL22 fusion protein with mouse ANGPTL3 was 1.040E-8, and the KD value with human ANGPTL3 was 5.572E-9, showing a similar affinity to the ANGPTL3 monoclonal antibody itself. (See Figure 3)

4. Thermal stability analysis to detect the melting point of fusion protein

Thermal stability analysis showed that the Tm1 values of ANGPTL3 monoclonal antibody-IL22 fusion protein, ANGPTL3 monoclonal antibody, and mIL22Fc were 66.1°C, 66.6°C, and 66.5°C respectively, indicating that the construction of the fusion protein did not affect its original melting point, showing Good thermal stability. (See Figure 4)

5. In vitro fluorescence experiments verify the in vitro activity of the fusion protein ANGPTL3 monoclonal antibody end

In vitro fluorescence experiments show that the ANGPTL3 monoclonal antibody-IL22 fusion protein is similar to the ANGPTL3 monoclonal antibody and can effectively block the binding of ANGPTL3 to podocyte surface integrin ανβ3 and inhibit the exposure of PSI epitopes, confirming the in vitro activity of its ANGPTL3 monoclonal antibody end. (See Figure 5)

6. WesternBlot to verify the in vitro activity of the IL-22 end of the fusion protein

Western Blot showed that ANGPTL3 monoclonal antibody-IL22 fusion protein could significantly increase the phosphorylation level of STAT3 2 hours after acting on mouse proximal renal tubular epithelial cells, confirming its in vitro activity at the IL-22 end. (See Figure 6)

7. Fusion protein intervention reduces urinary protein in mouse model of diabetic nephropathy

The diabetic nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. Compared with the model group and mIL22Fc treatment group, 24-hour urinary protein was significantly reduced (P<0.05). (See Figure 7)

8. Fusion protein intervenes to protect renal function in diabetic nephropathy mouse model

The diabetic nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks, and the serum urea nitrogen level was significantly reduced compared with the model group and ANGPTL3 monoclonal antibody treatment group (P<0.05). (See Figure 8)

9. Fusion protein intervention reduces blood sugar and blood lipids in diabetic nephropathy mouse model

The diabetic nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. Compared with the model group, blood glucose, serum triglyceride, and serum total cholesterol levels were significantly reduced (P<0.05). At the same time, it can be seen that ANGPTL3 monoclonal antibody -IL22 fusion protein shows certain advantages in reducing serum triglycerides compared with the combination of ANGPTL3 and IL22. (See Figure 9)

10. Fusion protein intervention reduces podocyte damage in diabetic nephropathy mouse model

The diabetic nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. Compared with the model group, ANGPTL3 monoclonal antibody treatment group, and mIL22Fc treatment group, foot process fusion and basement membrane thickening were significantly improved. (See Figure 10)

11. Fusion protein intervention inhibits renal fibrosis in diabetic nephropathy mouse model

The diabetic nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. Compared with the model group, ANGPTL3 monoclonal antibody treatment group, and mIL22Fc treatment group, collagen deposition in the glomerular and renal interstitial areas was significantly reduced, and renal fibrosis was improved. Significant improvement. (See Figure 11)

12. Fusion protein intervention reduces inflammatory response in diabetic nephropathy mouse model

The diabetic nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. Compared with the model group and the ANGPTL3 monoclonal antibody treatment group, the serum levels of inflammatory factors TNF-α, IL-6 and IL-1β were significantly reduced (P<0.05 ), and it can be seen that the ANGPTL3 monoclonal antibody-IL22 fusion protein shows significant advantages in reducing TNF-α and IL-1β compared with the combination of ANGPTL3 and IL22. (See Figure 12)

13. Fusion protein protects kidneys by inhibiting NF-κB/NLRP3 signaling pathway in diabetic nephropathy mouse model

After 8 weeks of diabetic nephropathy mouse model treatment with ANGPTL3 monoclonal antibody-IL22 fusion protein, compared with the model group and ANGPTL3 monoclonal antibody treatment group, the levels of NLRP3 and Pro-caspase-1 in the kidney tissue were significantly lower (P<0.05). It was confirmed that it exerts renal protective effects by inhibiting the NF-κB/NLRP3 signaling pathway. (See Figure 13)

14. Fusion protein intervention reduces urinary protein in mouse model of doxorubicin nephropathy

The doxorubicin nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. Compared with the model group and mIL22Fc treatment group, the urinary albumin/creatinine ratio was significantly reduced (P<0.05). At the same time, it can be seen that the ANGPTL3 monoclonal antibody- IL22 fusion protein showed significant advantages in reducing urinary albumin/creatinine ratio compared with the combination of ANGPTL3 and IL22. (See Figure 14)

15. Fusion protein intervenes to protect renal function in the mouse model of doxorubicin nephropathy.

The mouse model of doxorubicin nephropathy was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. The serum creatinine level was significantly lower than that of the model group (P<0.05). At the same time, it can be seen that the ANGPTL3 monoclonal antibody-IL22 fusion protein was significantly lower than that of ANGPTL3 and The combination of IL22 showed significant advantages in reducing serum creatinine. (See Figure 15)

16. Fusion protein intervention improves hypoalbuminemia in the mouse model of doxorubicin nephropathy

The doxorubicin nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks, and the serum albumin level was significantly increased compared with the model group (P<0.05). (See Figure 16)

17. Fusion protein intervention improves hyperlipidemia in the mouse model of doxorubicin nephropathy

The mouse model of doxorubicin nephropathy was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks, and the serum total cholesterol level was significantly lower than that of the model group (P<0.05). (See Figure 17)

18. Fusion protein intervention reduces podocyte damage in mouse model of doxorubicin nephropathy

The doxorubicin nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. Compared with the model group, pathological changes such as foot process fusion, basement membrane thickening, and microvilli degeneration were significantly improved. (See Figure 18)

19. Fusion protein intervention inhibits renal fibrosis in the mouse model of doxorubicin nephropathy

The doxorubicin nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. Compared with the model group, collagen deposition in the glomerular and renal interstitial areas was significantly reduced, and renal fibrosis was significantly improved. (See Figure 19)

20. Fusion protein protects kidneys by improving mitochondrial function in mouse model of doxorubicin nephropathy

The doxorubicin nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. Compared with the model group, pathological damage such as shortened mitochondrial length, disordered arrangement, and internal cristae rupture were significantly improved. (See Figure 20)

21. Fusion protein protects kidneys by improving lysosomal autophagy in mouse model of doxorubicin nephropathy

The doxorubicin nephropathy mouse model was treated with ANGPTL3 monoclonal antibody-IL22 fusion protein for 8 weeks. Compared with the model group, the pathological changes of increased number and larger volume of lysosomes were significantly alleviated, revealing the improvement of lysosomal autophagy behavior ( See Figure 21).

Claims (10)

1. A fusion protein ANGPTL3 monoclonal antibody-B comprising ANGPTL3 monoclonal antibody, wherein the fusion protein ANGPTL3 monoclonal antibody-B is composed of the fusion of ANGPTL3 monoclonal antibody and sequence B. 2. The fusion protein ANGPTL3 monoclonal antibody-B as claimed in claim 1, wherein the B is selected from the group consisting of cytokines, and the cytokines can be selected from the group consisting of interleukin (IL), interferon (IFN) and tumors. Necrosis factor (tumornecrosis factor, TNF). 3. The fusion protein ANGPTL3 monoclonal antibody-B as claimed in claim 2, the cytokine is selected from: IL-22, IL-1RA, IL-4, IL-10, IL-13, TGF-β and/or EPO. 4. The fusion protein ANGPTL3 monoclonal antibody-B according to any one of claims 1 to 3, characterized in that ANGPTL3 monoclonal antibody and sequence B can be connected through a linker, and the linker can be selected from (GGGGS)2, ( GGGGS)3, (GGGGS)4, (EAAAK)2 and or (EAAAK)3. 5. A pharmaceutical composition comprising the fusion protein ANGPTL3 monoclonal antibody-B according to any one of claims 1 to 4, further comprising a pharmaceutically acceptable carrier. 6. Polynucleotide encoding the fusion protein ANGPTL3 monoclonal antibody-B according to any one of claims 1 to 4. 7. A host cell expressing the polynucleotide of the fusion protein ANGPTL3 monoclonal antibody-B according to any one of claims 1 to 4. 8. The use of the fusion protein ANGPTL3 monoclonal antibody-B according to any one of claims 1 to 4 in the preparation of medicines for treating diseases or conditions mediated by it. 9. The use of the fusion protein ANGPTL3 monoclonal antibody-B as claimed in claim 8 in the preparation of medicines for treating diseases or disorders mediated by it, wherein the diseases or disorders include diabetic nephropathy, diabetic retinopathy, diabetic foot, Diabetic cardiovascular complications, diabetic cerebrovascular disease, cerebral arteriosclerosis, silent stroke, diabetic neuropathy, membranous nephropathy, IgA nephropathy, purpura kidney, lupus kidney. 10. The use of the fusion protein ANGPTL3 monoclonal antibody-B as claimed in claim 8 in the preparation of medicines for treating diseases or disorders mediated by it, wherein the diseases or disorders include hepatocellular carcinoma, renal cell carcinoma, ovarian cancer, Cervical cancer, oral cancer, esophageal cancer.