CN117677640A - Fusion protein and medical application thereof - Google Patents

Fusion protein and medical application thereof Download PDF

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CN117677640A
CN117677640A CN202280046816.0A CN202280046816A CN117677640A CN 117677640 A CN117677640 A CN 117677640A CN 202280046816 A CN202280046816 A CN 202280046816A CN 117677640 A CN117677640 A CN 117677640A
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fusion protein
protein
seq
fgf21
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毛东杰
谢岳峻
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Changzhou Hengbang Pharmaceutical Co ltd
Jiangsu Hansoh Pharmaceutical Group Co Ltd
Shanghai Hansoh Biomedical Co Ltd
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Jiangsu Hansoh Pharmaceutical Group Co Ltd
Shanghai Hansoh Biomedical Co Ltd
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Abstract

Provides a fusion protein composed of FGF21 protein and immunoglobulin Fc or fragments thereof, and a method for treating diabetes, obesity, dyslipidemia, metabolic syndrome, nonalcoholic fatty liver disease or nonalcoholic steatohepatitis and other related diseases by using the FGF21-Fc fusion protein and a pharmaceutical composition thereof.

Description

Fusion protein and medical application thereof Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a fusion protein of FGF21 and medical application thereof.
Background
Fibroblast growth factor 21 (Fibroblast growth factor, FGF 21) is a polypeptide consisting of 209 amino acids (SEQ ID NO: 1) with an amino acid sequence that has about 75% homology with murine FGF 21. FGF21 contains a 28 amino acid signal peptide at the N-terminus, so that mature FGF21 consists of 181 amino acids (SEQ ID NO: 3). Mature FGF21 has the native human FGF21 isoform (isoform) or allelic form (allelic form) with Leu replacing Pro at position 146 of SEQ ID NO. 3 herein. FGF21 is expressed primarily in the liver and pancreas, while also being present in adipose and muscle tissue. FGF21 can induce various signal paths and functional activities in liver, pancreas and adipose tissues through mediation of FGFR and assistance of transmembrane protein beta Klotho (KLB), so that the regulation of glycolipid metabolism and the physiological function of protecting islet beta cells are realized. FGF21 regulates glucose uptake by adipocytes by activating non-insulin dependent glucose uptake. In addition, studies have shown that FGF21 can be dose-dependently reduced in body weight and systemic body fat, such as administration of FGF21 to diabetic rhesus monkeys, and that there is a significant reduction in fasting plasma glucose, triglycerides and glucagon levels. Meanwhile, it was found that white adipose tissue lipase expression was increased and plasma β -hydroxybutyrate and free fatty acid levels were increased in FGF21 transgenic mice. This means that FGF21 may regulate lipid metabolism by promoting lipolysis and ketogenesis. FGF21 is capable of inhibiting glucose-mediated release of glucagon, stimulating insulin production, and preventing apoptosis of islet cells in islet cells and INS-1E cells, thereby improving pancreatic cell function. In addition, FGF21 also activates exocrine pancreatic cells and hepatocyte signaling pathways, inhibiting hepatic glycogen output.
FGF21 is a member of the FGF gene family, most FGFs have a broad spectrum of mitogenic capacity, and the results of the studies indicate that FGF21 has neither the capacity to promote cell proliferation nor antagonize the function of other members of the FGF family. Experiments prove that FGF21 transgenic mice (the in vivo FGF21 amount is about 150 times of that of normal mice) have no abnormal conditions such as tumor, tissue hyperplasia and the like in the whole life cycle. Meanwhile, its metabolic regulation is related to the metabolic level of the body, and the regulation only acts when the metabolism is abnormal, and even if FGF21 exceeds the pharmacological dose, hypoglycemia does not occur. However, the major drugs for treating diabetes (insulin, thiazolidinedione, etc.) on the market are prone to side effects when the dosage is incorrect, such as hypoglycemia caused by large doses of insulin, liver function damage and edema caused by thiazolidinedione, which are not found in animal experiments receiving FGF21, and this is also sufficient to prove that FGF21 is an ideal drug for treating diabetes, obesity, etc.
The physiological functions of FGF21 in the aspects of controlling blood sugar, reducing weight and the like bring hope for treating related diseases, but wild FGF21 serving as a small molecular protein is easy to hydrolyze by protease, and can also be filtered through glomeruli, the half-life period is only 0.5-2h, and the effective medicine action time is difficult to ensure. In view of such difficulties, the pharmaceutical industry has increased half-life of FGF21 by performing site-directed mutagenesis of an amino acid at the cleavage site, preparing a long-acting fusion protein, or attaching polyethylene glycol to a polypeptide backbone. In addition, significant challenges in developing FGF21 as a protein formulation also come from instability caused by aggregation itself. The ideal effect of the therapeutic protein of interest is to increase the tolerance to proteolysis and reduce the aggregation of the protein, thereby enhancing the half-life and stability of FGF21 protein formulations, enabling low frequency administration to patients. Some mutants based on human wild-type FGF21 polypeptide sequences have been described in WO2009/149171 and WO2017/074117, and there is a need in the art for mutants of FGF21 polypeptide sequences that are suitable for pharmaceutical use.
Disclosure of Invention
The invention provides FGF21 protein or variant thereof, which has the following general formula:
wherein:
X 166 selected from L or F;
X 175 selected from R or W.
A preferred embodiment of the present invention is the FGF21 protein of formula (II) or variant thereof, having the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.
In another aspect, the present invention also provides a fusion protein having the general formula:
F 1 -F 2 -F 3
(I)
wherein:
F 1 selected from FGF21 proteins or variants thereof as set forth in formula (II);
F 2 is a connecting peptide;
F 3 is a domain consisting of an Fc of an immunoglobulin or a fragment thereof.
In a preferred embodiment of the invention, F 1 The sequence is shown as SEQ ID NO. 5 or SEQ ID NO. 6.
In a preferred embodiment of the invention, F 1 The sequence is shown as SEQ ID NO. 5.
In a preferred embodiment of the invention, F 2 The sequence is shown as SEQ ID NO. 7.
In a preferred embodiment of the invention, said F 3 As shown in the general formula (III):
F 4 (GGGGGS) m (GGGGS) n F 4
(III)
wherein:
F 4 an Fc fragment that is an immunoglobulin;
m is selected from integers from 1 to 10; and is also provided with
n is an integer from 1 to 10.
In a preferred embodiment of the invention, for the general formula (III), wherein:
F 4 is SEQ ID NO:8, 8;
m is 1; and is also provided with
n is 9.
In a preferred embodiment of the invention, said F 3 Is SEQ ID NO:9.
in a preferred embodiment of the invention, the fusion protein is selected from the group consisting of SEQ ID NO:12 or SEQ ID NO:13.
the invention also relates to a polynucleotide which codes for a fusion protein comprising the general formula (I).
In a preferred embodiment of the invention, the coding of said nucleotide comprises the FGF21 protein SEQ ID NO:5.
in a preferred embodiment of the invention, the coding of said nucleotide comprises the FGF21 fusion protein SEQ ID NO:12-13.
The invention also relates to an expression vector comprising a polynucleotide as described above.
In addition, the present invention relates to a host cell into which or containing the expression vector as described above is introduced, wherein the host cell is a bacterium, preferably E.coli; or the host cell is saccharomycete, preferably pichia pastoris; alternatively, the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.
The invention also relates to a method for producing a fusion protein as described above, comprising the steps of:
1) Culturing a host cell as described above;
2) Isolating the protein from the culture;
3) And purifying the protein.
The invention further comprises a pharmaceutical composition comprising a fusion protein of general formula (I) and a pharmaceutically acceptable excipient, diluent or carrier.
The invention also relates to the application of the fusion protein or the pharmaceutical composition in preparing medicines for treating or preventing and treating related diseases such as diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.
The fusion protein provided by the invention has the effects of remarkably promoting the proliferation of fibroblasts, has good plasma stability, can induce glucose uptake and promote the phosphorylation of ERK1/2 protein, is beneficial to the preparation and preparation of therapeutic proteins, and has potential therapeutic effects on related diseases such as diabetes, obesity, dyslipidemia, metabolic syndrome, nonalcoholic fatty liver, nonalcoholic steatohepatitis and the like.
Detailed description of the invention
Unless stated to the contrary, the terms used in the specification and claims have the following meanings.
The amino acid position change in the FGF21 mutants of the present invention is determined from the amino acid position in the mature human wild-type FGF21 (SEQ ID NO: 3) polypeptide.
The amino acid sequences of the invention contain standard single-letter or three-letter codes of twenty amino acids.
The term "FGF21 polypeptide" refers to a naturally occurring wild-type polypeptide expressed in humans. Comprising a full length form encoded by SEQ ID NO. 2 consisting of SEQ ID NO. 1 and a mature form encoded by SEQ ID NO. 4 consisting of SEQ ID NO. 3.
The term "FGF21 mutant" refers to an FGF21 polypeptide modified based on the naturally occurring FGF21 amino acid (SEQ ID NO: 4) sequence. Such modifications include, but are not limited to, substitution of one or more amino acids, including, but not limited to, protease resistant FGF21 mutants, aggregation-reduced FGF21 mutants, and FGF21 combination mutants described herein.
The term "patient" is a mammal, preferably a human.
The term "treating" refers to slowing, reducing, or reversing the progression or severity of a symptom, disorder, or disease.
The term "Fc fragment" refers to the constant region of the heavy chain of an immunoglobulin.
The term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to deliver encoded information to a host cell.
The term "expression vector" refers to a vector suitable for transformation of a host cell and containing a nucleic acid sequence that directs and/or controls expression of an inserted heterologous nucleic acid sequence. Including but not limited to processes such as transcription, translation, and RNA splicing.
The term "host cell" is used to refer to a cell transformed or capable of being transformed with a nucleic acid sequence and then capable of expressing a selected gene of interest. The term includes progeny of a parent cell, whether or not the progeny is identical in morphology or genetic composition to the original parent, and the selected gene is predominantly present.
Detailed Description
The following specific embodiments are provided in order to illustrate the present invention in more detail, but the aspects of the present invention are not limited thereto.
1. Main experimental reagent:
2. main experimental instrument:
name of the name Branding Model number
Protein purifying instrument GE AKTA-Pure150
Enzyme label instrument MS Cereal molecule
EXAMPLE 1 preparation of FGF21 mutant proteins
FGF21 mutant protein (SEQ ID NO: 5-6) was expressed using the ExpiCHO system (Thermo Fisher#A 29133).
Specifically, the DNA sequence encoding the FGF21 mutant protein with the His tag at the C end is cloned into a pCDNA3.1 vector, and the plasmid for expressing the fusion protein is obtained through sequencing. Plasmid was transfected into the ExpiFectamine reagent into expcho-S cells, and after culturing the cells in 100 ml of expcho medium for 7 days, the supernatant was harvested; clarifying fermentation liquor by adopting a centrifugal filtration or deep filtration method.
The EQ buffer (PBS, pH 7.4) was prepared by taking a bag of PBS phosphate buffer powder, dissolving the powder in 2000ml of ultra pure water, and filtering with a 0.22 μm filter membrane.
The method of preparing the solution buffer (500 mM imidazole, pH 7.4) is to weigh 34g imidazole and 450ml EQ buffer, adjust ph to 7.4, fix volume to 500ml, and filter with 0.22 μm filter membrane for later use.
The harvested supernatant was purified by AKTA Pure instrument. Firstly, balancing the instrument by using the EQ buffer solution until the pH value and the conductivity value of the effluent liquid are consistent with those of the EQ buffer solution; samples were collected with the solution buffer.
The fusion Protein SEQ ID NO 10-16 was expressed in the same manner, purified using Protein A affinity chromatography column, and conditioned medium containing the expressed receptor was washed sequentially. The eluate was dialyzed against 10mM tris buffer saline, pH 7.2.
The concentration and purity of the protein prepared were determined by ultraviolet spectrophotometry.
Protein numbering Concentration (mg/L) Purity (%)
5 846 97
6 850 93
10 710 96
11 640 82
12 350 91
13 205 95
14 460 94
15 210 83
16 310 91
EXAMPLE 2 FGF21 mutant-induced glucose uptake function Studies
FGF21 protein mutants were evaluated for their modulatory effect on glucose uptake levels. The surface of differentiated mature adipocytes of 3T3-L1 mouse embryo fibroblasts expresses glucose transporter 1 (GLUT 1), and FGF21 protein regulates the glucose uptake level of adipocytes by regulating the expression level of GLUT 1.
Cultured 3T3-L1 (Nanjac Bai, cat# CBP 60758) mouse embryo fibroblasts were digested with trypsin (gibco, cat# 25200-056) to prepare a single cell suspension, and the cell density was adjusted to 1X10 6 Per mL, inoculated in a T75 flask, and cultured overnight at 37℃in a 5% carbon dioxide incubator. The original medium was aspirated, an induction medium was added, namely, 2. Mu.g/ml of a human insulin (Sinobiologics, cat# 11038-HNAY) solution, 1. Mu.M dexamethasone (Sigma, cat# D4902-25 MG) and 0.5mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma, cat# I7018-100 MG) were added to a complete medium of DMEM (gibco, cat# 11995-065) containing 10% fetal bovine serum (gibco, cat# 1009141C), 3T3-L1 cells were induced and cultured for 3 days, the number and size of intracellular fat particles were observed under a mirror to differentiate them into adipocytes, and then the differentiation medium was changed to a complete medium containing only 2. Mu.g/ml human insulinA base.
Digesting the induced differentiation mature adipocytes to prepare a single cell suspension, and adjusting the cell density to 1x10 with DMEM complete basal medium 6 Per mL,100 uL/well, inoculated in 96-well plate (Corning, cat# 3610), after cell attachment, the mutant protein to be tested (SEQ ID NO: 5) was diluted with DMEM basal medium to a final concentration of 5000nM in the experimental group, 100 uL/well was added to the plate, the control group was added with the same volume of DMEM basal medium, and incubated overnight at 37℃with 5% carbon dioxide. The following day, cells were starved in DMEM basal medium for 2 hours, then 100um 2-NBDG was added, incubated at 37 ℃ for 1 hour, washed 2 times with ph7.4pbs solution, trypsinized, single cell suspensions were prepared, cells were transferred to V-bottom 96-well plates, intracellular 2-NBDG signals were detected using ZE5 flow cytometry, glucose uptake was calculated using MFI, and the formula was calculated: % glucose uptake = (experimental MFI-control MFI)/control MFI 100%.
Regulation of glucose uptake levels by FGF21 protein mutants
Protein number/SEQ ID NO: glucose uptake 5000nM (%)
6 42.76
Experimental results show that the FGF21 mutant (SEQ ID NO: 6) obviously induces adipocytes to absorb glucose analog 2-NBDG, and has good induction efficiency.
EXAMPLE 3 cell function study of FGF21 mutant inducing phosphorylation of ERK1/2 protein
FGF21 protein regulates the phosphorylation level of ERK1/2 protein via the Ras/Raf/MAPK signaling pathway and transduces cellular signals into in vivo energy metabolism. Evaluation and comparison of the difference in phosphate level modulation of the FGF21 protein mutants on the ERK1/2 protein can evaluate the level of the transduced cell signals.
HuH-7 (cat# of the Chinese sciences: SCSP-526) was set at 1X10 5 inoculating/mL into 96-well plate, 100 uL/well, incubating at 37 ℃ with 5% carbon dioxide overnight, the next day starving cells in DMEM (gibco, cat# 11995-065) basic medium for 2 hours, adding to experimental group to dilute protein to be tested with DMEM basic medium, adjusting final concentration of mutant protein to be tested (SEQ ID NO: 5) to 50nM,100 uL/well adding to plate, adding to control group to same volume of DMEM basic medium, incubating at 37 ℃ for 20 minutes, adding fixing solution (BD Cytofix, cat# 554655) to fix at 37 ℃ for 30 minutes, centrifuging at 400g for 5 minutes at 4 ℃ and washing twice. Adding penetrating liquid (BD photoflow, cat# 558050) to penetrate for 1 hr at 4deg.C, adding Alexa647 labeled mouse anti-human pERK1/2 protein antibody (Biolegend, cat# 369504), incubation for 1 hour at 4℃and centrifugation at 400g,4℃for 5 min, washing twice, and 100 uL/well of resuspended cells in ZE5 (Bio-Rad) flow cytometer to detect Alexa with 2% FBS solution647 channel signal, pERK1/2 increase efficiency was calculated using Mean Fluorescence Intensity (MFI), calculated as: pERK 1/2% increase = (experimental MFI-control MFI)/control MFI × 100%.
Protein numbering pERK1/2 increase%
SEQ ID NO:5 13.13
SEQ ID NO:6 16.25
Experimental results show that FGF21 mutants (SEQ ID NO:5 and SEQ ID NO: 6) can up-regulate the phosphate level of the ERK1/2 protein in HuH-7 cells, and the effect is good.
Example 4 BLI detection of fusion proteins binding to Fc receptor
FGF21 fusion proteins consist of a functional FGF21 and an Fc fragment, wherein the extracellular domain of Fc-binding fcγr is bound by non-covalent bonds, activating an immunoreceptor tyrosine activation motif, and the immune activation effect induced by mediated ADCC (cytotoxicity) is detrimental to disease treatment. While Fc binding to FcRn (neonatal Fc receptor) favors a longer circulation of the fusion protein in vivo.
The potential immune effects and in vivo circulation properties mediated by the Fc fragment were assessed by affinity analysis between the fusion proteins and fcyri, fcyriia, fcyriiia, fcRn. Fusion protein 12 was diluted to 1108.0nM,553.8nM,276.9nM,138.4nM and 69.2nM using PBST (ph=7.4) solution; fcgammaRI (Sino, # 10256-H27H-B), fcgammaRIIA (Sino, # 10374-H27H-B), fcgammaRIIIa (Sino, 10389-H27H 1-B) and FcRn (Sino, # CT 009-H08H-B) were diluted to concentrations of 10 μg/mL,3 μg/mL,5 μg/mL and 3 μg/mL, respectively.
Capturing fcyri, fcyriia, fcyriiia with SA probes of a protein interaction instrument (PALL, # Red) in PBST (ph=7.4) solution; and then mixed with the fusion protein 12 for reaction, and the fully reacted solid phase conjugate is subjected to dissociation Analysis in PBST buffer, so that the result is analyzed by Data Analysis software. The same method, PBST (ph=6.0) solution was used for binding detection of fusion proteins to FcRn. The affinity constants obtained are shown in the following table:
the fusion protein mutant 12 has weaker combination with FcgammaR (FcgammaRI, fcgammaRIIa and FcgammaRIIIa), and the risk of side effects caused by immune effect is smaller; has strong binding with FcRn, and is beneficial to long-acting circulation in blood.
Implementation of the 5 FGF21 fusion protein mutant PK Studies
The human FcRn transgenic mouse model was used to evaluate the drug metabolism of FGF21 fusion protein mutants in mice.
Human FcRn transgenic mice with average body weights of 18-22g and 18-22 weeks of age were randomly grouped, 3 animals per group, the subject FGF21 fusion protein mutants 12 were dosed as 4mpk, s.c., single dose, PBS vehicle as negative control group, plasma was isolated by blood sampling at 0.5, 2, 4, 6, 8, 24, 48, 72, 96, 120 hours, respectively, frozen in-20 ℃ refrigerator, and then the concentration of FGF21 fusion protein mutants in mouse plasma was detected by human KLB protein-FGF 21 fusion protein mutant-Fc-HRP indirect ELISA, and PK parameters were analyzed by PK lever software non-atrioventricular model, intravascular dosing formula, as shown in the following table:
PK parameters Unit (B) Mutant 12
t1/2 h 71.3
Cmax ng/ml 1.49E+05
AUC 0-t ng/ml*h 5.43E+06
AUC 0-inf_obs ng/ml*h 6.95E+06
MRT 0-inf_obs h 93.4
Cl_obs (mg/kg)/(ng/ml)/h 5.8E-07
Comprehensive half-life t 1/2 FGF21 fusion protein mutant 12 showed good in vivo metabolic activity, as well as parameters such as exposure, peak time to peak of the highest concentration of the blood drug.
EXAMPLE 6 in vivo efficacy study of FGF21 fusion protein in DIO mice
And (3) using a high-fat diet to induce a C57BL/6 mouse obesity model, namely a DIO model mouse, and evaluating the effect of the FGF21 fusion protein mutant on weight reduction and liver weight reduction.
DIO male obese model mice weighing 30-50g,12 weeks old were randomly grouped into 8 animals each, and the tested FGF21 fusion protein mutants 12, 13 were dosed as 2mpk, s.c., q3d, 3 times in PBS vehicle as a negative control group. The body weight was weighed and administered on day 0, and then administered every 3 days and the body weight was measured and the food intake was accumulated. Weigh and fasted overnight on day 9 weigh and collect blood on day 10, take liver, and weigh liver.
DIO mouse body weight change (%) (mean.+ -. SD)
DIO mouse experimental endpoint (day 10) blood glucose level (mean±sd)
* The statistical difference of the test coefficients p <0.05 is detected, and one-way ANOVA is adopted for multi-group comparison analysis,
endpoint (day 10) liver weight changes in DIO mice experiments
* The test coefficients were statistically different for p <0.05 and analyzed using one-way ANOVA multi-group comparisons.
Comprehensive experimental results, FGF21 fusion protein mutants 12, 13 showed a significant and durable weight-loss effect with increasing feeding in mice given 2mpk, s.c., q3 d; compared with PBS in a solvent group, the composition has obvious statistical difference in reducing liver weight and blood sugar level, and the results show that the composition has better metabolic regulation effect in reducing liver fat accumulation and reducing blood sugar level.
EXAMPLE 7 in vivo efficacy study of FGF21 fusion protein in ob/ob mice
The FGF21 fusion protein mutant 12 was evaluated for four items, weight loss, blood glucose level, liver-to-body weight ratio, blood lipid, using a leptin knockout mouse diabetes model, ob/ob model mice: total Cholesterol (TC), triglycerides (TG), low Density Lipoproteins (LDL), high Density Lipoproteins (HDL) levels.
The ob/ob male obese model mice weighing 30-50g and 12 weeks old were randomly grouped, 6 animals per group, and the tested FGF21 fusion protein mutant 12 was administered 2mpk, s.c., q3d, 3 times in PBS vehicle as a negative control group. The body weight was weighed and administered on day 0, and then administered every 3 days and the body weight was measured and the food intake was accumulated. On day 8, the mixture was weighed and fasted overnight, and on day 9, the four items of blood glucose level and blood glucose were measured by blood sampling, liver was separated, liver and body weight were weighed, respectively, and the liver-body weight ratio was calculated.
The experimental result shows that the kit has obvious weight-reducing effect and blood sugar-reducing effect relative to the solvent group candidate molecules.
ob/ob mice body weight Change (%) (mean.+ -. SD)
* The test coefficients were statistically different for p <0.05 and analyzed using one-way ANOVA multi-group comparisons.
Endpoint of ob/ob mice experiments (day 9) blood glucose level Change (mmoL/L) (mean+ -SD)
* The test coefficients were statistically different for p <0.05 and analyzed using one-way ANOVA multi-group comparisons.
EXAMPLE 8 research on drug efficacy of FGF21 fusion protein on NASH model
CD57BL/6 mice were purchased from Nanjing Jixiaokang and diet-induced NASH model was performed after one week of adaptation in the laboratory. High fat content feeds, i.e. CDA HFD induction, with choline deficiency are used. Normal healthy mice were fed with conventional diet, vehicle (ph 7.4pb solution) control and experimental mice were fed with CDA/HFD diet containing 60% high fat, 2% cholesterol, and 10% fructose in drinking water, and induced for 9 weeks. The experimental mice were then randomized into 4 groups of 5 animals each, with dosing starting at week 3 (i.e., dosing after 2 weeks of induction, dosing for 7 weeks). The test drug was administered at 5mg/kg subcutaneously at q3d frequency for 7 weeks, after reaching the experimental end point, the body weight was weighed, the treated animals were euthanized, the livers were collected and weighed, and the livers were stored in 4% neutral formaldehyde for tissue sections to evaluate the effect of the test drug on the progression of NASH by detecting the index of fat deposition, stem cell balloon-like lesions, liver cell inflammation level, etc.
The experimental result shows that compared with the comparison of the healthy group, the effect of the tested medicament on the aspect of reducing the weight can obviously reach 24.92 percent, and the liver-weight ratio level is obviously reduced, which is equivalent to the level of the healthy group; there were statistical differences in improving NASH fibrosis levels relative to vehicle group candidate molecules.
NASH mice body weight change (%) (mean±sem)
Improvement of fibrosis level and NAS score by test drug
Influence of the test drug on the liver-to-body weight ratio

Claims (13)

  1. A fusion protein having the general formula:
    F 1 -F 2 -F 3
    (I)
    wherein:
    F 1 is FGF21 protein or variant thereof, and has a structure represented by the following general formula (II):
    HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSWDPX 166 SMVGGSQGX 175 SPSYES
    (II)
    X 166 selected from L or F;
    X 175 selected from R or W;
    F 2 is a connecting peptide;
    F 3 is a domain consisting of an Fc of an immunoglobulin or a fragment thereof.
  2. The fusion protein of claim 2, wherein F 1 The sequence is shown as SEQ ID NO. 5 or SEQ ID NO. 6.
  3. The fusion protein of claim 2, wherein F 2 The sequence is shown as SEQ ID NO. 7.
  4. A fusion protein according to any one of claims 1 to 3, wherein F 3 As shown in the general formula (III):
    F 4 (GGGGGS) m (GGGGS) n F 4
    (III)
    wherein:
    F 4 an Fc fragment that is an immunoglobulin;
    m is selected from integers from 1 to 10; and is also provided with
    n is an integer from 1 to 10.
  5. The fusion protein of claim 4, wherein the fusion protein is a protein that,
    F 4 is SEQ ID NO:8, 8;
    m is 1; and is also provided with
    n is 9.
  6. A fusion protein according to any one of claims 1 to 3, wherein F 3 Is SEQ ID NO:9.
  7. the fusion protein of any one of claims 1-6, wherein the fusion protein is selected from the group consisting of SEQ ID NOs: 12 or SEQ ID NO:13.
  8. a polynucleotide encoding a fusion protein comprising any one of claims 1-7.
  9. An expression vector comprising the polynucleotide of claim 8.
  10. A host cell into which or containing the expression vector of claim 9, wherein the host cell is selected from the group consisting of a bacterium, preferably e. The saccharomycete is preferably pichia pastoris; the mammalian cells are preferably CHO cells or HEK293 cells.
  11. A method of producing a protein comprising the steps of:
    culturing the host cell of claim 10;
    isolating the protein from the culture;
    and purifying the protein.
  12. A pharmaceutical composition comprising the fusion protein of any one of claims 1-7, and a pharmaceutically acceptable excipient, diluent or carrier.
  13. Use of the fusion protein according to any one of claims 1-7, or the pharmaceutical composition according to claim 12, for the manufacture of a medicament for the treatment or prevention of diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease or non-alcoholic steatohepatitis related diseases.
CN202280046816.0A 2021-07-05 2022-07-05 Fusion protein and medical application thereof Pending CN117677640A (en)

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AU2014202582A1 (en) * 2009-05-05 2014-06-05 Amgen Inc. FGF21 mutants and uses thereof
ES2927607T3 (en) * 2013-09-13 2022-11-08 Scripps Research Inst Modified therapeutic agents and compositions thereof
KR20160088656A (en) * 2015-01-16 2016-07-26 주식회사유한양행 Long-acting fgf21 fusion proteins and pharmaceutical composition comprising the same
KR102668200B1 (en) * 2015-10-28 2024-05-23 주식회사유한양행 Long-acting fgf21 fusion proteins and pharmaceutical composition comprising the same
CN107759694B (en) * 2016-08-19 2023-01-13 安源医药科技（上海）有限公司 Bispecific antibody, preparation method and application thereof
CN116143939A (en) * 2017-11-24 2023-05-23 浙江道尔生物科技有限公司 Multiple active protein for treating metabolic diseases
CN111153965A (en) * 2018-11-07 2020-05-15 浙江道尔生物科技有限公司 Artificial recombinant protein for improving performance of active protein or polypeptide and application thereof
TW202140525A (en) * 2020-01-08 2021-11-01 大陸商上海翰森生物醫藥科技有限公司 Fgf21 mutant protein and fusion protein thereof

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