WO2021139763A1 - Fgf21 mutant protein and fusion protein thereof - Google Patents

Abstract

The present invention provides an FGF21 mutant protein, a fusion protein containing an FGF21 protein or a mutant protein thereof and Fc, GDF-15 or a mutant protein thereof, and a method for treating related disease such as diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver or non-alcoholic steatohepatitis by using an FGF21 mutant protein, a fusion protein containing the FGF21 mutant protein, and a pharmaceutical composition thereof.

Description

FGF21 mutant protein and its fusion protein Technical field

The invention belongs to the field of biomedicine, and specifically relates to a mutant protein of FGF21, a mutant protein of FGF21 and a fusion protein with Fc and growth differentiation factor-15 (GDF15).

Background technique

Fibroblast growth factor 21 (FGF21) is a polypeptide composed of 209 amino acids (SEQ ID NO: 152), and its amino acid sequence has about 75% homology with mouse FGF21. The N-terminal of FGF21 contains a signal peptide composed of 28 amino acids, so the mature FGF21 is composed of 181 amino acids (SEQ ID NO: 154). Mature FGF21 has natural human FGF21 isoform or allelic form in which Leu replaces Pro at position 146 of SEQ ID NO:154 herein. FGF21 is mainly expressed in liver and pancreas, but also in fat and muscle tissues. Through the mediation of FGFR and the assistance of the transmembrane protein βKlotho (KLB), FGF21 can induce a variety of signaling pathways and functional activities in the liver, pancreas and adipose tissue, thereby realizing the regulation of glucose and lipid metabolism and the protection of pancreatic β cells Physiological function. FGF21 regulates the uptake of glucose by fat cells by activating non-insulin-dependent glucose absorption. In addition, studies have also shown that FGF21 can reduce body weight and total body fat in a dose-dependent manner. For example, when FGF21 is administered to diabetic rhesus monkeys, fasting plasma glucose, triglycerides and glucagon levels are significantly reduced. At the same time, it was found in FGF21 transgenic mice that the expression of lipase in white adipose tissue increased, and the levels of plasma β-hydroxybutyric acid and free fatty acids increased. This means that FGF21 may regulate lipid metabolism by promoting lipolysis and ketogenesis. In islet cells and INS-1E cells, FGF21 can inhibit glucose-mediated glucagon release, stimulate insulin production, and prevent islet cell apoptosis, thereby improving pancreatic cell function. In addition, FGF21 can also activate exocrine pancreatic cells and hepatocyte signaling pathways and inhibit hepatic glycogen output.

FGF21 is a member of the FGF gene family. Most FGFs have a broad spectrum of mitogenic ability. The results of the investigation show that FGF21 neither has the ability to promote cell proliferation, nor will it antagonize the functions of other members of the FGF family. Experiments have shown that FGF21 transgenic mice (the amount of FGF21 in the body is about 150 times that of normal mice) during the entire life cycle, no abnormal conditions such as tumors and tissue proliferation were found in the body. At the same time, its metabolic regulation is related to the body's metabolic level. The regulation only works when the metabolism is abnormal. Even if the pharmacological dose of FGF21 is exceeded, hypoglycemia will not occur. At present, the main diabetes medications on the market (insulin, thiazolidinedione, etc.) are prone to side effects at improper doses, such as hypoglycemia caused by high doses of insulin, liver damage and edema caused by thiazolidinedione, these Nothing was found in animal experiments that received FGF21 treatment, which is enough to prove that FGF21 is an ideal medicine for treating diabetes, obesity and other diseases.

The physiological functions of FGF21 in controlling blood sugar and reducing body weight bring hope to the treatment of related diseases, but wild-type FGF21, as a small molecule protein, is easily hydrolyzed by proteases and can also be filtered through the glomerulus, with a half-life of only 0.5-2h. It is difficult to guarantee the effective time of drug action. In the face of this difficulty, the pharmaceutical industry has improved the half-life of FGF21 by performing site-directed mutations of amino acids at the restriction site, preparing long-acting fusion proteins, or linking polyethylene glycol to the polypeptide backbone. In addition, the major challenge in the development of FGF21 as a protein formulation also comes from the instability caused by its own aggregation. The ideal effect of the target therapeutic protein is to increase the tolerance to proteolysis and reduce the aggregation of the protein, thereby enhancing the half-life and stability of the FGF21 protein preparation, and realizing low-frequency administration to patients.

Some mutants based on the human wild-type FGF21 polypeptide sequence have been described in WO2009/149171 and WO2017/074117.

Growth differentiation factor 15 (GDF-15) is a branch member of the TGF superfamily. It is also known as macrophage inhibitory cytokine I (MIC1), placental bone morphogenetic factor (PLAB), placental transforming growth factor β (PTGFB), prostate-derived factor (PDF) and non-steroidal anti-inflammatory drug activation gene (NAG- 1). The 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 usually only active when the body experiences acute or long-term stress (including contact with toxic substances that damage tissues, such as chemotherapy drugs, or during chronic diseases such as obesity or cancer). Therefore, the GDF15 pathway is expected to allow people to develop potential drugs for the treatment of diseases related to obesity and underweight. Based on relevant research results, researchers are developing GDF-15 or its variant protein as a drug for treating obesity. Obesity is an increasingly prevalent epidemic, which is estimated to affect 78 million adults in the United States.

Summary of the invention

One embodiment of the present invention is a polypeptide based on the wild-type human FGF21 sequence (SEQ ID NO: 154), wherein:

(1) Leu at position 98 from the N-terminus of wild-type FGF21 protein is replaced by Arg;

(2) Gly at position 170 from the N-terminus of wild-type FGF21 protein is replaced by Thr;

(3) Ser at position 172 from the N-terminus of wild-type FGF21 protein is replaced by Leu;

(4) Arg at position 175 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;

And (5) Ala at position 180 from the N-terminus of the wild-type FGF21 protein is replaced by Glu;

In another embodiment:

(1) Leu at position 98 from the N-terminus of wild-type FGF21 protein is replaced by Arg;

(2) Ser at position 163 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Gln or Thr;

(3) Gly at position 170 from the N-terminus of wild-type FGF21 protein is replaced by Thr;

(4) Ser at position 172 from the N-terminus of wild-type FGF21 protein is replaced by Leu;

And (5) Ala at position 180 from the N-terminus of the wild-type FGF21 protein is replaced by Glu;

A further preferred embodiment of the present invention, wherein:

(1) Leu at position 98 from the N-terminus of wild-type FGF21 protein is replaced by Arg;

(2) Ser at position 163 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Glu Ile, Asp or His;

(3) Gly at position 170 from the N-terminus of wild-type FGF21 protein is replaced by Thr;

(4) Ser at position 172 from the N-terminus of wild-type FGF21 protein is replaced by Leu;

(5) Arg at position 175 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;

(6) Ala at position 180 from the N-terminus of the wild-type FGF21 protein is replaced by Glu;

Another preferred embodiment of the present invention, wherein:

(1) Leu at position 98 from the N-terminus of 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 at position 175 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;

(4) Ala at position 180 from the N-terminus of wild-type FGF21 protein is replaced by Glu;

In another preferred embodiment of the present invention:

(1) Leu at position 98 from the N-terminus of wild-type FGF21 protein is replaced by Arg;

(2) Ser at position 163 from the N-terminus of 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) Ala at position 180 from the N-terminus of wild-type FGF21 protein is replaced by Glu;

In another preferred embodiment of the present invention:

(1) Leu at position 98 from the N-terminus of wild-type FGF21 protein is replaced by Arg;

(2) Ser at position 163 from the N-terminus of 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 at position 175 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;

(5) Ala at position 180 from the N-terminus of the wild-type FGF21 protein is replaced by Glu;

In another preferred embodiment of the present invention:

(1) Leu at position 98 from the N-terminus of wild-type F1.GF21 protein is replaced by Arg;

(2) Gly-Pro-Ser at positions 170-172 from the N-terminus of wild-type FGF21 protein is replaced by Thr-Gly-Leu;

(3) Arg at position 175 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;

(4) Ala at position 180 from the N-terminus of wild-type FGF21 protein is replaced by Glu;

In another preferred embodiment of the present invention:

(1) Leu at position 98 from the N-terminus of wild-type F1.GF21 protein is replaced by Arg;

(2) Ser at position 163 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Gln or Thr;

(3) Gly-Pro-Ser at positions 170-172 from the N-terminus of wild-type FGF21 protein is replaced by Thr-Gly-Leu;

(4) Ala at position 180 from the N-terminus of wild-type FGF21 protein is replaced by Glu;

In another preferred embodiment of the present invention:

(1) Leu at position 98 from the N-terminus of wild-type FGF21 protein is replaced by Arg;

(2) Ser at position 163 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Trp, Tyr, Leu, Glu, Ile, Asp or His;

(3) Gly-Pro-Ser at positions 170-172 from the N-terminus of wild-type FGF21 protein is replaced by Thr-Gly-Leu;

(4) Arg at position 175 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Glu, Trp, Leu, Tyr, or Ile;

(5) Ala at position 180 from the N-terminus of the wild-type FGF21 protein is replaced by Glu;

In another preferred embodiment of the present invention:

(1) Leu at position 98 from the N-terminus of wild-type FGF21 protein is replaced by Arg;

(2) Pro at position 146 from the N-terminus of wild-type FGF21 protein is replaced by Leu;

(3) Leu at position 166 from the N-terminus of wild-type FGF21 protein is replaced by Phe;

(4) Pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;

(5) Arg at position 175 from the N-terminus of wild-type FGF21 protein is replaced by Trp;

(6) Ala at position 180 from the N-terminus of the wild-type FGF21 protein is replaced by Glu;

In another preferred embodiment of the present invention:

(1) Leu at position 98 from the N-terminus of wild-type FGF21 protein is replaced by Arg;

(2) Pro at position 146 from the N-terminus of wild-type FGF21 protein is replaced by Leu;

(3) Leu at position 166 from the N-terminus of wild-type FGF21 protein is replaced by Phe;

(4) Gly-Pro-Ser at positions 170-172 from the N-terminus of wild-type FGF21 protein is replaced by Thr-Gly-Leu;

(5) Arg at position 175 from the N-terminus of wild-type FGF21 protein is replaced by Trp;

(6) Ala at position 180 from the N-terminus of the wild-type FGF21 protein is replaced by Glu;

In another preferred embodiment of the present invention:

(1) Leu at position 98 from the N-terminus of wild-type FGF21 protein is replaced by Arg;

(2) Pro at position 146 from the N-terminus of wild-type FGF21 protein is replaced by Leu;

(3) The Ser at position 163 from the N-terminus of the wild-type FGF21 protein is Phe, Trp, Asp, Leu, Glu

Or one of the substitutions in Ile;

(4) Leu at position 166 from the N-terminus of wild-type FGF21 protein is replaced by Phe;

(5) Pro at position 171 from the N-terminus of wild-type FGF21 protein is replaced by Gly;

(6) Arg at position 175 from the N-terminus of wild-type FGF21 protein is replaced by Trp;

(7) Ala at position 180 from the N-terminus of the wild-type FGF21 protein is replaced by Glu;

In another preferred embodiment of the present invention:

(1) Leu at position 98 from the N-terminus of wild-type FGF21 protein is replaced by Arg;

(2) Pro at position 146 from the N-terminus of wild-type FGF21 protein is replaced by Leu;

(3) Ser at position 163 from the N-terminus of wild-type FGF21 protein is replaced by one of Phe, Trp, His, Leu, Glu or Ile;

(4) Leu at position 166 from the N-terminus of wild-type FGF21 protein is replaced by Phe;

(5) Gly-Pro-Ser at positions 170-172 from the N-terminus of wild-type FGF21 protein is replaced by Thr-Gly-Leu;

(6) Arg at position 175 from the N-terminus of wild-type FGF21 protein is replaced by Trp;

(7) Ala at position 180 from the N-terminus of the wild-type FGF21 protein is replaced by Glu;

Another preferred embodiment of the present invention, wherein the FGF21 variant protein and the GDF-15 protein are linked by a connexin.

Another preferred embodiment of the present invention, wherein the FGF21 variant protein and the GDF-15 protein are connected through a connexin, and the connexin contains an Fc fragment of an immunoglobulin.

The present invention provides a FGF21 protein or a variant thereof, the general formula of which is as follows:

among them:

X ₁₄₆ is Pro or Leu; X _{163 is} selected from Asp, Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln, Ile or Thr; X ₁₆₆ is Leu or Phe; X ₁₇₀ is Gly or Thr; X ₁₇₁ is Pro or Gly; X ₁₇₂ is Ser or Leu; X _{175 is} selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.

An embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein X ₁₄₆ is Pro or Leu; X _{163 is} selected from Asp, Ser, Phe, Glu, His, Trp, Tyr, Leu , Gln, Ile or Thr; X ₁₆₆ is Leu or Phe; X ₁₇₀ is Gly; X ₁₇₁ is Pro or Gly; X ₁₇₂ is Ser; X _{175 is} selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.

One embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein:

X ₁₄₆ is Pro; X _{163 is} selected from Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr; X ₁₆₆ is Leu; X ₁₇₀ is Gly; X ₁₇₁ is Gly; X ₁₇₂ is Ser; X _{175 is} selected From Arg, Phe, Glu, Trp, Leu, Tyr or Ile.

One embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein:

X _{146 is} selected from Pro or Leu; X _{163 is} selected from Phe, Ile, Ser or Trp; X _{166 is} selected from Leu or Phe; X ₁₇₀ is Gly; X ₁₇₁ is Gly; X ₁₇₂ is Ser; X _{175 is} selected from Arg or Trp .

One embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein:

X _{146 is} selected from Pro or Leu; X ₁₆₃ is Trp; X ₁₆₆ is Leu; X ₁₇₀ is Gly; X ₁₇₁ is Gly; X ₁₇₂ is Ser; X _{175 is} selected from Arg or Trp. One embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein:

X ₁₄₆ is Leu; X _{163 is} selected from Asp, Ser, Phe, Glu, Trp, Leu or Ile; X ₁₆₆ is Phe; X ₁₇₀ is Gly; X ₁₇₁ is Pro or Gly; X ₁₇₂ is Ser; X _{175 is} selected from Arg , Phe, Glu, Trp, Leu, Tyr or Ile.

One embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein X ₁₄₆ is Leu; X _{163 is} selected from Asp, Ser, Phe, Glu, Trp, Leu or Ile; X ₁₆₆ is Phe; X ₁₇₀ is Gly; X ₁₇₁ is Pro or Gly; X ₁₇₂ is Ser; X _{175 is} selected from Trp.

Another embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein X ₁₄₆ is Pro or Leu; X ₁₆₃ is Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln , Ile or Thr; X ₁₆₆ is Leu or Phe; X ₁₇₀ is Thr; X ₁₇₁ is Pro or Gly; X ₁₇₂ is Leu; _{the amino acid at position X 175} is Arg, Phe, Glu, Trp, Leu, Tyr or Ile.

Another embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein X ₁₄₆ is Pro; X ₁₆₃ is Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr X ₁₆₆ is Leu; X ₁₇₀ is Thr; X ₁₇₁ is Pro or Gly; X ₁₇₂ is Leu; _{the amino acid at position X 175} is Arg, Phe, Glu, Trp, Leu, Tyr or Ile.

Another embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein:

X ₁₄₆ is Pro; X _{163 is} selected from Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr; X ₁₆₆ is Leu; X ₁₇₀ is Thr; X ₁₇₁ is Pro; X ₁₇₂ is Leu; X _{175 is} selected From Arg, Phe, Glu, Trp, Leu, Tyr or Ile.

Another embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein:

X ₁₄₆ is Leu; X ₁₆₃ is Ser, Phe, Glu, His, Trp, Leu or Ile; X ₁₆₆ is Phe; X ₁₇₀ is Thr; X ₁₇₁ is Pro; X ₁₇₂ is Leu; X ₁₇₅ is the amino acid of Arg, Phe, Glu, Trp, Leu, Tyr or Ile.

Another embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, wherein X ₁₄₆ is Leu; X ₁₆₃ is Ser, Phe, Glu, His, Trp, Leu or Ile; X ₁₆₆ is Phe; X ₁₇₀ is Thr; X ₁₇₁ is Gly; X ₁₇₂ is Leu; the amino acid at position _{X 175 is Trp.}

Another embodiment of the present invention is the FGF21 protein of general formula (I) or a variant thereof, which is selected from the sequence of SEQ ID NO: 1-144 or SEQ ID NO: 155-171.

The present invention also relates to a fusion protein, which contains the FGF21 protein of general formula (I) or a variant thereof, and its general formula is as follows:

F ₁ -F ₂ -F ₃

(II)

among them:

F ₁ is FGF21 protein or its variants; F ₂ is connexin; F ₃ is GDF15 protein or its variants.

Another embodiment of the present invention is the fusion protein of general formula (II), and the general formula of F2 connexin is:

F ₄ F ₅ (GGGGS) _m F ₅ (GGGGS) _n

(III)

among them:

F ₄ is SEQ ID NO: 145; F ₅ is the Fc fragment of immunoglobulin; m=1-20; and n=1-8.

Another embodiment of the present invention is the fusion protein of the general formula (II), and the connexin of F2 is the general formula (III), wherein F5 is SEQ ID NO: 146.

Another embodiment of the present invention is the fusion protein of the general formula (II), and the F2 connexin protein of the general formula (III), wherein m=10.

Another embodiment of the present invention is the fusion protein of the general formula (II), and the F2 connexin protein of the general formula (III), wherein n=4.

Another embodiment of the present invention is the fusion protein of the general formula (II), wherein the F2 connexin has the general formula (III), and the specific sequence is SEQ ID NO: 147.

Another embodiment of the present invention is the fusion protein of general formula (II), and the sequence of the GDF15 protein of F3 or a variant thereof is SEQ ID NO: 148.

Another embodiment of the present invention is the fusion protein described by the general formula (II), and the specific sequence is SEQ ID NO: 149 and 150.

The present invention also relates to a polynucleotide which encodes the FGF21 protein or variants thereof or the fusion protein described by the general formula (II).

The present invention also relates to an expression vector, which contains the polynucleotide as described above.

In addition, the present invention also relates to a host cell, which introduces or contains the expression vector as described above, wherein the host cell is a bacteria, preferably Escherichia coli; or the host cell is a yeast, preferably Pichia Yeast; or the host cell is a mammalian cell, preferably a CHO cell or HEK293 cell.

The present invention also relates to a method for producing protein, including the steps:

1) Culturing the host cell as described above;

2) Separate protein from culture;

3) And to purify the protein.

The present invention further comprises a pharmaceutical composition containing the FGF21 protein of general formula (I) or its variants or the fusion protein of general formula (II) and pharmaceutically acceptable excipients, diluents or carriers .

The present invention also relates to the use of the FGF21 protein or its variants, fusion proteins or the above-mentioned pharmaceutical composition in the preparation of medicines for the treatment or prevention of diabetes, obesity, dyslipidemia, and metabolic syndrome , Non-alcoholic fatty liver or non-alcoholic fatty liver disease and other related diseases.

The FGF21 mutant protein provided by the present invention has the functions of significantly inducing glucose uptake, promoting phosphorylation of ERK1/2 protein, and regulating blood sugar and body weight. The fusion protein of FGF21 mutant and Fc and GDF-15 protein provided by the present invention has It is significantly superior to the advantages of FGF21 and the known FGF21 mutants disclosed in the art, and can significantly and lastingly reduce blood sugar and body weight, and has a good metabolic regulation effect and good metabolic activity in the body. These features are beneficial to the preparation and formulation of therapeutic proteins, and have potential therapeutic effects on related diseases such as diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver or non-alcoholic steatohepatitis.

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 mutant of the present invention is determined from the amino acid position in the mature human wild-type FGF21 (SEQ ID NO: 154) polypeptide.

The amino acid sequence of the present invention contains standard one-letter or three-letter codes for twenty amino acids.

The term "FGF21 polypeptide" refers to a naturally-occurring wild-type polypeptide expressed in humans. Including SEQ ID NO: 152 constitutes the full-length form encoded by SEQ ID NO: 151 and SEQ ID NO: 154 constitutes the mature form encoded by SEQ ID NO: 153.

The term "FGF21 mutant" refers to an FGF21 polypeptide modified based on the naturally occurring FGF21 amino acid (SEQ ID NO: 154) sequence. Such modifications include, but are not limited to, substitution of one or more amino acids, including but not limited to the protease-resistant FGF21 mutants, FGF21 mutants with reduced aggregation, and FGF21 combined mutants described herein.

The term "conservative amino acid mutation" refers to the substitution of a natural amino acid residue so that the polarity or charge of the amino acid residue at this position has little influence.

According to the nature of the amino acid side chain, amino acids can be divided into the following categories:

(1) Hydrophobicity: Ala, Ile, Leu, Met, Phe, Pro and Trp;

(2) Neutral hydrophilicity: Cys, Ser or Thr;

(3) Acidity: Asp, Glu;

(4) Basic: Arg, Asn, Gln, His or Lys.

Conservative substitutions include the replacement of a member of one of these categories by another member of the same category; non-conservative substitutions include the replacement of a member of one of these categories by a member of another category.

The term "dyslipidemia" refers to disorders of lipoprotein metabolism, including overproduction or underproduction of lipoproteins. It can 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 concentration.

The term "patient" is a mammal, preferably a human.

The term "treatment" refers to slowing, reducing or reversing the progression or severity of symptoms, conditions, or diseases.

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 deliver encoded information to a host cell.

The term "expression vector" refers to a vector suitable for host cell transformation and containing nucleic acid sequences that direct and/or control the expression of inserted heterologous nucleic acid sequences. 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 by a nucleic acid sequence or capable of being transformed by the nucleic acid sequence and then capable of expressing a selected target gene. The term includes the progeny of the parent cell, regardless of whether the progeny is the same in morphology or genetic composition as the original parent, the selected genes are mainly present.

Detailed ways

In order to explain the present invention in more detail, this specification provides the following specific embodiments, but the scheme of the present invention is not limited to this.

1. Main experimental reagents:

name	Brand	Item No.
NI Sepharose excel	GE	17-3712-01
NaOH	Shanghai test	10019718
Imidazole	Shanghai test	10000218
PBS	Prosperous	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. Main experimental equipment:

name	Brand	model
Protein Purification Apparatus	GE	AKTA-Pure150
Micro spectrophotometer	Hangzhou Aosheng	Nano-300
Multifunctional microplate reader	Thermo	Lux

Example 1 Preparation of protein number 1

The ExpiCHO system (Thermo Fisher #A29133) is used to express the target protein.

Specifically, the DNA sequence encoding the numbered protein sequence (SEQ ID NO:1) with a His tag at the C-terminal was cloned into the pCDNA3.1 vector, and after sequencing, it was confirmed that a plasmid expressing the target protein was obtained. Use ExpiFectamine reagent to transfect the plasmid into ExpiCHO-S cells. After culturing the cells in 100 ml of ExpiCHO medium for 7 days, harvest the supernatant; use centrifugal filtration or deep filtration to clarify the fermentation broth.

The method of configuring EQ buffer (PBS, pH7.4) is to take a bag of PBS phosphate buffer powder, dissolve the powder in 2000ml ultrapure water, and filter it with a 0.22μm filter membrane for use.

The method to configure Elution buffer (500mM imidazole, pH7.4) is to weigh 34g of imidazole and 450ml of EQ buffer, adjust the pH to 7.4, fix the volume to 500ml, and filter with a 0.22μm filter membrane for use.

The harvested supernatant was purified by AKTA Pure instrument. First, equilibrate the instrument with EQ buffer until the pH and conductivity of the effluent are consistent with EQ buffer; then collect samples with Elution buffer.

1.3 Experimental results:

The concentration of protein number 1 determined by ultraviolet spectrophotometry was 847 mg/L, and the purity was 96%.

Example 2 Preparation of protein numbers 2-144 and 155-171

The protein numbers 2-144 and 155-171 were prepared by the method of Example 1, and the concentration and purity of the protein numbers 2-144 and 155-171 were determined by ultraviolet spectrophotometry, as shown in the following table:

Example 3 Preparation of protein numbers 149 and 150

The ExpiCHO system (Thermo Fisher #A29133) was used to express the fusion protein.

Specifically, the DNA sequence encoding the protein sequence No. 149 (SEQ ID NO: 149) and the protein sequence No. 150 (SEQ ID NO: 150) were cloned into the pCDNA3.1 vector, and after sequencing, it was confirmed that a plasmid expressing the fusion protein was obtained. The plasmid was transfected into ExpiCHO-S cells using ExpiFectamine reagent. After 7 days of culturing the cells in 100 ml of ExpiCHO medium, the supernatant was harvested. Use centrifugal filtration or deep filtration to clarify the fermentation broth.

The method of configuring EQ buffer (PBS, pH7.4) is to take a bag of PBS phosphate buffer powder, dissolve the powder in 2000ml ultrapure water, and filter it with a 0.22μm filter membrane for use.

The method to prepare Elution buffer (50mM HAc-NaAc, pH3.5) is to weigh 1.91g of sodium acetate trihydrate, add glacial acetic acid to adjust the pH to 3.5, dilute the volume to 1000ml, and filter with a 0.22μm filter membrane for use.

The harvested supernatant was purified by AKTA Pure instrument. First, equilibrate the instrument with EQ buffer until the pH and conductivity of the outflow liquid are consistent with the EQ buffer. Collect samples with Elution buffer.

Experimental results:

The protein concentration of No. 149 is 965 mg/L and the purity is 92%; the protein concentration of No. 150 is 893 mg/L and the purity is 87% by ultraviolet spectrophotometry.

Example 4 Study on the glucose uptake function induced by FGF21 mutant

To evaluate the regulatory effect of FGF21 protein mutants on the level of glucose uptake. Glucose transporter 1 (GLUT1) is expressed on the surface of adipocytes differentiated and matured from 3T3-L1 mouse embryonic fibroblasts, and FGF21 protein regulates the level of glucose uptake by adipocytes by regulating the expression level of GLUT1.

Digest the cultured 3T3-L1 (Nanjing Kebai, cat #: CBP60758) mouse embryonic fibroblasts with trypsin (gibco, cat #: 25200-056) to prepare a single cell suspension, and adjust the cell density to 1x10 ⁶ / mL, inoculated in a T75 culture flask, cultured overnight in a 37°C, 5% carbon dioxide incubator. Aspirate the original medium, add induction medium, that is, add 2μg/ml human insulin (Sinobiologics) to DMEM (gibco, cat #:11995-065) complete medium containing 10% fetal bovine serum (gibco, cat #: 1009141C) , Cat #:11038-HNAY) solution, 1μM dexamethasone (Sigma, cat #:D4902-25MG) and 0.5mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma, cat #:I7018- 100MG), induce and culture 3T3-L1 cells for 3 days, observe the number and size of the fat granules in the cells under a microscope to differentiate them into adipocytes, and then change the differentiation medium to a complete medium containing only 2μg/ml human insulin.

The induction of differentiation and maturation of adipocytes digestion, single cell suspensions, cell density was adjusted to 1x10 ⁶ / mL, 100uL / well was complete basal medium DMEM, seeded in 96-well plates (Corning, cat #: 3610) , until the cells To adhere to the wall, add DMEM basal medium to the test group to dilute the protein to be tested to a final concentration of 5000nM, and 100uL/well into the plate. Add the same volume of DMEM basal medium to the control group and incubate at 37°C, 5% carbon dioxide overnight. The next day, the cells were starved in DMEM basal medium for 2 hours, then 100uM 2-NBDG was added, and incubated at 37°C for 1 hour. The cells were washed twice with a pH 7.4 PBS solution and trypsinized to prepare a single cell suspension. Transfer to a V-bottom 96-well plate, use ZE5 flow cytometer to detect intracellular 2-NBDG signal, use MFI to calculate glucose uptake rate, calculation formula: glucose uptake rate% = (experimental group MFI-control group MFI)/control Group MFI*100%.

The experimental results are as follows:

Table 1 Regulatory effects of FGF21 protein mutants on glucose uptake

The experimental results show that the FGF21 mutant induces the uptake of the glucose analogue 2-NBDG by adipocytes at a concentration of 5000 nM, and the induction efficiency is good.

Example 5 Cell function study of FGF21 mutant inducing phosphorylation of ERK1/2 protein

FGF21 protein regulates the phosphorylation level of ERK1/2 protein through the Ras/Raf/MAPK signaling pathway, transducing cell signals to participate in energy metabolism in vivo. Evaluation and comparison of FGF21 protein mutants on ERK1/2 protein phosphate levels can be evaluated to evaluate the level of cell signal transduction.

The FGF21 protein mutant was incubated with liver cancer cell HuH-7 cells expressing FGF21 receptor FGFR and helper binding protein Klotho beta, and the cells were fixed to permeabilize the cells, and then fluorescein-labeled anti-pERK1/2 antibody was incubated with it, using flow The cytometer detects the intracellular signal intensity.

The HuH-7 (Chinese Academy of Sciences, cat #: SCSP-526) at 1x10 ⁵ / mL were seeded in 96-well plates, 100 uL / hole, at 37 ℃, 5% carbon dioxide were incubated overnight, the next day, the cells in DMEM (gibco, cat #:11995-065) Starvation in the basal medium for 2 hours, add DMEM basal medium to dilute the test protein in the experimental group, adjust the final concentration of the test protein to 50nM, add 100uL/well to the plate, and add the same volume of the control group DMEM basal medium, incubate at 37°C for 20 minutes, add fixative (BD Cytofix, cat #:554655), fix at 37°C for 30 minutes, centrifuge at 400g, 4°C for 5 minutes, and wash twice. Add permeate solution (BD Phosflow, cat #:558050) after permeabilization at 4℃ for 1 hour, add Alexa

647-labeled mouse anti-human pERK1/2 protein antibody (Biolegend, cat #: 369504), incubate at 4°C for 1 hour, centrifuge at 400g, 4°C for 5 minutes, wash twice, add 100uL/well to 2% FBS solution to resuspend the cells Detect Alexa on ZE5 (Bio-Rad) flow cytometer

For the 647 channel signal, the average fluorescence intensity (MFI) is used to calculate the growth efficiency of pERK1/2. The calculation formula is: pERK1/2 growth rate%=(experimental group MFI-control group MFI)/control group MFI*100%. The results are shown in the table below:

Table 2 Growth rate of pERK1/2

Protein number	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

The experimental results showed that the FGF21 mutant can up-regulate the phosphate level of ERK1/2 protein in HuH-7 cells at an induced concentration of 50 nM, and the effect is good. In addition, after testing, the variant protein has an isoform or allelic sequence of Leu instead of Pro at position 146 (for example, No. 61 protein), which has a similar growth rate of pERK1/2.

Example 6 In vivo efficacy of protein number 149

The effect of subcutaneous administration of No. 149 protein on blood glucose and body weight of db/db mice was tested and evaluated.

Male db/db mice weighing 45-55 grams and aged 10-12 weeks were purchased from Shanghai Jiesjie Experimental Animal Co., Ltd. A single subcutaneous injection of db/db mice was given the numbered protein 149 (SEQ ID NO: 149) 2 mg/kg body weight and the blood glucose and body weight of the mice were continuously observed. The specific method of measuring blood glucose level is to physically fix the mouse, expose the tail and cut off a little, squeeze the tail to make it bleed, discard the first drop of blood, and use the Roche Vigor blood glucose meter to test the blood glucose. At 0h, 24h, 48h, 78h, 96h, 120h, 144h and 168h, the blood glucose values of mice were detected. The weight values of mice were measured at 0h, 24h, 48h, 78h, 96h, 120h, 144h and 168h time points. Through the above experimental methods, the specific data of measuring the blood glucose value of mice is shown in the following table:

Table 3 Regulation effect of numbered protein on blood sugar of db/db mice.

Table 4 Regulation of numbered proteins on body weight of db/db mice.

The experimental results showed that the tested protein showed significant and long-lasting effects of reducing blood sugar and weight, indicating that it has the potential to become a long-acting drug that regulates metabolism.

Example 7 In vivo efficacy of numbered proteins 149 and 150

The high-fat diet was used to induce the obesity model of C57BL/6 mice, namely DIO model mice, to evaluate the candidate molecules in reducing body weight, fasting blood glucose level, liver weight ratio and mouse blood lipids: total cholesterol (TC), Triglycerides (TG), high-density lipoprotein cholesterol (HDL-cholesterol, HDL-C), low-density lipoprotein cholesterol (LDL-cholesterol, LDL-C) levels.

The 12-week-old DIO (Nanjing Jicui Yaokang) male obesity model mice weighing 30-50g were randomly divided into 4 groups with 7 animals in each group. The tested FGF21 fusion protein mutants 149 and 150, at 4mpk, sc , Q3d administration, a total of 3 administrations. The body weight was weighed and administered on day 0, and the body weight was measured every 3 days thereafter, and the food intake was accumulated. Weighed and fasted overnight on the 9th day. Weighed and collected blood on the 10th day. The liver was taken, and the liver and body weight were weighed. The blood glucose concentration at the end of the experiment was measured with a Roche blood glucose meter (viability, GB), and Hitachi 7060 was used Automatic biochemical analyzer/Olympus AU400 automatic biochemical analyzer blood lipid four indicators TG, TC, HDL, LDL, calculate the ratio of liver to body weight. The experimental results are shown in the following table:

Table 5 Body weight changes of DIO mice

Table 6 Cumulative food intake of DIO mice (g/mouse)

Table 7 Endpoint of mice (day 10) blood glucose and liver weight ratio

Table 8 Changes in four indexes of blood lipids at the end point of DIO mice (day 10)

The experimental results showed that with the increase in the food intake of the mice, the numbered proteins 149 and 150 showed significant and lasting effects on reducing blood sugar and weight; at the same time, the liver weight ratio decreased, indicating that the liver fat accumulation decreased; the four blood lipid indicators TG, TC, Compared with the HDL-C, LDL-C and PBS vehicle groups, there were statistical differences, and the tested protein showed a good metabolic regulation effect.

Example 8 PK study of numbered proteins 149 and 150

The human FcRn transgenic mouse model was used to evaluate the drug metabolism of FGF21 fusion protein mutants 149 and 150 in mice.

Human FcRn transgenic mice with an average weight of 18-22g and 18-22 weeks of age (Biocytometer) were randomly divided into 3 groups with 3 animals in each group. The tested FGF21 fusion protein mutants 149 and 150 were all at 4mpk. sc, single administration, PBS vehicle as a negative control group, blood was collected 0.5, 2, 4, 6, 8, 24, 48, 72, 96, 120 hours after administration to separate plasma, and stored frozen at -20 ℃ refrigerator, and then use the human Fc detection kit (Cisbio, 62HFCPEG) HTFR method to detect the concentration of the FGF21 fusion protein mutant in mouse plasma, and use the PK solver software non-compartment model, intravascular formula to analyze the PK parameters. The experimental results are shown in the following table:

Table 9 Numbered protein PK parameters

The main parameters	unit	149	150
t 1/2	h	212	143.2
C max	ug/ml	103.4	60.4
T max	h	twenty four	twenty four
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

Integrating half-life t _1/2 , exposure, time to peak blood drug concentration and other parameters, the numbered proteins 149 and 150 all show good metabolic activity in the body.

Claims (26)

1. A FGF21 protein or a variant thereof, its general formula is as follows:

   _{HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAX 146 PEPPGILAPQPPDVGSX 163 DPX 166 SMVX 170} X 171 X 172 QGX 175 SPSYES

   (I)

   among them:

   X ₁₄₆ is Pro or Leu;

   X _{163 is} selected from Asp, Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln, Ile or Thr;

   X ₁₆₆ is Leu or Phe;

   X ₁₇₀ is Gly or Thr;

   X ₁₇₁ is Pro or Gly;

   X ₁₇₂ is Ser or Leu;

   X _{175 is} selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
2. The FGF21 protein or variants thereof according to claim 1, wherein:

   X ₁₄₆ is Pro or Leu;

   X _{163 is} selected from Asp, Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln, Ile or Thr;

   X ₁₆₆ is Leu or Phe;

   X ₁₇₀ is Gly;

   X ₁₇₁ is Pro or Gly;

   X ₁₇₂ is Ser;

   X _{175 is} selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
3. The FGF21 protein or variants thereof according to claim 1, wherein:

   X ₁₄₆ is Pro;

   X _{163 is} selected from Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr;

   X ₁₆₆ is Leu;

   X ₁₇₀ is Gly;

   X ₁₇₁ is Gly;

   X ₁₇₂ is Ser;

   X _{175 is} selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
4. The FGF21 protein or variants thereof according to claim 1, wherein:

   X _{146 is} selected from Pro or Leu;

   X _{163 is} selected from Phe, Ile, Ser or Trp;

   X _{166 is} selected from Leu or Phe;

   X ₁₇₀ is Gly;

   X ₁₇₁ is Gly;

   X ₁₇₂ is Ser;

   X _{175 is} selected from Arg or Trp.
5. The FGF21 protein or variants thereof according to claim 4, wherein X _{146 is} selected from Pro or Leu;

   X ₁₆₃ is Trp;

   X ₁₆₆ is Leu;

   X ₁₇₀ is Gly;

   X ₁₇₁ is Gly;

   X ₁₇₂ is Ser;

   X _{175 is} selected from Arg or Trp.
6. The FGF21 protein or variants thereof according to claim 1, wherein:

   X ₁₄₆ is Leu;

   X _{163 is} selected from Asp, Ser, Phe, Glu, Trp, Leu or Ile;

   X ₁₆₆ is Phe;

   X ₁₇₀ is Gly;

   X ₁₇₁ is Pro or Gly;

   X ₁₇₂ is Ser;

   X _{175 is} selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
7. The FGF21 protein or variants thereof according to claim 1, wherein:

   X ₁₄₆ is Pro or Leu;

   X ₁₆₃ is Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln, Ile or Thr;

   X ₁₆₆ is Leu or Phe;

   X ₁₇₀ is Thr;

   X ₁₇₁ is Pro or Gly;

   X ₁₇₂ is Leu;

   X ₁₇₅ amino acid position Arg, Phe, Glu, Trp, Leu, Tyr , or Ile.
8. The FGF21 protein or variants thereof according to claim 1, wherein:

   X ₁₄₆ is Pro;

   X ₁₆₃ is Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr;

   X ₁₆₆ is Leu;

   X ₁₇₀ is Thr;

   X ₁₇₁ is Pro or Gly;

   X ₁₇₂ is Leu;

   X ₁₇₅ amino acid position Arg, Phe, Glu, Trp, Leu, Tyr , or Ile.
9. The FGF21 protein or variants thereof according to claim 1, wherein:

   X ₁₄₆ is Pro;

   X _{163 is} selected from Ser, Phe, Glu, His, Trp, Tyr, Leu, Gln or Thr;

   X ₁₆₆ is Leu;

   X ₁₇₀ is Thr;

   X ₁₇₁ is Pro;

   X ₁₇₂ is Leu;

   X _{175 is} selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
10. The FGF21 protein or variants thereof according to claim 1, wherein:

    X ₁₄₆ is Leu;

    X _{163 is} selected from Ser, Phe, Glu, His, Trp, Leu or Ile;

    X ₁₆₆ is Phe;

    X ₁₇₀ is Thr;

    X ₁₇₁ is Pro;

    X ₁₇₂ is Leu;

    X _{175 is} selected from Arg, Phe, Glu, Trp, Leu, Tyr or Ile.
11. The FGF21 protein or variants thereof according to claim 1, wherein:

    X ₁₄₆ is Leu;

    X _{163 is} selected from Ser, Phe, Glu, His, Trp, Leu or Ile;

    X ₁₆₆ is Phe;

    X ₁₇₀ is Thr;

    X ₁₇₁ is Gly;

    X ₁₇₂ is Leu;

    X ₁₇₅ is Trp.
12. The FGF21 protein or variants thereof according to claim 1, wherein:

    It is selected from the sequence of SEQ ID NO: 1-144 or SEQ ID NO: 155-171.
13. A fusion protein whose general formula is as follows:

    F ₁ -F ₂ -F ₃

    (II)

    among them:

    F _{1 is} selected from the FGF21 protein or variants thereof according to any one of claims 1-12;

    F ₂ is connexin;

    F ₃ is GDF15 protein or a variant thereof.
14. The fusion protein of claim 13, wherein the general formula of F2 is:

    F ₄ F ₅ (GGGGS) _m F ₅ (GGGGS) _n

    (III)

    among them:

    F ₄ is SEQ ID NO: 145;

    F ₅ is the Fc fragment of immunoglobulin;

    m=1-20; and

    n=1-8.
15. The fusion protein of claim 14, the F2 connexin general formula (III), wherein F5 is SEQ ID NO: 146.
16. The fusion protein of claim 14, wherein the F2 connexin has the general formula (III), wherein m=10.
17. The fusion protein of claim 14, wherein the F2 connexin has the general formula (III), wherein n=4.
18. The fusion protein according to claim 14, wherein the specific sequence of F2 is SEQ ID NO: 147.
19. The fusion protein according to claim 13, wherein the sequence of F3 is SEQ ID NO: 148.
20. The fusion protein of general formula (II) according to claim 13, the specific sequence is SEQ ID NO: 149 and 150.
21. A polynucleotide encoding the FGF21 protein or variants thereof according to any one of claims 1-12, or the fusion protein according to any one of claims 13-20.
22. An expression vector containing the polynucleotide of claim 21.
23. A host cell which is introduced into or contains the expression vector of claim 22, wherein the host cell is selected from bacteria, yeast or mammalian cells, and the bacteria is preferably Escherichia coli; and the yeast is preferably Bismuth Red yeast; the mammalian cells are preferably CHO cells or HEK293 cells.
24. A method of producing protein, including the steps:

    Culturing the host cell of claim 23;

    Isolate the protein from the culture;

    And to purify the protein.
25. A pharmaceutical composition comprising the FGF21 protein or variants thereof according to any one of claims 1-12, or the fusion protein according to any one of claims 13-20, and pharmaceutically acceptable excipients, Diluent or carrier.
26. Use of the FGF21 protein or variants 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 in the preparation of medicines The medicine is used to treat or prevent diabetes, obesity, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver or non-alcoholic steatohepatitis related diseases.