CN113366024A - FGF21 mutant protein and fusion protein thereof - Google Patents

FGF21 mutant protein and fusion protein thereof Download PDF

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CN113366024A
CN113366024A CN202180001894.4A CN202180001894A CN113366024A CN 113366024 A CN113366024 A CN 113366024A CN 202180001894 A CN202180001894 A CN 202180001894A CN 113366024 A CN113366024 A CN 113366024A
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phe
trp
glu
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花海清
余华星
毛东杰
包如迪
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Jiangsu Hansoh Pharmaceutical Group Co Ltd
Shanghai Hansoh Biomedical Co Ltd
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Abstract

The invention provides FGF21 mutant protein, a fusion protein comprising FGF21 protein or mutant protein thereof and Fc, GDF-15 or mutant protein thereof, an FGF21 mutant protein, a fusion protein comprising FGF21 mutant protein and a pharmaceutical composition thereof, which are used for treating related diseases such as diabetes, obesity, dyslipidemia, metabolic syndrome, nonalcoholic fatty liver disease or nonalcoholic fatty hepatitis.

Description

FGF21 mutant protein and fusion protein thereof Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a mutant protein of FGF21, a mutant protein of FGF21 and a fusion protein of the mutant protein and Fc and growth differentiation factor-15 (GDF 15).
Background
Fibroblast growth factor 21 (FGF 21) is a polypeptide consisting of 209 amino acids (SEQ ID NO:152) whose amino acid sequence has about 75% homology with mouse FGF 21. FGF21N contains a 28 amino acid signal peptide at the end, thus mature FGF21 consists of 181 amino acids (SEQ ID NO: 154). Mature FGF21 has a natural human FGF21 isoform (isoform) or allelic form (allelic form) with Leu instead of Pro at position 146 of SEQ ID NO:154 herein. FGF21 is expressed primarily in the liver and pancreas, as well as in adipose and muscle tissue. FGF21 induces multiple signaling pathways and functional activities in liver, pancreas, and adipose tissue, mediated by FGFR and aided by the transmembrane protein β klotho (klb), thereby fulfilling the physiological functions of regulation of glycolipid metabolism and protection of islet β cells. FGF21 regulates glucose uptake by adipocytes by activating non-insulin dependent glucose uptake. In addition, studies have shown that FGF21 reduces body weight and systemic body fat dose-dependently, as seen by administration of FGF21 to diabetic rhesus monkeys, with significant reductions in fasting plasma glucose, triglyceride, and glucagon levels. Meanwhile, the expression of white adipose tissue lipase is increased, and the levels of plasma beta-hydroxybutyric acid and free fatty acid are increased in FGF21 transgenic mice. This means that FGF21 may regulate lipid metabolism by promoting lipolysis and ketogenesis. FGF21 was able to inhibit glucose-mediated glucagon release, stimulate insulin production, and prevent islet cell apoptosis 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 export.
FGF21 is a member of the FGF gene family, most FGFs have a broad spectrum of mitogenic capabilities, and research results indicate that FGF21 neither has the ability to promote cell proliferation nor antagonizes the function of other members of the FGF family. Experiments prove that in vivo abnormal conditions such as tumors and tissue hyperplasia are not found in FGF21 transgenic mice (the amount of FGF21 in vivo is about 150 times that of normal mice) in the whole life cycle. Meanwhile, the metabolic regulation effect of the compound is related to the metabolic level of the organism, the regulation effect only plays a role in abnormal metabolism, and hypoglycemia does not occur even if the dose of FGF21 exceeds the pharmacological dose. However, the main drugs (insulin, thiazolidinedione and the like) for treating diabetes on the market at present are easy to produce side effects when the dosage is not proper, such as hypoglycemia caused by large dose of insulin, liver function damage and edema caused by thiazolidinedione are not found in the animal experiment treated by FGF21, which is also enough to prove that FGF21 is an ideal drug for treating diabetes, obesity and other diseases.
The physiological functions of FGF21 in aspects of controlling blood sugar and reducing weight and the like bring hopes for treating related diseases, but wild FGF21 as a small molecular protein is easy to hydrolyze by protease and can be filtered by glomeruli, the half-life period is only 0.5-2h, and the effective drug action time is difficult to guarantee. In the face of the difficulty, the pharmaceutical industry improves the half-life of FGF21 by performing site-directed mutagenesis on the amino acids at the restriction sites to prepare long-acting fusion proteins or connecting polyethylene glycol to a polypeptide backbone. Furthermore, a significant challenge in developing FGF21 as a protein formulation also comes from instability due to its own aggregation. The desired effect of the therapeutic protein of interest is to increase the resistance to proteolysis and decrease the aggregation of the protein, thereby enhancing the half-life and stability of the FGF21 protein formulation, allowing for low frequency administration to the patient.
Some mutants based on the human wild-type FGF21 polypeptide sequence have been described in WO2009/149171 and WO 2017/074117.
Growth differentiation factor 15(GDF-15) is a branched member of the TGF superfamily. It is also known as macrophage inhibitory cytokine i (micl), placental bone morphogenetic factor (PLAB), Placental Transforming Growth Factor Beta (PTGFB), Prostate Derived Factor (PDF), and nonsteroidal anti-inflammatory drug activating gene (NAG-1). Mature GDF15 peptide shares low homology with its family members.
GDF-15 has been reported to play an important role in regulating body weight. GDF-15 is generally only active when the body is subjected to acute or chronic stress, including contact with toxic substances that damage tissue, such as chemotherapeutic drugs, or during chronic diseases such as obesity or cancer. Therefore, the GDF15 pathway is expected to allow the development of drugs for treating diseases related to obesity and weight deficiency. Based on the results of the relevant studies, researchers are developing GDF-15 or its variant proteins as a drug for the treatment of obesity. Obesity is an increasingly prevalent epidemic that is estimated to affect 7800 million adults in the united states.
Disclosure of Invention
One embodiment of the present invention is a polypeptide based on the sequence of wild-type human FGF21(SEQ ID NO:154), wherein:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the Gly at the position 170 from the N end of the wild FGF21 protein is substituted by Thr;
(3) the Ser at position 172 from the N terminal of the wild-type FGF21 protein is replaced by Leu;
(4) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
and (5) replacement of Ala at position 180 from the N-terminus of wild-type FGF21 protein with Glu;
in yet another embodiment:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the 163 th Ser from the N terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Gln or Thr;
(3) the Gly at the position 170 from the N end of the wild FGF21 protein is substituted by Thr;
(4) the Ser at position 172 from the N terminal of the wild-type FGF21 protein is replaced by Leu;
and (5) replacement of Ala at position 180 from the N-terminus of wild-type FGF21 protein with Glu;
a further preferred embodiment of the present invention, wherein:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the 163 th Ser from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Glu Ile, Asp or His;
(3) the Gly at the position 170 from the N end of the wild FGF21 protein is substituted by Thr;
(4) the Ser at position 172 from the N terminal of the wild-type FGF21 protein is replaced by Leu;
(5) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
(6) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
another preferred embodiment of the present invention, wherein:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;
(3) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
(4) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the 163 th Ser from the N terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Gln or Thr;
(3) pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;
(4) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the 163 th Ser from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Glu, Ile, Asp or His;
(3) pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;
(4) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
(5) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in another preferred embodiment of the present invention:
(1) substitution of Arg for Leu at position 98 from the N-terminus of wild type F1.GF21 protein;
(2) the Gly-Pro-Ser at the position 170-172 from the N end of the wild FGF21 protein is substituted by Thr-Gly-Leu;
(3) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
(4) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in yet another preferred embodiment of the present invention:
(1) substitution of Arg for Leu at position 98 from the N-terminus of wild type F1.GF21 protein;
(2) the 163 th Ser from the N terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Gln or Thr;
(3) the Gly-Pro-Ser at the position 170-172 from the N end of the wild FGF21 protein is substituted by Thr-Gly-Leu;
(4) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in yet another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) the 163 th Ser from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Glu, Ile, Asp or His;
(3) the Gly-Pro-Ser at the position 170-172 from the N end of the wild FGF21 protein is substituted by Thr-Gly-Leu;
(4) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;
(5) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) pro at position 146 from the N-terminus of the wild-type FGF21 protein is replaced by Leu;
(3) the 166 th Leu is replaced by Phe from the N-terminal of wild FGF21 protein;
(4) pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;
(5) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by Trp;
(6) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) pro at position 146 from the N-terminus of the wild-type FGF21 protein is replaced by Leu;
(3) the 166 th Leu is replaced by Phe from the N-terminal of wild FGF21 protein;
(4) the Gly-Pro-Ser at the position 170-172 from the N end of the wild FGF21 protein is substituted by Thr-Gly-Leu;
(5) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by Trp;
(6) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in yet another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) pro at position 146 from the N-terminus of the wild-type FGF21 protein is replaced by Leu;
(3) the 163 th Ser from the N-terminus of the wild-type FGF21 protein is replaced by Phe, Trp, Asp, Leu, Glu
Or Ile;
(4) the 166 th Leu is replaced by Phe from the N-terminal of wild FGF21 protein;
(5) pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;
(6) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by Trp;
(7) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
in yet another preferred embodiment of the present invention:
(1) leu at position 98 from the N-terminus of the wild-type FGF21 protein is replaced by Arg;
(2) pro at position 146 from the N-terminus of the wild-type FGF21 protein is replaced by Leu;
(3) the 163 th Ser from the N-terminal of the wild-type FGF21 protein is replaced by one of Phe, Trp, His, Leu, Glu or Ile;
(4) the 166 th Leu is replaced by Phe from the N-terminal of wild FGF21 protein;
(5) the Gly-Pro-Ser at the position 170-172 from the N end of the wild FGF21 protein is substituted by Thr-Gly-Leu;
(6) arg 175 from the N-terminal of the wild-type FGF21 protein is replaced by Trp;
(7) the Ala at position 180 from the N-terminus of the wild-type FGF21 protein is substituted by Glu;
another preferred embodiment of the invention is one wherein the FGF21 variant protein is linked to the GDF-15 protein by a connexin.
Another preferred embodiment of the invention is one wherein the FGF21 variant protein is linked to the GDF-15 protein by a connexin, and the connexin comprises an Fc fragment of an immunoglobulin.
The invention provides FGF21 protein or a variant thereof, which has the following general formula:
Figure PCTCN2021070842-APPB-000001
wherein:
X 146pro or Leu; x163Selected from Asp, Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln, Ile or Thr; x166Is Leu or Phe; x170Is Gly or Thr; x171Is Pro or Gly; x172Is Ser or Leu; x175Selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
One embodiment of the present invention is a FGF21 protein or a variant thereof according to general formula (I), wherein X146Pro or Leu; x163Selected from Asp, Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln, Ile or Thr; x166Is Leu or Phe; x170Is Gly; x171Is Pro or Gly; x172Is Ser; x175Selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
One embodiment of the present invention is a FGF21 protein or a variant thereof according to general formula (I) wherein,
X 146is Pro; x163Selected from Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr; x166Is Leu; x170Is Gly; x171Is Gly; x172Is Ser; x175Selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
One embodiment of the present invention is a FGF21 protein or a variant thereof according to general formula (I) wherein,
X 146selected from Pro or Leu; x163Selected from Phe, Ile, Ser or Trp; x166Selected from Leu or Phe; x170Is Gly; x171Is Gly; x172Is Ser; x175Selected from Arg or Trp.
One embodiment of the present invention is a FGF21 protein or a variant thereof according to general formula (I) wherein,
X 146selected from Pro or Leu; x163Is Trp; x166Is Leu; x170Is Gly; x171Is Gly; x172Is Ser; x175Selected from Arg or Trp. One embodiment of the present invention is a FGF21 protein or a variant thereof according to general formula (I) wherein,
X 146is Leu; x163Selected from Asp, Ser, Phe, Glu, Trp, Leu or Ile; x166Is Phe; x170Is Gly; x171Is Pro or Gly; x172Is Ser; x175Selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
One embodiment of the present invention is a FGF21 protein or a variant thereof according to general formula (I), wherein X146Is Leu; x163Selected from Asp, Ser, Phe, Glu, Trp, Leu or Ile; x166Is Phe; x170Is Gly; x171Is Pro or Gly; x172Is Ser; x175Selected from Trp.
Another embodiment of the present invention is a FGF21 protein or a variant thereof according to formula (I), wherein X146Pro or Leu; x163Is Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln, Ile or Thr; x166Is Leu or Phe; x170Is Thr; x171Is Pro or Gly; x172Is Leu; x175The amino acid at the position is Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
Another embodiment of the present invention is a kitThe FGF21 protein or a variant thereof of formula (I), wherein X146Is Pro; x163Is Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr; x166Is Leu; x170Is Thr; x171Is Pro or Gly; x172Is Leu; x175The amino acid at the position is Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
Another embodiment of the present invention is a FGF21 protein or a variant thereof according to formula (I),
X 146is Pro; x163Selected from Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr; x166Is Leu; x170Is Thr; x171Is Pro; x172Is Leu; x175Selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
Another embodiment of the present invention is a FGF21 protein or a variant thereof according to formula (I),
X 146is Leu; x163Is Ser, Phe, Glu, His, Trp, Leu or Ile; x166Is Phe; x170Is Thr; x171Is Pro; x172Is Leu; x175The amino acid at the position is Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
Another embodiment of the present invention is a FGF21 protein or a variant thereof according to formula (I), wherein X146Is Leu; x163Is Ser, Phe, Glu, His, Trp, Leu or Ile; x166Is Phe; x170Is Thr; x171Is Gly; x172Is Leu; x175The amino acid at position is Trp.
Another embodiment of the invention is a FGF21 protein according to formula (I) or a variant thereof selected from the sequences SEQ ID NO 1-144 or SEQ ID NO 155-171.
The invention also relates to a fusion protein containing FGF21 protein according to the general formula (I) or a variant thereof, wherein the general formula is as follows:
F 1-F 2-F 3
(II)
wherein:
F 1is an FGF21 protein or a variant thereof; f2Is a connexin; f3Is a GDF15 protein or a variant thereof.
Another embodiment of the invention is a fusion protein of formula (II), wherein the connexin of F2 has the formula:
F 4F 5(GGGGS) mF 5(GGGGS) n
(III)
wherein:
F 4is SEQ ID NO: 145; f5An Fc fragment that is an immunoglobulin; m is 1-20; and n is 1-8.
Another embodiment of the invention is a fusion protein of formula (II), a connexin of formula (III) F2, wherein F5 is SEQ ID NO: 146.
another embodiment of the invention is a fusion protein of formula (II), said connexin of F2 of formula (III), wherein m is 10.
Another embodiment of the invention is a fusion protein of formula (II), said connexin of F2 of formula (III), wherein n-4.
Another embodiment of the invention is a fusion protein according to formula (II), wherein the connexin of F2 is of formula (III) and has the specific sequence of SEQ ID NO: 147.
another embodiment of the invention is a fusion protein of general formula (II), wherein the GDF15 protein of F3 or a variant thereof has the sequence of SEQ ID NO: 148.
another embodiment of the invention is a fusion protein according to general formula (II) having the sequence of SEQ ID NO:149 and 150.
The invention also relates to a polynucleotide encoding a polypeptide comprising the FGF21 protein of formula (I) or a variant thereof or a fusion protein of formula (II).
The invention also relates to an expression vector containing the polynucleotide as described above.
In addition, the present invention also relates to a host cell introduced or containing the expression vector as described above, wherein the host cell is a bacterium, preferably Escherichia coli; or the host cell is saccharomycete, preferably pichia pastoris; or the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.
The present invention also relates to a method for producing a protein 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 present invention further comprises a pharmaceutical composition comprising the FGF21 protein of formula (I) or a variant thereof or the fusion protein of formula (II) and a pharmaceutically acceptable excipient, diluent or carrier.
The invention also relates to application of the FGF21 protein or a variant thereof, a fusion protein or the pharmaceutical composition as described above in preparation of medicaments for treating or preventing diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease or non-alcoholic fatty hepatitis and other related diseases.
The FGF21 mutant protein provided by the invention has the effects of obviously inducing glucose uptake, promoting phosphorylation of ERK1/2 protein and regulating blood sugar and body weight, and the fusion protein of the FGF21 mutant and Fc and GDF-15 protein provided by the invention has the advantages of being obviously superior to FGF21 and known FGF21 mutants disclosed in the field, and can obviously and durably reduce blood sugar and body weight, and has good metabolic regulation effect and in-vivo metabolic activity. These features are advantageous for the preparation and formulation of therapeutic proteins and have potential therapeutic effects on diseases associated with diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, and the like.
Detailed description of the invention
Unless stated to the contrary, terms used in the specification and claims have the following meanings.
The amino acid position changes in the FGF21 mutants of the invention are determined from the amino acid positions in the mature human wild-type FGF21(SEQ ID NO:154) polypeptide.
The amino acid sequences of the present invention contain the standard single or three letter codes for twenty amino acids.
The term "FGF 21 polypeptide" refers to a naturally occurring wild-type polypeptide expressed in vivo in humans. Comprising SEQ ID NO 152 constituting the full-length form encoded by SEQ ID NO 151 and SEQ ID NO 154 constituting the mature form encoded by SEQ ID NO 153.
The term "FGF 21 mutant" refers to an FGF21 polypeptide modified based on the naturally occurring amino acid sequence of FGF21(SEQ ID NO: 154). 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, as described herein.
The term "conservative amino acid mutation" refers to a substitution by a natural amino acid residue such that the polarity or charge of the amino acid residue at that position has little effect.
Amino acids can be classified into the following groups according to the properties of their side chains:
(1) hydrophobicity: ala, Ile, Leu, Met, Phe, Pro, and Trp;
(2) neutral hydrophilicity: cys, Ser or Thr;
(3) acidity: asp and Glu;
(4) alkalinity: arg, Asn, Gln, His, or Lys.
Conservative substitutions include the replacement of a member of one of these classes by another member of the same class; non-conservative substitutions involve the replacement of a member of one of these classes by a member of the other class.
The term "dyslipidaemia" refers to disorders of lipoprotein metabolism, including overproduction or underproduction of lipoproteins. May be manifested as an increase in blood total cholesterol, low density lipoprotein cholesterol and triglyceride concentrations and/or a decrease in high density lipoprotein cholesterol concentrations.
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) used to convey encoded information to a host cell.
The term "expression vector" refers to a vector suitable for transformation of a host cell and containing nucleic acid sequences that direct and/or control the 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 with a nucleic acid sequence or capable of being transformed with said nucleic acid sequence and then capable of expressing a selected gene of interest. The term includes progeny of the parent cell, whether or not the progeny is identical in morphology or genetic makeup to the original parent, with the selected gene being predominantly present.
Detailed Description
The following specific embodiments are provided in order to explain the present invention in more detail, but the present invention is not limited thereto.
1. The main experimental reagents are as follows:
name (R) Brand Goods number
NI Sepharose excel GE 17-3712-01
NaOH Shanghai test 10019718
Imidazole Shanghai test 10000218
PBS Dingguosheng Changsheng BF-0012
75% ethanol Shanghai test 801769610
Eshmuno A MERCK 1.25161.0001
Acetic acid Shanghai test 10000218
Sodium acetate (trihydrate) Shanghai test 10018718
2. The main experimental apparatus:
name (R) Brand Model number
Protein purification instrument GE AKTA-Pure150
Micro-spectrophotometer Hangzhou Osheng Nano-300
Multifunctional enzyme mark instrument Thermo Lux
Example 1 preparation of protein No. 1
The target protein was expressed using the ExpicHO system (Thermo Fisher # A29133).
Specifically, the DNA sequence encoding the numbered protein sequence (SEQ ID NO:1) with His tag at the C-terminal was cloned into pCDNA3.1 vector, and the sequencing was performed to confirm that the plasmid expressing the target protein was obtained. The plasmid was transfected into expihcho-S cells using expifctamine reagent, and after culturing the cells in 100 ml of expihcho medium for 7 days, the supernatant was harvested; clarifying the fermentation liquor by adopting a centrifugal filtration or deep filtration method.
The EQ buffer (PBS, pH7.4) was prepared by taking a bag of PBS phosphate buffer powder, dissolving the powder in 2000ml of ultrapure water, and filtering the solution through a 0.22 μm filter for use.
The solution of Elution buffer (500mM imidazole, pH7.4) was prepared by weighing 34g of imidazole, adding 450ml of EQ buffer, adjusting ph to 7.4, diluting to 500ml, and filtering with 0.22 μm filter.
The harvested supernatant was purified by AKTA Pure instrument. Firstly, balancing an instrument by using an EQ buffer solution until the pH value and the electric conductivity value of an effluent liquid are consistent with those of the EQ buffer solution; samples were collected again with Elution buffer.
1.3 Experimental results:
the concentration of the protein number 1 measured by an ultraviolet spectrophotometry method is 847mg/L, and the purity is 96%.
Example 2 preparation of protein Nos. 2-144 and 155-171
The proteins numbered 2-144 and 155-171 were prepared by the method of example 1, and the concentrations and purities of the proteins numbered 2-144 and 155-171 were determined by UV spectrophotometry, as shown in the following table:
Figure PCTCN2021070842-APPB-000002
Figure PCTCN2021070842-APPB-000003
Figure PCTCN2021070842-APPB-000004
Figure PCTCN2021070842-APPB-000005
Figure PCTCN2021070842-APPB-000006
Figure PCTCN2021070842-APPB-000007
example 3 preparation of protein Nos. 149 and 150
The fusion protein was expressed using the ExpicHO system (Thermo Fisher # A29133).
Specifically, the DNA sequences encoding the 149 protein sequence (SEQ ID NO:149) and the 150 protein sequence (SEQ ID NO:150) were cloned into the pCDNA3.1 vector, and sequencing was performed to confirm that a plasmid expressing the fusion protein was obtained. The plasmid was transfected into ExpCHO-S cells using ExpFectamine reagent, and after culturing the cells in 100 ml of ExpCHO medium for 7 days, the supernatant was harvested. Clarifying the fermentation liquor by adopting a centrifugal filtration or deep filtration method.
The EQ buffer (PBS, pH7.4) was prepared by taking a bag of PBS phosphate buffer powder, dissolving the powder in 2000ml of ultrapure water, and filtering the solution through a 0.22 μm filter for use.
The solution buffer (50mM HAc-NaAc, pH3.5) was prepared by weighing 1.91g of sodium acetate trihydrate, adjusting the pH to 3.5 with glacial acetic acid, diluting to 1000ml, and filtering with 0.22 μm filter.
The harvested supernatant was purified by AKTA Pure instrument. The instrument was first equilibrated with EQ buffer until the pH and conductivity of the effluent was consistent with EQ buffer. Samples were collected again with Elution buffer.
The experimental results are as follows:
the protein concentration 965mg/L and purity 92% of number 149 were determined by UV spectrophotometry; the protein of number 150 was 893mg/L in concentration and 87% pure.
Example 4 functional study of FGF21 mutant induced glucose uptake
The FGF21 protein mutants were evaluated for their modulating effect on glucose uptake levels. Glucose transporter 1(GLUT1) is expressed on the surface of adipose cells differentiated and matured by 3T3-L1 mouse embryonic fibroblasts, and the FGF21 protein regulates the glucose uptake level of the adipose cells by regulating the expression level of GLUT 1.
The cultured 3T3-L1 (Nanjing Kebai, cat #: CBP60758) mouse embryonic fibroblasts were digested with trypsin (gibco, cat #:25200-056) to prepare a single cell suspension, and the cell density was adjusted to 1X106The cells were inoculated in T75 flask and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. The original culture medium was aspirated, the induction medium was added, i.e., 2. mu.g/ml human insulin (Sinobiologics, cat #:11038-HNAY) solution was added to DMEM (gibco, cat #: 11995-HNAY) complete medium containing 10% fetal bovine serum (gibco, cat #:1009141C),mu.M dexamethasone (Sigma, cat #: D4902-25MG) and 0.5mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma, cat #: I7018-100MG), induced culture of 3T3-L1 cells for 3 days, microscopic observation of the number and size of intracellular adipocytes, to differentiate into adipocytes, after which the differentiation medium was changed to a complete medium containing only 2. mu.g/ml human insulin.
Digesting the fat cells induced to differentiate and mature to prepare single cell suspension, and adjusting the cell density to 1x10 by using DMEM complete basic medium6100 uL/well, inoculating to 96-well plate (Corning, cat #:3610), adding DMEM basal medium to dilute the protein to be tested to 5000nM in the experimental group when the cells adhere to the wall, adding 100 uL/well into the plate, adding DMEM basal medium to the control group in the same volume, and incubating overnight at 37 ℃ and 5% carbon dioxide. The next day, the 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 pbs solution at ph7.4, trypsinized, single cell suspension was prepared, the cells were transferred to V-bottom 96-well plate, intracellular 2-NBDG signal was detected using ZE5 flow cytometer, glucose uptake rate was calculated using MFI, and the formula was calculated: glucose uptake (% experimental MFI-control MFI)/control MFI 100%.
The experimental results are as follows:
TABLE 1 Regulation of glucose uptake levels by FGF21 protein mutants
Figure PCTCN2021070842-APPB-000008
Figure PCTCN2021070842-APPB-000009
The experiment result shows that the FGF21 mutant induces the uptake of the glucide analogue 2-NBDG by fat cells under the action concentration of 5000nM, and the induction efficiency is good.
Example 5 cellular functional Studies of the FGF21 mutant inducing phosphorylation of ERK1/2 protein
FGF21 protein regulates phosphorylation level of ERK1/2 protein through Ras/Raf/MAPK signal path to transduce cell signals to participate in energy metabolism in vivo. Evaluation of the differences in the regulation of phosphate levels of the mutant FGF21 protein on the ERK1/2 protein was compared to evaluate the level of cell signaling transduced therewith.
The FGF21 protein mutant is incubated with liver cancer cell HuH-7 cells expressing FGF21 receptor FGFR and auxiliary binding protein Klotho beta, cell permeabilization is fixed, then fluorescein-labeled anti-pERK 1/2 antibody is incubated with the cell permeabilization, and the intracellular signal intensity is detected by a flow cytometer.
HuH-7 (Chinese academy, cat #: SCSP-526) at 1x105and/mL, inoculating in a 96-well plate, 100 uL/well, incubating overnight at 37 ℃ and 5% carbon dioxide, starving the cells for 2 hours in a DMEM (gibco, cat #: 11995-. Adding permeation liquid (BD Photoflow, cat #:558050), permeating for 1 hour at 4 deg.C, and adding Alexa
Figure PCTCN2021070842-APPB-000010
647 mouse anti-human pERK1/2 protein antibody (Biolegend, cat #:369504), incubated at 4 ℃ for 1 hour, centrifuged at 400g and 4 ℃ for 5 minutes, washed twice, 100 uL/well with 2% FBS solution to resuspend the cells in ZE5(Bio-Rad) flow cytometer for Alexa detection
Figure PCTCN2021070842-APPB-000011
647 channel signals, and calculating pERK1/2 increase efficiency by using Mean Fluorescence Intensity (MFI), the formula: pERK1/2 increase rate ═ 100% MFI of experimental group-control group/control group. The results are shown in the following table:
TABLE 2 pERK1/2 growth rates
Protein numbering 50nM(％)
54 8.75
61 13.13
155 16.88
156 7.50
157 8.75
158 4.38
159 1.25
160 4.38
161 5.63
162 20
163 14.38
164 16.25
165 5.0
166 1.88
168 10
169 11.25
170 2.5
Experimental results show that the FGF21 mutant can up-regulate the phosphate level of ERK1/2 protein in HuH-7 cells under the induction concentration of 50nM, and the effect is good. In addition, it was verified by testing that the variant protein has a sequence of an isoform or allelic form of Leu instead of Pro at position 146 (e.g., protein No. 61), which has a similar growth rate of pERK 1/2.
Example 6 in vivo efficacy of protein 149
Test and evaluation the regulation of blood glucose and body weight in db/db mice by subcutaneous administration of accession number 149.
Male db/db mice weighing 45-55 grams and aged 10-12 weeks were purchased from Shanghai Jitsie laboratory animals, Inc. Mice were observed for blood glucose and body weight after 2 mg/kg body weight of numbered protein 149(SEQ ID NO:149) administered to db/db mice by a single subcutaneous injection. The specific method for measuring blood sugar value is to fix the mouse by physical method, expose the tail and cut off a little, squeeze the tail to bleed, discard the 1 st drop of blood and then measure the blood sugar by Roche vitality type blood sugar meter. The blood glucose values of the mice were measured at time points 0h, 24h, 48h, 78h, 96h, 120h, 144h and 168 h. The body weight values of the mice were measured at time points 0h, 24h, 48h, 78h, 96h, 120h, 144h and 168 h. Through the above experimental method, the specific data for measuring the blood sugar value of the mouse are shown in the following table:
TABLE 3 Regulation of blood glucose in db/db mice by numbered proteins.
Figure PCTCN2021070842-APPB-000012
TABLE 4 Regulation of body weight in db/db mice by the numbered proteins.
Figure PCTCN2021070842-APPB-000013
The experimental result shows that the tested protein shows obvious and durable effects of reducing blood sugar and weight, and the tested protein has the potential of becoming a long-acting medicament for regulating metabolism.
Example 7 in vivo efficacy of proteins 149 and 150
The candidate molecules were evaluated in four terms of reduction of body weight, fasting blood glucose level, liver body weight ratio and mouse blood lipid using a high fat diet induced C57BL/6 mouse obesity model, i.e., DIO model mouse: total Cholesterol (TC), Triglycerides (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C).
DIO (Nanjing Collection drug kang) male obesity model mice with the weight of 30-50g and the age of 12 weeks were randomly divided into 4 groups of 7 animals each, and the FGF21 fusion protein mutants 149 and 150 to be tested were administered in the form of 4mpk, s.c., q3d for 3 times. Body weight was weighed and administered on day 0, and body weight was administered and measured every 3 days thereafter, and food intake was accumulated. Weighing on day 9, fasting overnight, weighing and blood sampling on day 10, taking the liver, weighing the liver and the weight respectively, detecting the blood glucose concentration at the end point of the experiment by using a Roche glucometer (vitality, GB), and calculating the liver-body weight ratio by using the four blood lipid indexes TG, TC, HDL and LDL of the Hitachi 7060 full-automatic biochemical analyzer/Orlinbus AU400 full-automatic biochemical analyzer. The results of the experiments are shown in the following table:
TABLE 5 DIO mouse weight changes
Figure PCTCN2021070842-APPB-000014
Figure PCTCN2021070842-APPB-000015
TABLE 6 cumulative food intake for DIO mice (g/mouse)
Figure PCTCN2021070842-APPB-000016
TABLE 7 mouse endpoint (day 10) blood glucose and liver weight ratio
Figure PCTCN2021070842-APPB-000017
TABLE 8 DIO mice endpoint (day 10) four index changes in blood lipid
Figure PCTCN2021070842-APPB-000018
The experimental result shows that the numbered proteins 149 and 150 show obvious and lasting effects of reducing blood sugar and weight with the increase of the food intake of the mouse; meanwhile, the weight ratio of the liver is reduced, which indicates that the accumulated fat of the liver is reduced; the four indexes of blood fat TG, TC, HDL-C, LDL-C are all statistically different from the PBS solvent group, and the tested protein shows good metabolic regulation effect.
Example 8 PK Studies of numbered proteins 149, 150
The drug metabolism of FGF21 fusion protein mutants 149, 150 in mice was evaluated using a human FcRn transgenic mouse model.
Human FcRn transgenic mice (jacobi) with an average body weight of 18-22g and an age of 18-22 weeks were randomly divided into 3 groups of 3 animals each, tested FGF21 fusion protein mutants 149, 150 were administered at 4mpk, s.c., in a single dose, PBS vehicle as a negative control group, blood was collected and plasma was isolated at 0.5, 2, 4, 6, 8, 24, 48, 72, 96, 120 hours after administration, frozen in a-20 ℃ freezer, and then the concentration of FGF21 fusion protein mutants in mouse plasma was determined using the human Fc assay kit (Cisbio, 62HFCPEG) HTFR method, and PK parameters were analyzed using the PK solver software non-compartmental model, intravascular administration formula. The results of the experiments are shown in the following table:
TABLE 9 numbering of protein PK parameters
Principal parameters Unit of 149 150
t 1/2 h 212 143.2
C max ug/ml 103.4 60.4
T max h 24 24
AUC 0-t ug/ml*h 10415 5854.7
AUC 0-inf_obs ug/ml*h 32211.4 13180.2
MRT 0-inf_obs h 307.5 207.9
Cl_obs (mg/kg)/(ug/ml)/h 1.24E-10 3.03E-10
Integrated half-life t1/2Exposure, peak time of the highest blood concentration and other parameters, and the numbered proteins 149 and 150 show good in vivo metabolic activity.
Figure PCTCN2021070842-APPB-000019
Figure PCTCN2021070842-APPB-000020
Figure PCTCN2021070842-APPB-000021
Figure PCTCN2021070842-APPB-000022
Figure PCTCN2021070842-APPB-000023
Figure PCTCN2021070842-APPB-000024
Figure PCTCN2021070842-APPB-000025
Figure PCTCN2021070842-APPB-000026
Figure PCTCN2021070842-APPB-000027
Figure PCTCN2021070842-APPB-000028
Figure PCTCN2021070842-APPB-000029
Figure PCTCN2021070842-APPB-000030
Figure PCTCN2021070842-APPB-000031
Figure PCTCN2021070842-APPB-000032
Figure PCTCN2021070842-APPB-000033
Figure PCTCN2021070842-APPB-000034

Claims (26)

  1. An FGF21 protein or variant thereof, having the formula:
    HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAX 146PEPPGILAPQPPDVGSX 163DPX 166SMVX 170X 171X 172QGX 175SPSYES
    (I)
    wherein:
    X 146pro or Leu;
    X 163selected from Asp, Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln, Ile or Thr;
    X 166is Leu or Phe;
    X 170is Gly or Thr;
    X 171is Pro or Gly;
    X 172is Ser or Leu;
    X 175selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
  2. The FGF21 protein or a variant thereof according to claim 1,
    X 146pro or Leu;
    X 163selected from Asp, Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln, Ile or Thr;
    X 166is Leu or Phe;
    X 170is Gly;
    X 171is Pro or Gly;
    X 172is Ser;
    X 175selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
  3. The FGF21 protein or a variant thereof according to claim 1,
    X 146is Pro;
    X 163selected from Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr;
    X 166is Leu;
    X 170is Gly;
    X 171is Gly;
    X 172is Ser;
    X 175selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
  4. The FGF21 protein or a variant thereof according to claim 1,
    X 146selected from Pro or Leu;
    X 163selected from Phe, Ile, Ser or Trp;
    X 166selected from Leu or Phe;
    X 170is Gly;
    X 171is Gly;
    X 172is Ser;
    X 175selected from Arg or Trp.
  5. The FGF21 protein or variant thereof according to claim 4, wherein X is146Selected from Pro or Leu;
    X 163is Trp;
    X 166is Leu;
    X 170is Gly;
    X 171is Gly;
    X 172is Ser;
    X 175selected from Arg or Trp.
  6. The FGF21 protein or a variant thereof according to claim 1,
    X 146is Leu;
    X 163selected from Asp, Ser, Phe, Glu, Trp, Leu or Ile;
    X 166is Phe;
    X 170is Gly;
    X 171is Pro or Gly;
    X 172is Ser;
    X 175selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
  7. The FGF21 protein or a variant thereof according to claim 1,
    X 146pro or Leu;
    X 163is Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln, Ile or Thr;
    X 166is Leu or Phe;
    X 170is Thr;
    X 171is Pro or Gly;
    X 172is Leu;
    X 175the amino acid at the position is Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
  8. The FGF21 protein or a variant thereof according to claim 1,
    X 146is Pro;
    X 163is Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr;
    X 166is Leu;
    X 170is Thr;
    X 171is Pro or Gly;
    X 172is Leu;
    X 175the amino acid at the position is Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
  9. The FGF21 protein or a variant thereof according to claim 1,
    X 146is Pro;
    X 163selected from Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr;
    X 166is Leu;
    X 170is Thr;
    X 171is Pro;
    X 172is Leu;
    X 175selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
  10. The FGF21 protein or a variant thereof according to claim 1,
    X 146is Leu;
    X 163selected from Ser, Phe, Glu, His, Trp, Leu or Ile;
    X 166is Phe;
    X 170is Thr;
    X 171is Pro;
    X 172is Leu;
    X 175selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
  11. The FGF21 protein or a variant thereof according to claim 1,
    X 146is Leu;
    X 163selected from Ser, Phe, Glu, His, Trp, Leu or Ile;
    X 166is Phe;
    X 170is Thr;
    X 171is Gly;
    X 172is Leu;
    X 175is Trp.
  12. The FGF21 protein or a variant thereof according to claim 1,
    selected from the sequences SEQ ID NO 1-144 or SEQ ID NO 155-171.
  13. A fusion protein of the general formula:
    F 1-F 2-F 3
    (II)
    wherein:
    F 1selected from the group consisting of the FGF21 protein of any one of claims 1-12, or a variant thereof;
    F 2is a connexin;
    F 3is a GDF15 protein or a variant thereof.
  14. The fusion protein of claim 13, wherein F2 has the formula:
    F 4F 5(GGGGS) mF 5(GGGGS) n
    (III)
    wherein:
    F 4is SEQ ID NO: 145;
    F 5an Fc fragment that is an immunoglobulin;
    m is 1-20; and is
    n＝1-8。
  15. The fusion protein of claim 14, wherein the connexin of F2 is of formula (III) wherein F5 is SEQ ID NO: 146.
  16. the fusion protein of claim 14, wherein the connexin of F2 is of formula (III), wherein m is 10.
  17. The fusion protein of claim 14, wherein the connexin of F2 has general formula (III), wherein n-4.
  18. The fusion protein of claim 14, wherein the specific sequence of F2 is SEQ ID NO: 147.
  19. the fusion protein of claim 13, wherein the sequence of F3 is SEQ ID NO: 148.
  20. the fusion protein of claim 13, of the general formula (II) having the sequence of SEQ ID NO:149 and 150.
  21. A polynucleotide encoding the FGF21 protein or variant thereof of any one of claims 1-12, or the fusion protein of any one of claims 13-20.
  22. An expression vector comprising the polynucleotide of claim 21.
  23. A host cell into which or containing the expression vector of claim 22 is introduced, wherein the host cell is selected from a bacterium, a yeast or a mammalian cell, preferably escherichia coli; the yeast is preferably pichia pastoris; the mammalian cell is preferably a CHO cell or a HEK293 cell.
  24. A method of producing a protein comprising the steps of:
    culturing the host cell of claim 23;
    isolating the protein from the culture;
    and purifying the protein.
  25. A pharmaceutical composition comprising the FGF21 protein or variant thereof of any one of claims 1-12, or the fusion protein of any one of claims 13-20, and a pharmaceutically acceptable excipient, diluent, or carrier.
  26. Use of the FGF21 protein or a variant thereof according to any one of claims 1-12, or the fusion protein according to any one of claims 13-20, or the pharmaceutical composition according to claim 25, for the preparation of a medicament for the therapeutic or prophylactic treatment of diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease or a non-alcoholic steatohepatitis-related disease.
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