Fusion proteins containing ANGPTL3 monoclonal antibodies

Abstract

The present application discloses a fusion protein, ANGPTL3 monoclonal antibody-B, comprising an ANGPTL3 monoclonal antibody, wherein the fusion protein, ANGPTL3 monoclonal antibody-B, comprises a fusion of an ANGPTL3 monoclonal antibody with sequence B. The fusion protein, ANGPTL3 monoclonal antibody-B, disclosed in the present application is used to treat diseases or conditions mediated thereby, for example, 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 nephritis, and lupus nephritis, and tumor-related diseases such as hepatocellular carcinoma, renal cell carcinoma, ovarian cancer, cervical cancer, oral cancer, and esophageal cancer.

Description

[Technical Field]

[0001] The present invention relates to the fields of medical immunology and molecular biology, and more particularly to a fusion protein containing a monoclonal antibody against angiopoietin-like protein 3 (ANGPTL3) and its medical applications. [Background technology]

[0002] Angiopoietin-like protein 3 (ANGPTL3) is a secreted glycoprotein containing an amino-terminal folded helix domain (CCD), a secretory signal peptide, a short linker peptide, and a carboxy-terminal globular fibrinogen-like domain (FLD). The amino-terminal folded helix domain of ANGPTL3 inhibits the activity of lipoprotein esterase (LPL), thereby increasing plasma triglyceride (TG) levels. The carboxy-terminal FLD (207-460aa) promotes angiogenesis by binding to the integrin αvβ3 receptor (NPL 1). The amino-terminal helix domain is involved in the regulation of lipid metabolism (NPL 2), and also plays an important role in podocyte injury (NPL 3), insulin resistance (NPL 4), atherosclerosis (NPL 5), tumorigenesis and metastasis (NPL 6), obesity, diabetes, and familial hypobetalipidemia. Furthermore, studies have shown a correlation between ANGPTL3 and the development of other metabolic diseases such as kidney disease (Non-Patent Document 7), and while ANGPTL3 is expressed only in trace amounts in functionally normal kidneys, its expression is significantly increased in various kidney diseases in which proteinuria is the main symptom (Non-Patent Document 8).

[0003] Inflammatory factors are a type of highly active, low-molecular-weight soluble protein secreted by immune cells and tissue cells that plays a role in mutual regulation between cells. Common types of inflammatory factors include interleukins (IL), interferons (IFN), and tumor necrosis factors (TNF). These control and influence the development and differentiation of immune cells, and are involved in intrinsic and adaptive immune responses and immune regulation, playing an important role in inflammatory responses (Non-Patent Document 9).

[0004] Diabetes is a systemic disease that causes various complications, including diabetic nephropathy, a renal lesion secondary to diabetes. In China, the incidence of end-stage renal disease due to diabetic nephropathy has been the highest for the past decade (Non-Patent Documents 10 and 11). In recent years, several studies have indicated that serum inflammatory factors play an important role in the development of diabetic nephropathy. This is because inflammatory cytokines act on the body, inducing the production of inflammatory cytokines and promoting inflammatory factor responses, which ultimately promote the development of diabetic nephropathy (Non-Patent Document 12).

[0005] Podocyte injury is a common pathological change in nephrotic syndrome (NS). Damage to podocytes, a key component of the glomerular filtration barrier, leads to proteinuria, which is the primary cause of NS. When proteinuria reaches a certain threshold, symptoms such as hypoproteinemia and edema appear (Non-Patent Document 13). Proteinuria is a common clinical manifestation of kidney disease, and chronic kidney disease may lead to renal fibrosis at some stage. Renal fibrosis can be induced by various factors, including severe kidney trauma or infection, obstruction of renal blood circulation, and immune responses. After renal tissue is damaged by various factors, numerous collagen fibers are deposited in the interstitium, resulting in scar formation, which leads to changes in renal structure and function and leads to renal fibrosis. The mechanisms of renal fibrosis are not yet fully understood.

[0006] Currently, the pathological diagnosis of NS mainly relies on kidney biopsy, but its widespread use is limited due to its invasive nature. The basic drugs used to treat NS, hormones and immunosuppressants, have numerous toxic side effects. In Patent Document 1, a mouse-derived monoclonal antibody against ANGPTL3-FLD was prepared, and its therapeutic effects in vivo and in vivo were shown to alleviate podocyte injury and reduce proteinuria. However, it did not address the important issue of renal fibrosis in the treatment of diabetic nephropathy. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Chinese Patent Application No. 201911177503.5 [Non-patent literature]

[0008] [Non-Patent Document 1] Camenisch G., Pisabarro MT, Sherman D., et al.ANGPTL3 stimulates endothelial cell adhesion and migration via integrin αvβ3 and induces blood vessel formation in vivo., J Biol Chem, 2002, 277(19): 17281~17290. [Non-patent document 2] Ono M., Shimizugawa T., Shimamura M., et al., Protein region important for regulation of lipid metabolism inangiopoietin-like 3 (ANGPTL3): ANGPTL3 is cleaved and activated in vivo[ J].J. Biol. Chem., 2003, 278(43): 41804~41809.

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[0009] Currently, there is a need for the development of therapeutic methods and agents that can achieve multidimensional and multitemporal pharmacological effects, such as protecting podocytes, reducing urinary protein, alleviating renal inflammation, and suppressing renal fibrosis, and the present invention can meet such needs. [Means for solving the problem]

[0010] The present application discloses a fusion protein ANGPTL3 monoclonal antibody-B comprising an ANGPTL3 monoclonal antibody, wherein the fusion protein ANGPTL3 monoclonal antibody-B comprises a fusion of an ANGPTL3 monoclonal antibody with a sequence B, wherein the B is selected from a cytokine.

[0011] Furthermore, the cytokine may be selected from interleukin (IL), interferon (IFN), tumor necrosis 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. More preferably, it is human-derived IL-22 (SEQ ID No: 5) and / or mouse-derived IL-22 (SEQ ID No: 6). In some embodiments, the ANGPTL3 monoclonal antibody and sequence B can be connected by a linker, and the linker may be selected from (GGGGS)2, (GGGGS)3, (GGGGS)4, (EAAAK)2, and / or (EAAAK)3, etc.

[0012] The present application further discloses a pharmaceutical composition, which comprises the disclosed fusion protein ANGPTL3 monoclonal antibody-B and a pharmaceutically acceptable vector.

[0013] The present application further discloses isolated nucleotides encoding the fusion protein ANGPTL3 monoclonal antibody-B disclosed herein, and the invention further discloses vectors comprising the disclosed nucleotides, and host cells encoding the vectors or the polynucleotides of the fusion protein ANGPTL3 monoclonal antibody-B of the invention.

[0014] The present application further discloses the use of the fusion protein ANGPTL3 monoclonal antibody-B in the preparation of a medicament for treating a disease or condition mediated thereby, which use comprises administering to a patient in need thereof a pharmaceutical composition comprising the fusion protein ANGPTL3 monoclonal antibody-B disclosed herein. [Effects of the Invention]

[0015] The present application discloses a fusion protein ANGPTL3 monoclonal antibody-B, which is bound to cytokines via ANGPTL3 monoclonal antibody, and can protect podocytes, reduce urinary protein, alleviate renal inflammation, and inhibit renal fibrosis. [Brief explanation of the drawings]

[0016] [Figure 1] The molecular weights of the fusion proteins detected by SDS-PAGE are shown. [Figure 2] The purity of the fusion protein as detected by SEC-HPLC is shown. [Figure 3] The affinity of the fusion protein detected by SPR is shown. [Figure 4] The melting temperatures of the fusion proteins detected by thermostability analysis are shown. [Figure 5] The in vitro activity of the fusion protein ANGPTL3 monoclonal antibody terminal was verified by in vitro fluorescence experiments. [Figure 6] The in vitro activity of the fusion protein IL-22 terminal was verified by Western Blot. [Figure 7]1 shows the reduction of urinary protein by intervention with the fusion protein in a mouse model of diabetic nephropathy. [Figure 8] Figure 1 shows protection of renal function by fusion protein intervention in a mouse model of diabetic nephropathy. [Figure 9] Figure 1 shows reduction in glucose and blood lipids by intervention with the fusion protein in a mouse model of diabetic nephropathy. [Figure 10] Figure 1 shows that fusion protein intervention attenuates podocyte injury in a mouse model of diabetic nephropathy. [Figure 11] 1 shows the suppression of renal fibrosis by intervention with the fusion protein in a mouse model of diabetic nephropathy. [Figure 12] Figure 1 shows that fusion protein intervention attenuates inflammatory responses in a mouse model of diabetic nephropathy. [Figure 13] This shows the renal protection of the fusion protein by inhibiting the NF / κB / NLRP3 signaling pathway in a mouse model of diabetic nephropathy. [Figure 14] 1 shows the reduction of urinary protein by intervention with the fusion protein in a mouse model of adriamycin nephropathy. [Figure 15] Figure 1 shows protection of renal function by fusion protein intervention in a mouse model of adriamycin nephropathy. [Figure 16] 1 shows the improvement of hypoalbuminemia by intervention with the fusion protein in an adriamycin nephropathy mouse model. [Figure 17] 1 shows the improvement of hyperlipidemia by intervention with the fusion protein in an adriamycin nephropathy mouse model. [Figure 18] 1 shows that fusion protein intervention attenuates podocyte injury in a mouse model of adriamycin nephropathy. [Figure 19] 1 shows the suppression of renal fibrosis by intervention of the fusion protein in a mouse model of adriamycin nephropathy. [Figure 20] Figure 1 shows the renal protection of the fusion protein by improving mitochondrial function in a mouse model of adriamycin nephropathy. [Figure 21]Figure 1 shows the renal protection of the fusion protein by improving lysosomal autophagy in a mouse model of adriamycin nephropathy. DETAILED DESCRIPTION OF THE INVENTION

[0017] Definitions and General Techniques Unless otherwise defined herein, technical terms and techniques used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Generally, 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 those well known and routinely used in the art.

[0018] As described herein, the term ANGPTL3 is an important member of the angiopoietin-like protein (ANGPTL) family, a secreted glycoprotein structurally similar to angiopoietin and playing an important role in regulating angiogenesis. The structure of ANGPTL3 includes an amino-terminal folded helix-like domain, a secretory signal peptide, a short linker peptide, or a carboxy-terminal globular fibrinogen-like domain (FLD). The term "fusion protein ANGPTL3 monoclonal antibody-B" refers to a fusion of a monoclonal antibody prepared using the ANGPTL3 protein or its protein fragment (amino-terminal folded helix-like domain, secretory signal peptide, a short linker peptide, or a carboxy-terminal globular fibrinogen-like domain) as an antigen with another sequence. The ANGPTL3 monoclonal antibody can target the ANGPTL3 protein or a protein fragment thereof, such as the amino-terminal folded helix-like domain, the secretory signal peptide, the short linker peptide, or the carboxy-terminal globular fibrinogen-like domain FLD, and preferably targets the FLD domain. Currently available 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 applicable to the present invention.

[0019] As used interchangeably herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length, and may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified, and chemically or biologically modified or derivatized amino acids, and peptides with modified peptide backbones. The terms include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusion proteins with heterologous and homologous precursor sequences, and the like.

[0020] The fusion protein ANGPTL3 monoclonal antibody-B can be obtained by fusing an ANGPTL3 monoclonal antibody to a heterologous sequence. As described herein, the heterologous sequence can be a polymer containing or not containing an amino acid sequence. The heterologous sequence can be directly fused to the ANGPTL3 monoclonal antibody, fused to the ANGPTL3 monoclonal antibody chemically or by recombinant expression of a single polynucleotide, or conjugated via a linker or conjugation molecule. The peptidyl linker or conjugation 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). The linker or conjugation molecule can also be designed to have a cleavage site for a DNA restriction endonuclease or protease to allow separation of the fused moieties.

[0021] As the term "linker" is used herein, a linker may (or may not) be employed in forming the fusion proteins of the present invention. A linker is composed of amino acids linked by peptide bonds (i.e., a peptidyl linker). In some embodiments of the present invention, the linker is composed of 1 to 20 amino acids linked by peptide bonds, wherein these amino acids are selected from the 20 naturally occurring amino acids. In some embodiments, suitable linkers include (GGGGS)2, (GGGGS)3, (GGGGS)4, (EAAAK)2, (EAAAK)3, and the like.

[0022] As described herein, the term "pharmaceutical composition" is prepared by mixing the fusion protein ANGPTL3 monoclonal antibody-B of the present invention with a pharmaceutically acceptable vector of desired purity, the vector being in the form of a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable vectors are generally formulated for use in liquid, solid, aerosol, or other oral ingestion forms, and are non-toxic to recipients at the dosage and concentration used. In some aspects, the pharmaceutical compositions provided in the present disclosure can be used in methods for treating diseases in subjects, wherein the method comprises administering to the subject a vector comprising a fusion protein and a polynucleotide encoding the fusion protein, a modified host cell expressing the fusion protein, or a pharmaceutical composition thereof, wherein the disease is associated with the presence of an antigen to which the fusion protein binds.

[0023] As used herein, the term "cytokine" refers to various cytokines involved in inflammatory responses, and the cytokines described herein are primarily one or more of interleukins (IL), interferons (IFN), and tumor necrosis factors (TNF). In one example, the cytokine is further selected from IL-22, IL-1RA, IL-4, IL-10, IL-13, TGF-β, EPO, etc.

[0024] The term "vector," as used herein, also refers to a nucleic acid molecule capable of transporting another nucleic acid. A vector may be, for example, a plasmid, mucoid, virus, phage, RNA vector, or linear or circular DNA or RNA molecule, and may include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules. In some embodiments, a vector is a "plasmid," i.e., a circular double-stranded DNA loop into which other DNA segments can be ligated. Furthermore, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0025] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which an exogenous nucleic acid and / or recombinant vector has been introduced. It should be understood that the terms "recombinant host cell" and "host cell" refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in the progeny as a result of mutation or environmental influences, the progeny may differ from the parent cell, yet still be included within the scope of the term "host cell" as used herein. Transformed host cells can include, but are not limited to, prokaryotic and eukaryotic cells, as well as cells of mammalian, plant, insect, fungal, or bacterial origin. Mammalian cells include, but are not limited to, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells, and HEK293T cells. However, mammalian cells are not limited to these, and any cells known to those skilled in the art to function as mammalian host cells can be used.

[0026] As used herein, terms such as "treatment" refer to obtaining a desired pharmacological and / or physiological effect. The effect may be a prophylactic effect, meaning that a disease or its symptoms are completely or partially prevented, and / or a therapeutic effect, meaning that a disease and / or side effects resulting from the disease are partially or completely cured. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject susceptible to the disease but not yet diagnosed with the disease, (b) inhibiting the disease, i.e., arresting its progression, and (c) relieving the disease, i.e., mitigating the disease.

[0027] The term "patient," which may be used interchangeably herein, is intended to include humans and non-human animals, e.g., mammals, such as mice, rats, guinea pigs, dogs, cats, rabbits, cows, horses, sheep, goats, and pigs. The term also includes birds, fish, reptiles, amphibians, and the like. It should be understood that a more particular patient is a human. Similarly, a more particular patient or subject is a non-human mammal, such as a mouse, rat, or dog.

[0028] The term "administration," as used interchangeably herein, refers to an amount of a fusion protein disclosed herein sufficient to ameliorate one or more symptoms of the disease being treated, and a statistically significant method of administration. In one example, 5-100 mg / kg, 20-100 mg / kg, 30-100 mg / kg, 40-100 mg / kg, 50-100 mg / kg, 60-100 mg / kg, 70-100 mg / kg, 80-100 mg / kg, or 90-100 mg / kg of an ANGPTL3 monoclonal antibody / IL-22 fusion protein is administered by injection.

[0029] As used herein, the term "monoclonal antibody" generally refers to a population of essentially homogeneous antibodies, the individual antibodies of which may be identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed directly against a single antigenic site.

[0030] "Isolated nucleotide" refers to a gene-free nucleic acid (e.g., DNA), and in the context of the present invention, this nucleotide refers to the nucleic acid molecule of the fusion protein ANGPTL3 monoclonal antibody-B.Therefore, this term includes, for example, recombinant DNA incorporated into a vector; recombinant DNA incorporated into an autonomously replicating plasmid or virus; recombinant DNA incorporated into the genomic DNA of a prokaryotic or eukaryotic organism; or recombinant DNA incorporated as a separate molecule that exists independently of other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion).In addition, this term also includes RNA molecules transcribed from DNA molecules, and recombinant DNA that encodes a portion of a hybrid gene to which a polypeptide sequence is added.

[0031] As used herein, the term "variable light chain" (VL) refers to an antibody light chain variable binding region. The variable binding region is composed of discrete, well-defined subregions called "complementarity-determining regions" (CDRs, also known as HVRs (hypervariable regions)) and "framing regions" (FRs). CDRs are amino acids within the variable region of an antibody that confer antigen specificity and / or binding affinity and are separated by FRs. Each antibody light chain variable region (LCDR1, LCDR2, LCDR3) has three CDRs, and each antibody heavy chain variable region (HCDR1, HCR2, HCDR3) has three CDRs.

[0032] As used herein, the term "CL" refers to an "immunoglobulin light chain constant region" or "light chain constant region," i.e., a constant region from an antibody light chain.

[0033] As used herein, the term "expression" refers to the process of producing a polypeptide based on a coding sequence of a nucleic acid molecule, such as a gene. This process can include transcription, post-transcriptional regulation, post-transcriptional modification, translation, post-translational regulation, post-translational modification, or any combination thereof.

[0034] As used herein, the term "disease or condition" includes 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 IgA nephropathy, purpura nephritis, lupus nephritis, membranous nephropathy, and tumor-related diseases such as hepatocellular carcinoma, renal cell carcinoma, ovarian cancer, cervical cancer, oral cancer, and esophageal cancer.

[0035] As used herein, the term "transient transfection" refers to the introduction of a constructed plasmid into a mammalian cell in a certain manner, where the foreign gene on the plasmid is not integrated into the cell's own genome, and the entire process of rapid transfection to obtain a protein is called transient transfection expression. [Example]

[0036] Example 1 Expression and purification of fusion proteins 1.1 Construct two separate plasmids using pTT5 as a vector 1.1.1 Insert the monoclonal antibody heavy chain variable region and constant region sequence (VH+CH1-CH3), the junction sequence linker (GGGGS)3, and the mouse-derived IL-22 sequence (mIL-22) into the HindIII and BamHI cleavage sites. 1.1.2 Insert the monoclonal antibody light chain variable region and constant region sequences (VL+CL) into the EcoRI and BamHI cleavage sites as shown in Figure 1. After the constructed plasmid has been sequenced correctly by Sanger analysis, proceed to the next step.

[0037] 1.2 Transformation of receptor cells DH5α with amplification plasmid The plasmid and receptor cells DH5α were mixed in a common ice bath, and then heated in a 42°C water bath. The mixture was then quickly transferred to ice and allowed to stand. LB liquid medium was added and the mixture was revived on a shaker at 37°C. A small amount of the revived liquid was applied to an LB solid medium plate and cultured overnight in an upside-down culture at 37°C. A single clone was selected and placed in a shaker flask for developing the culture medium. The plasmid was then extracted using a spare part Tiangen Daikatsu kit.

[0038] 1.3 Co-transfect the amplified two plasmids into transiently transfected ExpiCHO-S cells to express the protein ExpiCHO-S cells were cultured in a 5% CO2 incubator at a density of 3–6 × 10 6 After culturing until the cell concentration reached 3.5 × 10 6 cells / mL, and then under the same culture conditions, the cell density was adjusted to 7–9 × 10 6 cells / mL, and culture for 18–20 h until the viability exceeded 95%. 6 Dilute to 100 cells / mL and use Thermo Fisher ExpiFectamine TM Using a CHO transfection kit, the plasmid and transfection reagent were mixed and left to stand for 1 minute, then slowly added to the cells. The flask was shaken while adding the reagent (to prevent the local concentration from becoming too high), and the mixture was mixed well. The cells were then cultured under the same conditions as before. Supplementary medium was added on days 1 and 5, and the cell density during this period was monitored. After culturing for 12 days, the supernatant was centrifuged and collected for subsequent protein purification.

[0039] 1.4 Protein purification and ultrafiltration concentration Proteins were purified using a Protein A affinity chromatography column. Buffer A (1x TBS solution) and Buffer B (0.1 M glycine solution) were prepared, the purifier was turned on, and a 1 mL Protein A affinity chromatography column was equilibrated with 10 column volumes of Buffer A. The cell culture supernatant was then loaded onto the column, rinsed with at least 10 column volumes of Buffer A, and then eluted with 5 column volumes of Buffer B. The eluate was collected and neutralized with Tris-HCl buffer. The eluate was ultrafiltered using a 50KD ultrafiltration tube, brought to 15 mL with PBS, and centrifuged three times at 3500 rpm for 10 min. Finally, 600–1000 μL of the liquid in the ultrafiltration tube was collected into a clean 1.5 mL EP tube and stored at -80°C.

[0040] Example 2: Methods for characterizing fusion proteins 2.1 Molecular weight detection of fusion proteins by SDS-PAGE A 10% Tris-glycine gel was set up, and 20 μl of the sample was electrophoresed under reducing and non-reducing conditions, concentrated at 80 V, and separated at 120 V. After electrophoresis, the gel was removed and stained with Caulobacter Brilliant Blue for 10 minutes, then rinsed overnight with a destaining solution consisting of 30% methanol and 10% acetic acid. Images were taken with a Biorad gel imager, and the molecular weights were determined from the positions of the bands.

[0041] 2.2 Purity detection of fusion proteins by SEC-HPLC Using an Agilent 1260 Infinity II SFC system, 100 μg of purified protein was diluted with PBS buffer and injected into a TOSOH TSKgel G3000WXL chromatography column (7.8 mm × 30 cm, 5 μm) at a flow rate of 1.0 ml / min. The column was then incubated at 37°C for 30 minutes under 280 nm UV light to determine the degree of aggregation and degradation of the fusion protein and its purity.

[0042] 2.3 Fusion protein affinity detection by SPR SPR analysis was performed using a Biacore T200 system (GE Healthcare, USA). A human IgG capture antibody was pre-immobilized on a CM5 chip (GE Healthcare, USA). The capture time was adjusted to capture the fusion protein on the chip for approximately 60 RU. The antigen hANGPTL3 (S17-E460) or antigen mANGPTL3 (S17-T455) was then flowed over the chip, binding for 120 seconds and dissociation for 600 seconds. The affinity was calculated from the binding and dissociation parameters generated by the system.

[0043] 2.4 Determining the melting point of fusion proteins by thermal stability analysis Using a Nano Temper PR.48 instrument (Nano Temper Scientific, Germany), the fusion protein was diluted to 1.03 mg / ml, 0.5 mg / ml, and 2.2 mg / ml in PBS buffer within the temperature range of 20 to 95°C. Changes in fluorescence intensity due to small changes in tryptophan residues were monitored, and the raw data were fitted to a sigmoidal curve. The inflection point was taken as the melting point, thereby determining the thermal stability of the fusion protein.

[0044] 2.5 Verification of in vitro activity of fusion protein ANGPTL3 monoclonal antibody terminal by in vitro fluorescence experiment Under high glucose conditions, podocytes highly express ANGPTL3, which binds to integrin ανβ3, exposing the PSI epitope, which can be bound and displayed by the specific antibody AP5. To measure the activity of the fusion protein ANGPTL3 monoclonal antibody terminal, glomerular podocytes (MPC5) were plated at a density of 1 × 10 in 6-well plates. 6 The cells were inoculated overnight at 1 / ml at 37°C with 5% CO2, and after bringing them into close proximity with the wall, 126 ng / ml of fusion protein was added and incubated for 1-2 hours. A 30 mM high glucose solution was then added to allow injury intervention for 4-6 hours. Finally, the PSI structural domain-specific antibody AP5 was used to detect whether the fusion protein could competitively bind to ANGPTL3 and inhibit the exposure of the PSI epitope.

[0045] 2.6 Verification of in vitro activity of IL-22 fusion protein by Western Blot IL-22 can induce STAT3 phosphorylation in mouse proximal renal tubular epithelial cells (mPTCs). First, mPTCs were cultured in a 6-well cell culture plate at a density of 1 x 10. 6 The cells were inoculated with 100 μl of fusion protein at 37°C under 5% CO2 overnight, and then treated with 20 μm of fusion protein for 2, 4, and 6 h. After harvesting, the cells were lysed with 50 μl of weak RIPA for 30 min. After centrifugation at 12,000 rpm at 4°C for 15 min, the lysate supernatant was collected, and finally Western blot detection was performed using rabbit anti-mouse phosphorylated STAT3 antibody (Biolegend) and HPR-labeled goat anti-rabbit antibody (Merck).

[0046] Example 3: Functional verification during animal testing 3.1 Construction of diabetic nephropathy mouse model and administration method 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 housed under specific pathogen-free (SPF) conditions. The control group of db / m mice was fed a normal diet, and the model group of db / db mice was fed a high-fat (40% fat) diet for 4-5 weeks. After about 4 weeks, the DN mouse model was successfully established, and intraperitoneal injections of the drug were initiated twice a week for 8 weeks. The normal control and model groups were injected with an equal volume of saline. The experimental groups were: control group (7 mice), model group (7 mice), ANGPTL3 monoclonal antibody-treated group (7 mice, ANGPTL3 monoclonal antibody 20 mg / kg), IL-22Fc-treated group (7 mice, IL-22Fc 12 mg / kg), ANGPTL3 monoclonal antibody / IL-22 fusion protein-treated group (7 mice, fusion protein 25.3 mg / kg), ANGPTL3 monoclonal antibody / IL-22Fc combination-treated group (7 mice, ANGPTL3 monoclonal antibody 20 mg / kg + IL-22Fc 12 mg / kg), and positive drug cloxacillin-treated group (7 mice, cloxacillin 20 mg / kg / day, administered orally daily).

[0047] 3.2 Construction of an adriamycin nephropathy mouse model and drug administration method A total of 48 6-week-old male Balb / c mice were selected and housed under specific pathogen-free (SPF) conditions with free access to water and food throughout the study. After 1 week of breeding (7 weeks of age), mice were injected with a single dose of 10.5 mg / kg of adriamycin diluted to 2 mg / ml in saline via the tail vein for modeling. Starting on the second day after modeling, the mice were intraperitoneally injected twice weekly for 12 weeks. A normal control group and a model group were injected with the same volume of saline. Experimental grouping: control group (8 animals), model group (8 animals), ANGPTL3 monoclonal antibody-treated group (8 animals, ANGPTL3 monoclonal antibody 20 mg / kg), IL-22Fc-treated group (8 animals, IL-22Fc 12 mg / kg), ANGPTL3 monoclonal antibody / IL-22 fusion protein-treated group (8 animals, fusion protein 25.3 mg / kg), ANGPTL3 monoclonal antibody / IL-22Fc combination-treated group (8 animals, ANGPTL3 monoclonal antibody 20 mg / kg + IL-22Fc 12 mg / kg).

[0048] 3.3 Detection of individual biomolecules or metabolite levels in blood and urine samples Blood samples were collected at different time points after administration from the capillaries at the corners of the eyes, and urine samples were collected from the mouse metabolic cages for 24 hours. These samples were then stored in a refrigerator at -80°C. Related assay kits (Jiancheng, Nanjing, China) were used to detect serum creatinine, serum urea nitrogen, serum total cholesterol, serum triglycerides, blood glucose, urinary creatinine, and 24-hour urinary protein levels. ELISA kits (Jiancheng, Nanjing, China) were used to detect urinary microalbumin, serum TNF-α, IL-6, and IL-1β.

[0049] 3.4 Observation of renal histomorphological changes by light or electron microscopy After mouse sacrifice, kidneys were removed, cut longitudinally, and fixed overnight in 4% paraformaldehyde. They were then embedded in paraffin wax and cut into 4 μm-thick slices. Histopathological damage and fibrosis were assessed under a 200x and 400x microscope using H&E, PAS, and Masson staining, respectively. The percentage of positive areas in the entire image was quantified and analyzed using Image J software. To examine changes in glomerular ultrastructure, kidney tissues of appropriate size were collected, fixed overnight at 4°C, and then examined under an electron microscope at 60 kV. Renal tissues from three to five mice per group were used to examine podocyte ultrastructure, glomerular basement membrane thickness, mitochondria, and lysosomes.

[0050] Example 4: Experimental Results 1. Molecular weight detection of fusion proteins by SDS-PAGE As a result of SDS-PAGE, the molecular weight of the ANGPTL3 monoclonal antibody-IL22 fusion protein under reducing conditions was 72.39 KD for the heavy chain and 25.94 KD for the light chain, indicating high purity as expected (Fig. 1).

[0051] 2. Purity detection of fusion proteins by SEC-HPLC SEC-HPLC revealed that the main protein peak was located at 5.583 min, with only a weak, small peak at around 8 min, further confirming that the ANGPTL3 monoclonal antibody-IL22 fusion protein showed no obvious aggregation or dissociation and was highly pure (see Figure 2).

[0052] 3. Fusion protein affinity detection by SPR SPR analysis revealed that the KD value between the ANGPTL3 monoclonal antibody-IL22 fusion protein and mouse-derived ANGPTL3 was 1.040E-8, and the KD value between the ANGPTL3 monoclonal antibody and human-derived ANGPTL3 was 5.572E-9, demonstrating affinity similar to that of the ANGPTL3 monoclonal antibody itself (see Figure 3).

[0053] 4. Melting Point Detection of Fusion Proteins by Thermal Stability Analysis Thermal stability analysis revealed that the Tm1 values ​​of the three proteins, 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 native melting point and demonstrated good thermal stability (see Figure 4).

[0054] 5. Verification of in vitro activity of fusion protein ANGPTL3 monoclonal antibody terminal by in vitro fluorescence experiment In vitro fluorescence experiments showed that the ANGPTL3 monoclonal antibody-IL22 fusion protein was similar to the ANGPTL3 monoclonal antibody and effectively inhibited the binding of ANGPTL3 to integrin ανβ3 on the surface of podocytes, suppressing the exposure of the PSI epitope, confirming the in vitro activity of the ANGPTL3 monoclonal antibody terminus (see Figure 5).

[0055] 6. Western Blot analysis to verify in vitro activity of IL-22 fusion protein Western blot results showed that the ANGPTL3 monoclonal antibody-IL-22 fusion protein significantly increased the phosphorylation level of STAT3 after 2 hours of treatment in mouse proximal renal tubular epithelial cells, confirming the in vitro activity of the IL-22 terminal (see Figure 6).

[0056] 7. Reduction of urinary protein by intervention of fusion protein in a mouse model of diabetic nephropathy When the ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of diabetic nephropathy for 8 weeks, a significant reduction in 24-hour urinary protein was observed compared to the model group and the mIL22Fc-treated group (P<0.05) (see Figure 7).

[0057] 8. Renal function protection by fusion protein intervention in a mouse model of diabetic nephropathy When the ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of diabetic nephropathy for eight weeks, a significant decrease in serum urea nitrogen concentration was observed compared to the model group and the ANGPTL3 monoclonal antibody-treated group (P<0.05) (see Figure 8).

[0058] 9. Reduction of glucose and blood lipids by fusion protein intervention in a mouse model of diabetic nephropathy When ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a diabetic nephropathy mouse model for 8 weeks, significant reductions in blood glucose, serum triglycerides, and serum total cholesterol levels were observed compared to the model group (P<0.05). At the same time, the ANGPTL3 monoclonal antibody-IL22 fusion protein showed a certain advantage in lowering serum triglycerides compared to the combination of ANGPTL3 and IL22 (see Figure 9).

[0059] 10. Fusion protein intervention attenuates podocyte injury in a mouse model of diabetic nephropathy When ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of diabetic nephropathy for eight weeks, pedicle process fusion and basement membrane thickening were significantly improved compared to the model group, the ANGPTL3 monoclonal antibody-treated group, and the mIL22Fc-treated group (see Figure 10).

[0060] 11. Fusion protein intervention suppresses renal fibrosis in a mouse model of diabetic nephropathy When ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of diabetic nephropathy for eight weeks, collagen deposition in the glomeruli and renal interstitial regions was significantly reduced, and renal fibrosis was significantly improved compared to the model group, the ANGPTL3 monoclonal antibody-treated group, and the mIL22Fc-treated group (see Figure 11).

[0061] 12. Fusion protein intervention attenuates inflammatory responses in a mouse model of diabetic nephropathy When ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of diabetic nephropathy for 8 weeks, serum levels of inflammatory factors TNF-α, IL-6, and IL-1β were significantly reduced compared to the model group and the ANGPTL3 monoclonal antibody-administered group (P<0.05). At the same time, the ANGPTL3 monoclonal antibody-IL22 fusion protein showed a significant advantage in reducing TNF-α and IL-1β compared to the combination of ANGPTL3 and IL22 (see Figure 12).

[0062] 13. Nephroprotection of the fusion protein by inhibiting the NF-κB / NLRP3 signaling pathway in a mouse model of diabetic nephropathy When ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of diabetic nephropathy for eight weeks, the levels of NLRP3 and Pro-caspase-1 in kidney tissue were significantly reduced (P<0.05) compared to the model group and the ANGPTL3 monoclonal antibody-treated group, confirming the renal protective effect of inhibiting the NF-κB / NLRP3 signaling pathway (see Figure 13).

[0063] 14. Reduction of urinary protein by intervention of fusion protein in a mouse model of adriamycin nephropathy When ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of adriamycin nephropathy for 8 weeks, the urinary albumin / creatinine ratio was significantly reduced compared to the model group and the mIL22Fc-treated group (P<0.05). At the same time, the ANGPTL3 monoclonal antibody-IL22 fusion protein showed a significant advantage in reducing the urinary albumin / creatinine ratio compared to the combination of ANGPTL3 and IL22 (see Figure 14).

[0064] 15. Renal function protection by fusion protein intervention in a mouse model of adriamycin nephropathy When ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of adriamycin-induced nephropathy for 8 weeks, serum creatinine levels were significantly reduced compared to the model group (P<0.05). At the same time, the ANGPTL3 monoclonal antibody-IL22 fusion protein showed a significant advantage in reducing serum creatinine compared to the combination of ANGPTL3 and IL22 (see Figure 15).

[0065] 16. Improvement of hypoalbuminemia by fusion protein intervention in a mouse model of adriamycin nephropathy When the ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of adriamycin-induced nephropathy for 8 weeks, serum albumin levels were significantly elevated compared to the control group (P<0.05) (see Figure 16).

[0066] 17. Improvement of hyperlipidemia by fusion protein intervention in a mouse model of adriamycin nephropathy When the ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of adriamycin-induced nephropathy for 8 weeks, serum total cholesterol levels were significantly reduced compared to the control group (P<0.05) (Figure 17).

[0067] 18. Fusion protein intervention attenuates podocyte injury in a mouse model of adriamycin nephropathy When the ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of adriamycin-induced nephropathy for eight weeks, pathological changes such as pedicle process fusion, basement membrane thickening, and microvilli degeneration were significantly improved compared to the control group (see Figure 18).

[0068] 19. Fusion protein intervention suppresses renal fibrosis in a mouse model of adriamycin nephropathy When the ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of adriamycin-induced nephropathy for 8 weeks, collagen deposition in the glomeruli and renal interstitial regions was significantly reduced, and renal fibrosis was significantly improved compared to the control group (see Figure 19).

[0069] 20. Fusion protein protects kidneys by improving mitochondrial function in a mouse model of adriamycin-induced nephropathy When the ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of adriamycin-induced nephropathy for eight weeks, significant improvements in pathological damage symptoms, such as mitochondrial shortening, disorganization, and internal cristae damage, were observed compared to the model group (see Figure 20).

[0070] 21. Fusion protein protects kidneys by improving lysosomal autophagy in a mouse model of adriamycin-induced nephropathy When the ANGPTL3 monoclonal antibody-IL22 fusion protein was administered to a mouse model of adriamycin nephropathy for eight weeks, the number of lysosomes increased, the pathological changes of increased volume were significantly alleviated, and the behavior of lysosomal autophagy was improved compared to the model group (see Figure 21).

Claims

1. A fusion protein ANGPTL3 monoclonal antibody-B comprising an ANGPTL3 monoclonal antibody, said fusion protein ANGPTL3 monoclonal antibody-B comprising a fusion of an ANGPTL3 monoclonal antibody with sequence B.

2. The fusion protein ANGPTL3 monoclonal antibody-B according to claim 1, wherein B is selected from cytokines, and the cytokines are selected from interleukin (IL), interferon (IFN), and tumor necrosis factor (TNF).

3. The fusion protein ANGPTL3 monoclonal antibody-B according to claim 2, wherein 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 the ANGPTL3 monoclonal antibody and sequence B can be linked by 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, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable vector.

6. A 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. Use of the fusion protein ANGPTL3 monoclonal antibody-B according to any one of claims 1 to 4 in the preparation of a medicament for treating a disease or condition mediated thereby.

9. The disease or condition includes diabetic nephropathy, diabetic retinopathy, diabetic foot disease, diabetic cardiovascular complications, diabetic cerebrovascular disease, cerebral arteriosclerosis, silent stroke, diabetic neuropathy, membranous nephropathy, IgA nephropathy, purpura nephritis, and lupus nephritis. The use of the fusion protein ANGPTL3 monoclonal antibody-B according to claim 8 in the preparation of a medicament for treating a disease or condition mediated thereby.

10. The disease or condition includes hepatocellular carcinoma, renal cell carcinoma, ovarian cancer, cervical cancer, oral cancer, and esophageal cancer. The use of the fusion protein ANGPTL3 monoclonal antibody-B according to claim 8 in the preparation of a drug for treating a disease or condition mediated thereby.

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