CN117603361A - Fusion proteins comprising ANGPTL3 mab - Google Patents
Fusion proteins comprising ANGPTL3 mab Download PDFInfo
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- CN117603361A CN117603361A CN202211009188.7A CN202211009188A CN117603361A CN 117603361 A CN117603361 A CN 117603361A CN 202211009188 A CN202211009188 A CN 202211009188A CN 117603361 A CN117603361 A CN 117603361A
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- fusion protein
- angptl3
- mab
- diabetic
- monoclonal antibody
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Abstract
The application discloses a fusion protein ANGPTL3 monoclonal antibody-B containing ANGPTL3 monoclonal antibody, wherein the fusion protein ANGPTL3 monoclonal antibody-B is formed by fusing ANGPTL3 monoclonal antibody and a sequence B; the fusion protein ANGPTL3 monoclonal antibody-B disclosed in the application is used for treating diseases or symptoms mediated by the fusion protein ANGPTL3 monoclonal antibody-B, such as diabetes and complications thereof, such as: diabetic nephropathy, diabetic retinopathy, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular diseases (cerebral arteriosclerosis, asymptomatic cerebral apoplexy), diabetic neuropathy, kidney-related diseases such as membranous nephropathy, igA nephropathy, purpura kidney, lupus kidney, etc., tumor-related diseases such as hepatocellular carcinoma, renal cell carcinoma, ovarian cancer, cervical cancer, oral cancer, esophageal cancer, etc.
Description
Technical Field
The present invention relates to the fields of medical immunology and molecular biology. In particular to a fusion protein containing monoclonal antibody of angiopoietin-like protein 3 (ANGPTL 3) and medical application thereof.
Background
Angiopoietin-like protein 3 (ANGPTL 3) is a secreted glycoprotein whose structure includes an amino-terminal coiled-coil domain (CCD), a secretory signal peptide, a short connecting peptide, and a carboxy-terminal globular fibrinogen-like domain (FLD). The amino-terminal coiled coil region of ANGPTL3 increases plasma Triglyceride (TG) levels by inhibiting the activity of lipoprotein esterase LPL, and the binding of carboxy-terminal FLD (207-460 aa) to integrin αvβ3 receptor promotes angiogenesis (Camenisch G, pisabaro MT, shaerman D, et al, ANGPTL3 stimulates endothelial cell adhesion and migration via integrin αvβ 3and induces blood vessel formation in vivo.J Biol Chem,2002,277 (19): 17281-17290), amino-terminal helical domains are involved in regulating lipid metabolism (Ono M, shimizugawa T, shimamura M, et al protein region important for regulation of lipid metabolism inangiopoietin-like 3 (ANGPTL 3): ANGPTL3 is cleaved and activated in vivo [ J ]. J Biol Chem,2003,278 (43): 41804-41809), while metastasis occurs in podocyte lesions (Liu J, gao X, zhai Y, et al A novel role of angiopoietin-like-3associated with podocyte injury[J ]. Pediatr Res,2015,77 (6): 732-739.), insulin resistance (Robcic MR, maranghi M, lahikainen A, et al Angptl3 deficiency is associated with increased insulin sensitivity, lipoprotein lipase activity, and decreased serum free fatty acids [ J ]. Arterioscler Thromb Vasc Biol,2013,33 (7): A-1713 ], atherosclerosis (Dewey FE, guseaova V, dunbar RL, et al genetic 3923 [ J ] [ J3 ] N, 7, 37A, 37 mg 37/37, 37 mg 37A, 37 mg 37, 37 mg 37A), 2015,4 (5): 759-769), obesity, diabetes, familial hypobetalipoproteinemia, etc. In addition, studies have shown that ANGPTL3 has a certain correlation with the occurrence and progression of other metabolic diseases such as kidney disease (Liu J, gao X, zhai Y, et al, A novel role of angiopoietin-like-3associated with podocyte injury.Pediatr Res,2015,77 (6): 732-739), ANGPTL3 is only expressed in trace amounts in normal functioning kidneys, and the expression levels are significantly increased in various kidney diseases mainly represented by proteinuria (Zhang Jiang, yin Weidong, tang dynasty, and the research progress of ANGPTL3 with metabolic disorder related diseases [ J ] chemistry of life, 2018,38 (1): 30-34).
Inflammatory factors are a highly potent class of small-molecule soluble proteins secreted by immune cells or tissue cells that exert an interplay regulatory effect between cells, common types of inflammatory factors are Interleukins (IL), interferons (IFN), tumor Necrosis Factors (TNF), etc., which regulate developmental differentiation affecting immune cells, are involved in innate and adaptive immune responses, immunomodulation, and play an important role in inflammatory responses (EKTanYX.Chao, A.WestLL.ChanWPoeweJ.JankovicParkinson diseaseand the immune system-associates, mechanisms and therapeuticsJ ]. Nature return nerve 202016 (6): 303-318).
Diabetes is a systemic disease that can cause a variety of complications, with diabetic nephropathy being one of the complications of diabetes, which is a kidney disorder secondary to diabetes. The incidence of end stage renal disease caused by diabetic nephropathy has been the leading cause in China for the last decade (Zhao Yanxiang. Analysis of changes in serum TNF-a, IL-6 from early diabetic nephropathy patients and their correlation with urine albumin/creatinine [ U ]. Chinese medical Innovation, 2020,17 (4): 121-124.)) Shao Changjuan, shen Meng, sun Lili. Changes in serum IL-6, TNF-a, hypersensitive C-reactive protein levels in diabetic nephropathy patients [ J ]. Chinese sanitary 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, and that proinflammatory cytokines are induced to produce, promote inflammatory factor responses, and further promote the development of diabetic nephropathy (Cao Juan effects of hemodialysis combined with blood perfusion on serum inflammatory factor levels performed by patients with diabetic nephropathy in the end stage [ J ]. Clinical study, 2020,28 (3): 76-77.).
Podocyte injury is a common pathological change in Nephrotic Syndrome (NS). Foot cells, which are important components of the glomerular filtration barrier, cause proteinuria, which is a major contributor to the development of N S, and when it reaches a certain threshold, it develops hypoalbuminemia, edema, etc. (macec, chugh ss. Nephrotic syndrome: components, connections, and angiogenin-like-4-related therapeutics [ J ]. J Am Soc neprol, 2014,25 (11): 2393-2398), which is a common clinical manifestation of kidney disease; chronic kidney disease progresses to a stage that can cause renal fibrosis. Renal fibrosis can be induced by a variety of factors including severe trauma and infection of the kidney, blockage of renal blood circulation, or immune response. After damage to kidney tissue caused by various factors, a large amount of collagen fibers are deposited in the interstitium and scar formation, resulting in changes in the structure and function of the kidney, thereby causing renal fibrosis, and the occurrence mechanism of which has not been completely clarified so far.
At present, NS pathological diagnosis mainly depends on kidney biopsy, but the wide use of the NS pathological diagnosis is limited by invasiveness. The hormone and immunosuppressant are used as basic drugs for treating NS, and have a plurality of toxic and side effects. Although a murine monoclonal antibody against ANGPTL3-FLD was prepared in an anti-ANGPTL 3 monoclonal antibody and its use in the preparation of a medicament for treating nephrotic syndrome (patent number: 201911177503.5), it was effective in reducing podocyte injury and reducing proteinuria in vivo and in vitro, but did not have a major key problem of renal fibrosis in the course of tissue diabetic nephropathy treatment.
There is a need to develop a therapeutic method and a pharmaceutical agent capable of achieving multi-dimensional, multi-temporal pharmacological effects of protecting podocytes, reducing urine proteins, reducing kidney inflammation, and inhibiting kidney fibrosis, and the present invention meets this need.
Disclosure of Invention
The application discloses a fusion protein ANGPTL3 monoclonal antibody-B containing ANGPTL3 monoclonal antibody, wherein the fusion protein ANGPTL3 monoclonal antibody-B is formed by fusing ANGPTL3 monoclonal antibody and a sequence B; the B is selected from cytokines.
Further, the cytokine may be selected from the group consisting of Interleukin (IL), interferon (IFN), tumor Necrosis Factor (TNF), and the like. In some embodiments, the cytokine is further selected from the group consisting of: IL-22, IL-1RA, IL-4, IL-10, IL-13, TGF-beta and/or EPO, etc. Further preferred are human IL-22 (SEQ ID No: 5) and/or murine IL-22 (SEQ ID No: 6), and in some embodiments, the ANGPTL3 mab may be linked to sequence B via a linker selected from the group consisting of (GGGGS) 2, (GGGGS) 3, (GGGGS) 4, (EAAAK) 2, and/or (EAAAK) 3, etc.
The application also discloses a pharmaceutical composition comprising the disclosed fusion protein ANGPTL3 mab-B and a pharmaceutically acceptable carrier.
The present application also discloses isolated nucleotides encoding the fusion protein ANGPTL3 mab-B disclosed herein, vectors comprising the disclosed nucleotides, and host cells comprising the vectors or polynucleotides encoding the fusion protein ANGPTL3 mab-B of the present invention are also encompassed by the present invention.
Also disclosed is the use of the fusion protein ANGPTL3 mab-B in the manufacture of a medicament for treating a disease or disorder mediated thereby, the use comprising administering to a patient in need thereof a pharmaceutical composition comprising the fusion protein ANGPTL3 mab-B disclosed herein.
Drawings
FIG. 1SDS-PAGE for detection of molecular weight of fusion proteins
FIG. 2SEC-HPLC detection of the purity of fusion proteins
FIG. 3 affinity of SPR to detect fusion proteins
FIG. 4 melting Point of fusion protein detected by thermal stability analysis
FIG. 5 in vitro fluorescence experiments to verify the in vitro activity of the fusion protein ANGPTL3 mab end
FIG. 6Western Blot to verify the in vitro Activity of the IL-22 terminus of fusion proteins
FIG. 7 fusion protein intervention to reduce urine protein in a mouse model of diabetic nephropathy
FIG. 8 fusion protein intervention to preserve renal function in a mouse model of diabetic nephropathy
FIG. 9 fusion protein intervention to reduce blood glucose and blood lipid in a mouse model of diabetic nephropathy
FIG. 10 fusion protein intervention to mitigate podocyte damage in a mouse model of diabetic nephropathy
FIG. 11 fusion protein intervention to inhibit renal fibrosis in a mouse model of diabetic nephropathy
FIG. 12 fusion protein intervention to reduce inflammatory response in a mouse model of diabetic nephropathy
FIG. 13 protection of kidney by fusion proteins inhibiting NF/- κB/NLRP3 signaling pathway in mouse model of diabetic nephropathy
FIG. 14 fusion protein intervention to reduce urine protein in mouse model of Adriamycin nephropathy
FIG. 15 fusion protein intervention to protect renal function in a mouse model of Adriamycin nephropathy
FIG. 16 improvement of hypoalbuminemia by fusion protein intervention in a mouse model of Adriamycin nephropathy
FIG. 17 improvement of hyperlipidemia by fusion protein intervention in mouse model of Adriamycin nephropathy
FIG. 18 fusion protein intervention to mitigate podocyte injury in a mouse model of doxorubicin nephropathy
FIG. 19 fusion protein intervention to inhibit renal fibrosis in a mouse model of Adriamycin nephropathy
FIG. 20 protection of kidneys by improving mitochondrial function of fusion proteins in mouse models of Adriamycin nephropathy
FIG. 21 protection of kidney by improving lysosomal autophagy of fusion proteins in a mouse model of doxorubicin nephropathy
Detailed Description
The application discloses a fusion protein ANGPTL3 monoclonal antibody-B, which is connected with a cytokine through the ANGPTL3 monoclonal antibody, and can protect podocyte, reduce urine protein, alleviate kidney inflammation and inhibit kidney fibrosis.
Definition and general techniques
Unless defined otherwise herein, technical and scientific terms used in connection with the present invention should have the meaning commonly understood by one of ordinary skill in the art. Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and the techniques thereof, are those well known and commonly used in the art.
The term ANGPTL 3as described herein is an important constituent member of the angiopoietin-like protein (ANGPTL) family, a secreted glycoprotein structurally similar 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 linker peptide, or a carboxy-terminal globular fibrinogen-like domain (FLD), and the term "fusion protein ANGPTL3 mab-B" refers to fusion of an ANGPTL3 protein or protein fragment thereof (amino-terminal coiled-coil domain, secretion signal peptide, short linker peptide, or carboxy-terminal globular fibrinogen-like domain) as an antigen-producing mab with other sequences. The ANGPTL3 mab is capable of targeting to ANGPTL3 proteins or protein fragments thereof, such as amino-terminal coiled-coil domains, secretion signal peptides, short connecting peptides, or carboxy-terminal globular fibrinogen-like domains FLD, preferably ANGPTL3 mab targets to FLD domains. The existing ANGPTL3 monoclonal antibodies shown as SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3SEQ ID No. 4 are also suitable for the invention.
The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include genetically and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusions having heterologous and homologous leader sequences, and the like.
The fusion protein ANGPTL3 monoclonal antibody-B can be obtained by fusing a heterologous sequence with the ANGPTL3 monoclonal antibody. As described herein, the heterologous sequence may be a polymer containing amino acid sequences or no amino acid sequences. The heterologous sequence may be fused directly to the ANGPTL3 mab, chemically or by recombinant expression from a single polynucleotide to the ANGPTL3 mab, or it may be joined via a linker or linker molecule. The peptidyl linker or linker molecule may be one or more amino acid residues (or mers), for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 residues (or mers). The linker or linker molecule may also be designed with cleavage sites for DNA restriction endonucleases or proteases to allow isolation of the fused portions.
The term "linker" as used herein may (but need not) be employed when forming the fusion proteins of the invention. The linker may be comprised of amino acids linked together by peptide bonds (i.e., peptidyl linkers). In some embodiments of the invention, the linker is comprised of 1 to 20 amino acids linked 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, and the like.
The term "pharmaceutical composition" as described herein is prepared by mixing the fusion protein ANGPTL3 mab-B of the invention of the desired purity with a pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution. The pharmaceutically acceptable carrier is generally non-toxic to the recipient at the dosage and concentration used and is formulated for administration in liquid, solid, aerosol or other oral ingestion forms. In certain aspects, the pharmaceutical compositions provided in the present disclosure may be used in a method of treating a disease in a subject, wherein the method comprises administering to the subject: fusion proteins and vectors comprising a polynucleotide encoding a 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.
The term "cytokine" as used herein refers to a variety of cytokines involved in inflammatory reactions, and herein the cytokines are mainly one or more of Interleukins (IL), interferons (IFN) and Tumor Necrosis Factors (TNF). In one embodiment, the cytokine is further selected from the group consisting of: IL-22, IL-1RA, IL-4, IL-10, IL-13, TGF-beta, EPO, and the like.
The term "vector" as described herein may also be a nucleic acid molecule capable of transporting another nucleic acid. The vector may be, for example, a plasmid, cosmid, virus, phage, RNA vector, or a linear or circular DNA or RNA molecule, which may include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules. In some embodiments, the vector is a "plasmid," i.e., a circular double stranded DNA loop that can be ligated to other DNA segments. Furthermore, certain 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").
The term "recombinant host cell" (or simply "host cell") as described herein means a cell that directs an exogenous nucleic acid and/or recombinant vector. It will be understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell, but also to the progeny of such a cell. Since certain modifications may occur in offspring due to mutation or environmental effects, in fact, such offspring may be different from the parent cell, but are still 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. Further, for mammalian cells, CHO cells, F2N cells, CSO cells, BHK cells, bowes melanoma (Bowes melanoma) cells, heLa cells, 911 cells, AT1080 cells, a549 cells, HEK293T cells, and the like can be used. However, the mammalian cells are not limited thereto, and any cell known to those skilled in the art to be useful as a mammalian host cell may be used.
The term "treatment" or the like as used herein refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or partially or completely curing a disease and/or adverse effects attributable to the disease may be therapeutic. 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 who may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.
The term "patient" as used interchangeably herein is intended to include humans and non-human animals, such as mammals, e.g., 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 appreciated that the more specific patient is a human. Likewise, more specific patients and subjects are non-human mammals, such as mice, rats and dogs.
The term "administering" as used interchangeably herein refers to the amount and manner of administration of the fusion protein of the present disclosure sufficient to ameliorate one or more symptoms of the disease being treated, in a statistically significant manner. In one embodiment, the ANGPTL3 mab/IL-22 fusion protein is administered by injection at 5-100mg/kg, 20-100mg/kg, 30-100mg/kg, 40-100mg/kg, 50-100mg/kg, 60-100 mg/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, each antibody included in the population may be identical except for possible naturally occurring mutations that occur in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site.
An "isolated nucleotide" refers to a nucleic acid (e.g., DNA) that does not contain a gene, which in the present invention refers to a nucleic acid molecule of the fusion protein ANGPTL3 mab-B. Thus, the term includes, for example, recombinant DNA incorporated into a vector; a plasmid or virus that enters autonomous replication; or into the genomic DNA of a prokaryote or eukaryote; or as a separate molecule (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion) independent of other sequences. Furthermore, the term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA encoding a portion of a hybrid gene for an additional polypeptide sequence.
The term "variable light chain" (VL) as used herein refers to an antibody light chain variable binding region. Variable binding regions consist of discrete, well-defined sub-regions, known as "complementarity determining regions" (CDRs, also known as HVRs (hypervariable regions)) and "framework regions" (FR). CDRs refer to amino acids within the antibody variable region that confer antigen specificity and/or binding affinity, separated by FR. Three CDRs are present in each antibody light chain variable region (LCDR 1, LCDR2, LCDR 3) and three CDRs are present in each antibody heavy chain variable region (HCDR 1, HCR2, HCDR 3).
The term "CL" as used herein refers to an "immunoglobulin light chain constant region" or "light chain constant region," i.e., a constant region 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, e.g., a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.
The term "disorder or disease" as used herein includes diabetes and its complications such as: diabetic nephropathy, diabetic retinopathy, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular diseases (cerebral arteriosclerosis, asymptomatic cerebral apoplexy), diabetic neuropathy, kidney-related diseases such as membranous nephropathy, igA nephropathy, purpura kidney, lupus kidney, etc., 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 a mammalian cell in such a way that the foreign gene on the plasmid does not integrate into the genome of the cell itself, and this entire rapid transfection to protein is termed transient transfection expression.
EXAMPLE 1 fusion protein expression and purification
1.1 construction of 2 plasmids with pTT5 as vector
1.1.1 insertion of Single heavy chain variable and constant region sequences (VH+CH1-CH 3), linker sequence linker (GGGGS) in HindIII and BamHI cleavage sites 3、 Murine IL-22 sequence (mIL-22);
1.1.2 insertion of monoclonal antibody light chain variable and constant region sequences (VL+CL) in EcoRI and BamHI cleavage sites, as shown in FIG. 1. The constructed plasmid was sequenced by Sanger and subjected to the next step.
1.2 transformation competent cell DH 5. Alpha. Amplification plasmid
Mixing plasmid and competent cell DH5 alpha, cooling with water bath at 42deg.C, rapidly transferring to ice, standing, adding LB liquid culture medium, resuscitating on shaking table at 37deg.C, collecting small amount of resuscitated liquid, spreading on LB solid culture medium plate, culturing overnight at 37deg.C, selecting monoclonal, culturing in shake flask, and extracting plasmid with radix et rhizoma Nardostachyos kit.
1.3 Co-transient transfection of amplified 2 plasmids into ExpiCHO-S cells to express proteins
ExpiCHO-S cells were cultured to a density of 3-6X10 in a 5% CO2 incubator 6 cell/mL was adjusted to 3.5X10 cell concentration 6 cells/mL, followed by culturing under the same culture conditions for 18-20h to a cell density of 7-9X10 6 Diluting cells to 6X 10 with preheated medium when cell/mL has a viability of greater than 95% 6 cells/mL using Semerle off-pictamine TM The CHO transfection reagent kit is characterized in that plasmids and a transfection reagent are mixed and kept stand for 1min, then the plasmids and the transfection reagent are slowly added into cells, a shake flask is shaken while local concentration is avoided to be too high, the culture is continued under the previous condition after the plasmids and the transfection reagent is fully mixed, a feed medium is added on the first day and the fifth day, cell density is detected during the culture, and after 12 days of culture, the supernatant is collected centrifugally for subsequent protein purification.
1.4 protein purification and ultrafiltration concentration
Protein was purified using a Protein a affinity column, buffer a (1×tbs solution) and Buffer B (0.1M glycine solution) were prepared, the purifier was turned on, 1mL of Protein a affinity column was equilibrated with Buffer a for 10 column volumes, the cell culture supernatant was sampled, at least 10 column volumes were washed with Buffer a after the end of the loading, then 5 column volumes were eluted with Buffer B, the eluate was collected, and the eluate was neutralized with Tris-HCl Buffer. The eluate was ultrafiltered using a 50KD ultrafilter tube, supplemented with PBS to 15ml, centrifuged 3 times at 3500rpm for 10min, and the final 600-1000ul of the liquid in the ultrafilter tube was collected into a clean 1.5ml EP tube and stored at-80 ℃.
Example 2 characterization of fusion proteins
2.1SDS-PAGE detection of molecular weight of fusion proteins
Preparing 10% Tris-glycine gel, loading 20ul under reducing and non-reducing conditions for electrophoresis, setting a voltage of 80V for concentration and 120V for separation, taking out the gel after electrophoresis, dyeing for 10min by using coomassie brilliant blue, rinsing overnight by using a decolorization solution composed of 30% methanol and 10% acetic acid, shooting and imaging by a Biorad gel imager, and determining the molecular weight according to the position of the band.
2.2SEC-HPLC detection of the purity of fusion proteins
100 μg of purified protein was diluted with PBS buffer using the Agilent 1260 affinity II SFC system to
A TOSOH TSKgel G3000WXL column (7.8 mm. Times.30 cm,5 μm) was injected at a flow rate of 1.0ml/min, and the purity was judged by knowing the aggregation and degradation degree of the fusion protein by detection under ultraviolet irradiation at 37℃and 280nm for 30 minutes.
2.3SPR detection of affinity of fusion proteins
Human IgG capture antibodies were pre-immobilized on CM5 chips (GE Healthcare, USA) using the Biacore T200 system (GE Healthcare, USA) SPR assay, fusion proteins were captured on the chip at about 60RU by adjusting the capture time, followed by on-chip passage over antigen hANGPTL3 (S17-E460) or antigen hANGPTL3 (S17-T455), binding for 120 seconds, dissociation for 600 seconds, and affinity was calculated by the system generated binding and dissociation parameters.
2.4 thermal stability analysis to detect melting Point of fusion protein
The fusion protein was diluted to 1.03mg/ml, 0.5mg/ml and 2.2mg/ml with PBS buffer at 20-95℃using a Nano sample PR.48 instrument (Nano Temper Scientific, germany), and the heat stability of the fusion protein was judged by fitting the raw data to an S-shaped curve whose inflection point was the melting point by monitoring the change in fluorescence intensity due to the slight change in tryptophan residues.
2.5 in vitro fluorescence experiments to verify the in vitro activity of the fusion protein ANGPTL3 monoclonal antibody end
Under high sugar conditions, podocytes highly express ANGPTL3, can bind with integrin ανβ3, resulting in exposure of PSI epitopes, and can be bound and displayed by specific antibody AP 5. To determine the fusion protein ANGPTL3 mab end activity, glomerular podocytes (MPC 5) were seeded in 6-well plates at a density of 1 x 106/ml at 37 ℃ at 5% co2 overnight, and after cell attachment 126ng/ml of fusion protein was added for incubation for 1-2 hours, followed by injury intervention with 30mM high sugar solution for 4-6 hours, and finally detection of whether the fusion protein competed for binding with ANGPTL3 using PSI domain specific antibody AP5 to block PSI epitope exposure.
2.6WesternBlot verification of the in vitro Activity of the IL-22 end of fusion proteins
IL-22 is capable of inducing phosphorylation of STAT3 in mouse proximal tubular epithelial cells (mPTC). First, mPTC was inoculated into a 6-well cell culture plate at a density of 1X 106/ml at 37℃and a concentration of 5% CO2 overnight, then cells were treated with 20mm fusion protein for 2h, 4h and 6h, after which cells were collected, lysed with 50ul of weak RIPA for 30min, centrifuged at 12000rpm for 15min at 4℃and the lysate supernatant was collected, and finally Western blot detection was performed with rabbit anti-mouse phosphorylated STAT3 antibody (Biolegend) and HPR labeled goat anti-rabbit antibody (Merck).
Example 3 functional verification in animal experiments
3.1 construction of diabetic nephropathy mouse model and dosing regimen
Db/m mice and db/db mice (C57 BKS/Lepr, male, 6-7 weeks old) produced by Cavins animal experiments Co Ltd, changzhou were selected and fed under specific pathogen-free (SPF) conditions, the control group was db/m mice, the normal diet was fed, the model group was db/db mice, and the high fat (fat content 40%) diet was fed for 4-5 weeks. DN mice were successfully modeled for about 4 weeks, starting administration, 2 intraperitoneal injections per week, 8 weeks continuously, and normal control and model groups were injected with the same dose of physiological saline. Experimental grouping: control group (7), model group (7), ANGPTL3 mab-treated group (7, ANGPTL3 mab 20 mg/kg), IL-22 Fc-treated group (7, IL-22Fc 12 mg/kg), ANGPTL3 mab/IL-22 fusion protein-treated group (7, fusion protein 25.3 mg/kg), ANGPTL3 mab/IL-22 Fc-combined-treated group (7, ANGPTL3 mab 20mg/kg+il-22Fc 12 mg/kg), positive drug losartan-treated group (7, losartan 20mg/kg/d, daily gavage).
3.2 construction of mouse models of Adriamycin nephropathy and dosing regimen
A total of 48 male Balb/c mice of 6 weeks old were selected and kept free to feed and eat during the test under Specific Pathogen Free (SPF) conditions. After 1 week (7 weeks of age) of feeding, doxorubicin was diluted to 2mg/ml with physiological saline and was once injected into the tail vein for 10.5mg/kg molding. Administration was started on day 2 after molding, 2 times per week of intraperitoneal injection, and 12 weeks of continuous injection, and normal control group and model group were injected with the same dose of physiological saline. Experimental grouping: control group (8), model group (8), ANGPTL3 mab-treated group (8, ANGPTL3 mab 20 mg/kg), IL-22 Fc-treated group (8, IL-22Fc 12 mg/kg), ANGPTL3 mab/IL-22 fusion protein-treated group (8, fusion protein 25.3 mg/kg), ANGPTL3 mab/IL-22 Fc-combined treated group (8, ANGPTL3 mab 20mg/kg+IL-22Fc 12 mg/kg).
3.3 detection of the level of various biological macromolecules or metabolites in blood and urine samples
Samples were collected at different time points after dosing, blood was collected via the inner canthus capillaries, urine was collected via the 24 hour mouse metabolism cage, and stored in a-80 ℃ refrigerator. Subsequently, detecting serum creatinine, serum urea nitrogen, serum total cholesterol, serum triglyceride, blood sugar, urinary creatinine, 24-hour urinary protein level and the like by using a related detection kit (built by Nanjing, china); urine microalbumin, serum TNF-alpha, IL-6, IL-1 beta and the like are detected by using an ELISA kit (built in Nanjing, china).
3.4 viewing of morphological changes in renal tissue with the aid of an optical or electronic microscope
After mice were sacrificed, kidneys were taken for longitudinal cutting, fixed overnight in 4% paraformaldehyde solution, paraffin embedded, cut into 4 μm thick sections, and kidney tissue pathological lesions and fibrosis levels were observed under 200×, 400× microscope using H & E staining, PAS staining, masson staining, respectively, and the percentage of positive area in the whole Image was quantified and analyzed using Image J software. To observe changes in glomerular ultrastructure, kidney tissue of appropriate size was fixed overnight at 4 ℃, followed by electron microscopy at 60kv, 3-5 mouse kidney tissues were taken per group, and changes in podocyte ultrastructure, glomerular basal membrane thickness, mitochondria, and lysosomes were observed.
Example 4: experimental results
SDS-PAGE for detection of molecular weight of fusion proteins
SDS-PAGE showed that the molecular weight of the ANGPTL3 mab-IL 22 fusion protein under reducing conditions was 72.39KD for the heavy chain and 25.94KD for the light chain, and that it was highly pure as expected (FIG. 1)
SEC-HPLC detection of the purity of fusion proteins
SEC-HPLC shows that the main peak of the protein is located at 5.583min, and only a slightly weak small peak exists before and after 8min, which shows that the ANGPTL3 monoclonal antibody-IL 22 fusion protein does not have obvious aggregation and dissociation, and further proves that the purity is good. (see FIG. 2)
SPR detection of affinity of fusion proteins
SPR analysis shows that the KD value of the ANGPTL3 monoclonal antibody-IL 22 fusion protein and the murine ANGPTL3 is 1.040E-8, the KD value of the fusion protein and the human ANGPTL3 is 5.572E-9, and the fusion protein and the murine ANGPTL3 have similar affinity with each other. (see FIG. 3)
4. Thermal stability analysis to detect melting point of fusion proteins
Thermal stability analysis shows that Tm1 values of the ANGPTL3 monoclonal antibody-IL 22 fusion protein, the ANGPTL3 monoclonal antibody and the mIL22Fc are 66.1 ℃ and 66.6 ℃ and 66.5 ℃ respectively, and the construction of the fusion protein does not influence the original melting point and has good thermal stability. (see FIG. 4)
5. In vitro fluorescence experiment verifies the in vitro activity of the fusion protein ANGPTL3 monoclonal antibody terminal
In vitro fluorescence experiments show that the ANGPTL3 monoclonal antibody-IL 22 fusion protein is similar to the ANGPTL3 monoclonal antibody, can effectively block the combination of the ANGPTL 3and podocyte surface integrin alpha v beta 3 to inhibit the exposure of PSI epitope, and proves the in vitro activity of the ANGPTL3 monoclonal antibody end. (see FIG. 5)
WesternBlot to verify IL-22 end in vitro Activity of fusion proteins
WesternBlot shows that the IL-22 fusion protein of the ANGPTL3 monoclonal antibody can obviously raise the phosphorylation level of STAT3 after acting on mouse proximal renal tubular epithelial cells for 2 hours, and the in vitro activity of the IL-22 end of the protein is proved. (see FIG. 6)
7. Fusion protein intervention to reduce urine protein in diabetic nephropathy mouse model
The diabetic nephropathy mice model significantly decreased urine protein (P < 0.05) after 8 weeks of treatment with ANGPTL3 mab-IL 22 fusion protein compared to the model group and the mll 22 Fc-treated group at 24 hours. (see FIG. 7)
8. Fusion protein intervention protection of kidney function in diabetic nephropathy mouse model
The serum urea nitrogen levels of the diabetic nephropathy mice model were significantly reduced (P < 0.05) after 8 weeks of treatment with ANGPTL3 mab-IL 22 fusion protein compared to the model group and ANGPTL3 mab-treated group. (see FIG. 8)
9. Fusion protein intervention in diabetic nephropathy mouse model for reducing blood sugar and blood fat
The diabetic nephropathy mouse model has significantly reduced blood glucose, serum triglyceride and serum total cholesterol levels (P < 0.05) compared with the model group after 8 weeks of treatment by using the ANGPTL3 monoclonal antibody-IL 22 fusion protein, and meanwhile, the ANGPTL3 monoclonal antibody-IL 22 fusion protein shows a certain advantage in reducing serum triglyceride compared with the combination of the ANGPTL 3and IL 22. (see FIG. 9)
10. Fusion protein intervention for reducing podocyte injury in diabetic nephropathy mouse model
Compared with the model group, the ANGPTL3 monoclonal antibody treatment group and the mIL22Fc treatment group, the diabetic nephropathy mouse model is obviously improved in foot process fusion and basement membrane thickening after being treated by the ANGPTL3 monoclonal antibody-IL 22 fusion protein for 8 weeks. (see FIG. 10)
11. Fusion protein intervention for inhibiting kidney fibrosis in diabetic nephropathy mouse model
The collagen deposition of glomerulus and renal interstitial regions of a diabetic nephropathy mouse model is obviously reduced after the model is treated by the ANGPTL3 monoclonal antibody-IL 22 fusion protein for 8 weeks compared with that of a model group, an ANGPTL3 monoclonal antibody treatment group and an mIL22Fc treatment group, and the renal fibrosis is obviously improved. (see FIG. 11)
12. Fusion protein intervention to reduce inflammatory response in a mouse model of diabetic nephropathy
The serum inflammatory factor TNF-alpha, IL-6 and IL-1 beta levels of a diabetic nephropathy mouse model are significantly reduced (P < 0.05) after 8 weeks of treatment with the ANGPTL3 mab-IL 22 fusion protein compared with the model group and the ANGPTL3 mab-treatment group, and meanwhile, the ANGPTL3 mab-IL 22 fusion protein can be seen to show significant advantages in reducing TNF-alpha and IL-1 beta compared with the combination of ANGPTL 3and IL 22. (see FIG. 12)
13. Fusion protein for protecting kidney by inhibiting NF- κB/NLRP3 signal path in diabetic nephropathy mouse model
Compared with the model group and the ANGPTL3 monoclonal antibody treatment group, the diabetic nephropathy mouse model has significantly reduced levels of NLRP 3and Pro-caspase-1 (P < 0.05) after being treated by the ANGPTL3 monoclonal antibody-IL 22 fusion protein for 8 weeks, and the kidney tissue has proved that the diabetic nephropathy mouse model plays a role in kidney protection by inhibiting NF- κB/NLRP3 signaling pathway. (see FIG. 13)
14. Fusion protein intervention to reduce urine protein in mouse model of doxorubicin nephropathy
The mice model of doxorubicin nephropathy showed a significant decrease in urinary albumin/creatinine ratio (P < 0.05) after 8 weeks of treatment with ANGPTL3 mab-IL 22 fusion protein compared to the model and the mll 22 Fc-treated groups, while it can be seen that ANGPTL3 mab-IL 22 fusion protein showed a significant advantage in decreasing urinary albumin/creatinine ratio compared to ANGPTL 3and IL22 in combination. (see FIG. 14)
15. Fusion protein intervention protection of renal function in mouse model of doxorubicin nephropathy
The mice model of doxorubicin nephropathy showed a significant decrease in serum creatinine levels (P < 0.05) compared to the model group after 8 weeks of treatment with ANGPTL3 mab-IL 22 fusion protein, while it can be seen that ANGPTL3 mab-IL 22 fusion protein showed a significant advantage in reducing serum creatinine compared to ANGPTL 3and IL22 in combination. (see FIG. 15)
16. Intervention of fusion protein in mice model of doxorubicin nephropathy to improve hypoalbuminemia
The mice model of doxorubicin nephropathy showed significantly elevated serum albumin levels (P < 0.05) after 8 weeks of treatment with ANGPTL3 mab-IL 22 fusion protein compared to the model group. (see FIG. 16)
17. Fusion protein intervention improvement of hyperlipidemia in mouse model of doxorubicin nephropathy
The mice model of doxorubicin nephropathy showed a significant decrease in serum total cholesterol levels (P < 0.05) compared to the model group after 8 weeks of treatment with ANGPTL3 mab-IL 22 fusion protein. (see FIG. 17)
18. Fusion protein intervention in doxorubicin nephropathy mouse model to reduce podocyte injury
Compared with the model group podophyllotoxin fusion, the pathological changes such as basement membrane thickening, microvilli degeneration and the like of the doxorubicin nephropathy mouse model after 8 weeks of treatment by using the ANGPTL3 monoclonal antibody-IL 22 fusion protein are obviously improved. (see FIG. 18)
19. Fusion protein intervention inhibition of kidney fibrosis in mouse model of doxorubicin nephropathy
The doxorubicin nephropathy mouse model is significantly reduced in collagen deposition in glomerulus and renal interstitial regions compared with the model group after 8 weeks of treatment with ANGPTL3 mab-IL 22 fusion protein, and the renal fibrosis is significantly improved. (see FIG. 19)
20. Fusion protein in mouse model of doxorubicin nephropathy to protect kidney by improving mitochondrial function
Compared with the model group, the doxorubicin nephropathy mouse model has obviously improved pathological damage performance such as shortened mitochondrial length, disordered arrangement, broken internal ridge and the like after 8 weeks of treatment by using the ANGPTL3 monoclonal antibody-IL 22 fusion protein. (see FIG. 20)
21. Fusion protein for protecting kidney by improving lysosomal autophagy in mouse model of doxorubicin nephropathy
The pathological changes of increased lysosome numbers and increased volume in the mice model of doxorubicin nephropathy were significantly alleviated after 8 weeks of treatment with ANGPTL3 mab-IL 22 fusion protein compared to the model group, revealing an improvement in lysosomal autophagy behavior (see fig. 21).
Claims (10)
1. A fusion protein ANGPTL3 mab-B comprising ANGPTL3 mab, wherein the fusion protein ANGPTL3 mab-B is formed by fusing ANGPTL3 mab with sequence B.
2. The fusion protein ANGPTL3 mab-B of claim 1, said B selected from the group consisting of cytokines selected from the group consisting of Interleukins (IL), interferons (IFN), and Tumor Necrosis Factors (TNF).
3. The fusion protein ANGPTL3 mab-B of claim 2, said cytokine selected from the group consisting of: IL-22, IL-1RA, IL-4, IL-10, IL-13, TGF-beta and/or EPO.
4. The fusion protein ANGPTL3 mab-B of any one of claims 1-3, wherein ANGPTL3 mab and sequence B may be linked by a linker, said linker may be selected from (GGGGS) 2, (GGGGS) 3, (GGGGS) 4, (EAAAK) 2 and or (EAAAK) 3.
5. A pharmaceutical composition comprising the fusion protein ANGPTL3 mab-B of any one of claims 1-4, said pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
6. A polynucleotide encoding the fusion protein ANGPTL3 mab-B of any one of claims 1-4.
7. A host cell expressing a polynucleotide of the fusion protein ANGPTL3 mab-B of any one of claims 1-4.
8. Use of the fusion protein ANGPTL3 mab-B of any one of claims 1-4 in the manufacture of a medicament for treating a disease or disorder mediated thereby.
9. Use of the fusion protein ANGPTL3 mab-B of claim 8 in the manufacture of a medicament for treating a disease or condition mediated thereby, wherein the disease or condition comprises diabetic nephropathy, diabetic retinopathy, diabetic foot, diabetic cardiovascular complications, diabetic cerebrovascular disease, cerebral arteriosclerosis, asymptomatic stroke, diabetic neuropathy, membranous nephropathy, igA nephropathy, purpura kidney, lupus kidney.
10. Use of the fusion protein ANGPTL3 mab-B of claim 8 in the manufacture of a medicament for treating a disease or disorder mediated thereby, wherein the disease or disorder comprises hepatocellular carcinoma, renal cell carcinoma, ovarian carcinoma, cervical carcinoma, oral cancer, esophageal carcinoma.
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| PCT/CN2023/106045 WO2024041236A1 (en) | 2022-08-22 | 2023-07-06 | Fusion protein comprising angptl3 monoclonal antibody |
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| CR20200327A (en) * | 2018-01-26 | 2020-11-05 | Genentech Inc | Il-22 fc fusion proteins and methods of use |
| PL3650037T3 (en) * | 2018-11-07 | 2022-06-06 | Applied Molecular Transport Inc. | Delivery constructs for transcytosis and related methods |
| CN110938144B (en) * | 2019-11-27 | 2022-07-26 | 复旦大学附属儿科医院 | anti-ANGPTL 3 monoclonal antibody and application thereof in preparation of medicament for treating nephrotic syndrome |
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