CN114591415B - GLP-1/GCG double receptor agonist polypeptide and fusion protein thereof - Google Patents

GLP-1/GCG double receptor agonist polypeptide and fusion protein thereof Download PDF

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CN114591415B
CN114591415B CN202111474768.9A CN202111474768A CN114591415B CN 114591415 B CN114591415 B CN 114591415B CN 202111474768 A CN202111474768 A CN 202111474768A CN 114591415 B CN114591415 B CN 114591415B
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蒋鹏
肖�琳
周林俊
王慧
凌伊
陈小锋
李文佳
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Guangdong HEC Pharmaceutical
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Abstract

The application relates to a GLP-1/GCG double-receptor agonist polypeptide, and further relates to a fusion protein comprising the GLP-1/GCG double-receptor agonist polypeptide. The GLP-1/GCG double-receptor agonist polypeptide and the fusion protein have different mutation sites compared with the existing disclosed polypeptide or fusion protein, have good blood glucose and weight reducing effect and long in-vivo half-life, and are more effective long-acting weight reducing and sugar controlling medicines.

Description

GLP-1/GCG double receptor agonist polypeptide and fusion protein thereof
Technical Field
The invention relates to the field of biological medicine, in particular to GLP-1/GCG double-receptor agonist polypeptide, fusion protein, nucleic acid, construct, recombinant cell, pharmaceutical composition and pharmaceutical application.
Background
Obesity is closely related to type II diabetes, hyperlipidemia, hypertension, etc. At present, the first-line treatment of the second type diabetes is mainly used for reducing blood sugar, but the weight reduction effect is very limited, and how to develop the medicine with good blood sugar and weight reduction effect is a current research and development hot spot in the field.
Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted by the intestinal L-cells after eating, which stimulates islet beta cells to secrete insulin, thereby stabilizing postprandial blood glucose fluctuations. The effect of reducing blood sugar has glucose concentration dependence, and the risk of hypoglycemia is greatly reduced while regulating blood sugar. GLP-1 based drugs such as liraglutide, dolraglutide and cable Ma Lutai have gradually gained a very important role in diabetes drugs in recent years. The GLP-1 medicine has the effect of reducing weight when reducing blood sugar, and the mechanism is that GLP-1 acts on the gastrointestinal tract to delay gastric emptying and intestinal peristalsis, acts on the central nervous system to inhibit appetite and the like, so that the purpose of reducing ingestion is achieved. Liraglutide has been approved as a weight loss agent before, and its upgraded product, rope Ma Lutai (trade name wegovy), has better weight loss effect and has been approved by the FDA for marketing at 6 of 2021.
However, clinical trials have demonstrated that the disadvantages of GLP-1 receptor agonists are also evident, mainly in the following respects: firstly, the short half-life period leads to dense injection frequency and brings inconvenience to patients; secondly, the weight and blood glucose reducing effect is still limited, and the clinical requirements which are not met are still met. Preclinical studies have shown that glucagon-like peptide 1 and glucagon (GLP-1/GCG) dual receptor agonists exhibit better weight and glucose reduction relative to single target GLP-1 agonists, with great development potential.
Oxyntomodulin (OXM) has been studied as a dual receptor agonist for activating glucagon-like peptide-1 (GLP-1) and glucagon (GCG), and has demonstrated good hypoglycemic and weight-reducing effects in animal models, significantly superior to existing GLP-1 drugs, such as liraglutide. However, oxyntomodulin (OXM) has problems such as poor stability, low receptor activity, etc., which result in a large dosage, and it is difficult to achieve the optimal effect of controlling sugar and reducing weight.
Disclosure of Invention
In order to solve the problems, the inventor optimizes and reforms the OXM, improves the stability, prolongs the half life in vivo, improves the activity of the receptor, optimizes the balance among the receptors, and achieves the optimal effect of controlling sugar and reducing weight.
Based on the optimized transformation result, the technical scheme adopted by the invention is as follows:
In a first aspect of the invention, the invention provides a GLP-1/GCG dual receptor agonist polypeptide. According to an embodiment of the invention, the polypeptide has an amino acid sequence ：5'X1SQGT FTSDY SKYLD EKX18AK X21FX23EW LX27X28X29X30 3', as shown below wherein X 1 is H or Y; x 18 is R, A or K; x 21 is E or D; x 23 is V or I; x 27 is L or I; x 28 is A or E; x 29 is A or G; x 30 is gps gapppps or absent; when X27 is L, X28 is E.
According to an embodiment of the present invention, the above polypeptide may further have at least one of the following additional technical features:
According to an embodiment of the invention, the polypeptide has an amino acid sequence ：5'X1SQGT FTSDY SKYLD EKX18AK EFX23EW LX27X28GX30 3', as shown below, wherein X 1 is H or Y, X 18 is R, A or K, X 23 is V or I, X 27 is L or I, and X 28 is A or E.
According to an embodiment of the invention, the polypeptide has an amino acid sequence as shown below: HSQGT FTSDY SKYLD EKX 18AK EFX23EW LX27X28 G;
Wherein X 18 is R, A or K, X 23 is V or I, X 27 is L or I, and X 28 is A or E.
According to an embodiment of the invention, the polypeptide has the amino acid sequence as described above, and at least one of X23 or X27 is I in the amino acid sequence of the polypeptide.
According to an embodiment of the invention, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs 1 to 5.
HSQGT FTSDY SKYLD EKAAK EFIEW LLEG(SEQ ID NO:1)。
HSQGT FTSDY SKYLD EKRAK EFIEW LIAG(SEQ ID NO:2)。
HSQGT FTSDY SKYLD EKRAK EFVEW LIAG(SEQ ID NO:3)。
HSQGT FTSDY SKYLD EKKAK EFIEW LIAG(SEQ ID NO:4)。
HSQGT FTSDY SKYLD EKKAK EFVEW LIAG(SEQ ID NO:5)。
The inventor discovers that the GLP-1/GCG double-receptor agonist polypeptide with the amino acid sequence shown in SEQ ID NO. 1-5 has stronger in-vitro bioactivity on GLP-1R and GCGR cells, and meanwhile, the in-vivo experimental research result shows that the GLP-1/GCG double-receptor agonist polypeptide with the amino acid sequence shown in SEQ ID NO. 1-5 has remarkable effects in controlling weight and reducing blood sugar.
In a second aspect of the invention, the invention provides a fusion protein. According to an embodiment of the invention, the fusion protein comprises the polypeptide, a connecting peptide and an IgG Fc fragment as described above, wherein the connecting peptide is arranged between the polypeptide and the head and the tail of the IgG Fc fragment. The fusion protein provided by the embodiment of the invention has good hypoglycemic and weight-reducing effects and long in-vivo half-life, and is an effective long-acting weight-reducing and weight-controlling medicament.
According to an embodiment of the present invention, the above fusion protein may further include at least one of the following additional technical features:
According to an embodiment of the invention, the N-terminus of the linker peptide is linked to the C-terminus of the polypeptide and the C-terminus of the linker peptide is linked to the N-terminus of the IgG Fc fragment.
According to an embodiment of the invention, the connecting peptide has an amino acid sequence shown in any one of SEQ ID NO. 6-8.
GGGGSGGGGSGGGGS(SEQ ID NO:6)。
GGGGSGGGGSGGGGSA(SEQ ID NO:7)。
GGGGSGGGGS(SEQ ID NO:8)。
The IgG Fc fragment used in the present invention is derived from the Fc region of human IgG1, igG2 or IgG4 or a mutant thereof. Preferably an Fc region from human IgG4 or a mutant thereof.
According to an embodiment of the invention, the IgG Fc fragment is derived from an Fc region mutant of human IgG4 comprising the amino acid sequence:
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:9).
Wherein, the IgG4 Fc mutant has three site mutations (EU numbering) of S228P, F234A and L235A, which correspond to the 10 th, 16 th and 17 th sites in SEQ ID NO. 9, respectively, compared with the Fc of the human IgG4 wild type. The mutations described above may further eliminate effector functions of IgG4 Fc, such as ADCC and CDC, to improve the safety of the fusion protein. Meanwhile, the K447 lysine at the C terminal is deleted, so that the heterogeneity of the fusion protein is avoided.
According to an embodiment of the invention, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs 10 to 14.
HSQGTFTSDYSKYLDEKAAKEFIEWLLEGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:10).
HSQGTFTSDYSKYLDEKRAKEFIEWLIAGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:11).
HSQGTFTSDYSKYLDEKRAKEFVEWLIAGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:12).
HSQGTFTSDYSKYLDEKKAKEFIEWLIAGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:13).
HSQGTFTSDYSKYLDEKKAKEFVEWLIAGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:14).
In a third aspect of the invention, the invention provides a nucleic acid. According to an embodiment of the invention, the nucleic acid encodes a polypeptide as described above or a fusion protein as described above.
In a fourth aspect of the invention, the invention provides a construct. According to an embodiment of the invention, the construct carries a nucleic acid as described previously.
According to an embodiment of the present invention, the construct may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the vector of the construct is pxc17.4.
In a fifth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell expresses the polypeptide as described above or the fusion protein as described above.
In a sixth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell contains a construct or genome as described above with the nucleic acid as described above integrated therein.
According to an embodiment of the present invention, the recombinant cell may further include at least one of the following additional technical features:
according to an embodiment of the invention, the recombinant cell is a CHO cell.
In a seventh aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises the polypeptide as described above or the fusion protein as described above.
According to an embodiment of the present invention, the above pharmaceutical composition may further include at least one of the following additional technical features:
according to an embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
According to an embodiment of the invention, the pharmaceutical composition further comprises a further antidiabetic agent comprising at least one selected from the group consisting of insulin, biguanides, sulfonylureas, rosiglitazone or pioglitazone, an alpha-glucosidase inhibitor, and an aminodipeptidyl IV inhibitor.
In an eighth aspect of the invention, the invention provides a protein formulation. According to an embodiment of the invention, the polypeptide as described above or the fusion protein as described above is comprised. The protein preparation according to the embodiment of the invention is an effective long-acting weight-reducing saccharide-controlling drug.
In a ninth aspect of the invention, the invention provides the use of a polypeptide as described above or a fusion protein as described above for the manufacture of a medicament for the treatment or prevention of a metabolic disorder.
According to an embodiment of the present invention, the metabolic disease includes at least one selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), diabetes, obesity.
Drawings
FIG. 1 is a graph of blood glucose concentration versus time at various time points following administration of sugar loaded mice according to an embodiment of the present invention;
FIG. 2 is a graph of blood glucose concentration versus time at various time points following administration of sugar loaded mice according to an embodiment of the present invention;
FIG. 3 is a graph showing experimental results of the effect of a fusion protein on glucose tolerance of DIO mice according to an embodiment of the present invention;
FIG. 4 is a graph showing experimental results of the effect of fusion proteins on DIO mouse body weight according to an embodiment of the present invention;
FIG. 5 is a graph showing experimental results of glucose tolerance of the fusion protein HEC-C70 to DIO mice according to an embodiment of the present invention;
FIG. 6 is a graph of experimental results of the effect of the fusion protein HEC-C70 on DIO mouse body weight according to an embodiment of the invention;
FIG. 7 is a graph of experimental results of the fusion protein HEC-C70 versus random blood glucose in db/db mice according to an embodiment of the present invention;
FIG. 8 is a graph showing experimental results of the effect of the fusion protein HEC-C70 on db/db mouse body weight according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Definition of terms
The terms "protein" and "polypeptide" are used interchangeably and in their broadest sense refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other linkages, such as esters, ethers, amino groups, and the like. The protein or polypeptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can make up the protein or peptide sequence. The term "amino acid" as used herein refers to natural and/or unnatural or synthetic amino acids, including the D and L optical isomers of amino acids, e.g., glycine and D and L optical isomers, amino acid analogs, and peptidomimetics.
The "Fc fragment" as described herein consists of the hinge region, CH2 and CH3 constant region structures of an antibody. Antibodies comprise two functionally independent parts, a variable domain that binds antigen called "Fab" and a constant domain called "Fc" that is involved in effector functions (e.g., complement activation and attack by phagocytes). Fc has a long serum half-life, whereas Fab is of short life (Capon et al 1989,Nature 337:525-31). The Fc domain may provide a longer half-life when linked to a therapeutic protein, or incorporate such functions as Fc receptor binding, protein A binding, complement binding, or perhaps even placenta transfer (Capon et al, 1989). The term "Fc" as used herein refers to wild-type Fc sequences from natural antibodies (e.g., human IgG1, igG2, igG3, or IgG 4), and also includes variants thereof. Variants may include one or more amino acid substitutions, additions and/or deletions that have been disclosed. In some embodiments, the Fc variant has wild-type Fc activity, such as binding to an Fc receptor.
The amino acid numbering of the IgG4 Fc fragment described herein is according to the EU numbering system, e.g., the "S228P" means that serine at position 228, numbered according to the EU numbering system, is replaced with proline; "K447" means the absence or absence of lysine at position 447 numbered according to the EU numbering system.
The term "fusion protein" generally refers to a protein that results from the fusion of two or more proteins or polypeptides. The genes or nucleic acid molecules encoding the two or more proteins or polypeptides may be linked to each other to form a fusion gene or fused nucleic acid molecule, which may encode the fusion protein. Translation of the fusion gene results in a single polypeptide having the properties of at least one, and even each, of the two or more proteins or polypeptides prior to fusion. Recombinant fusion proteins are created artificially by recombinant DNA technology for biological research or therapy. Recombinant fusion proteins are proteins created by genetic engineering of fusion genes. The present invention relates to recombinant fusion proteins, and the terms fusion protein and recombinant fusion protein are used herein in the same sense. The fusion proteins described herein generally comprise at least two domains (a and C), and optionally a third component, a linker between the two domains. The generation of recombinant fusion proteins is known in the art and generally involves removing the stop codon from the cDNA sequence encoding a first protein or polypeptide and then attaching the cDNA sequence of a second protein in-frame by ligation or overlap extension PCR. The DNA sequence is then expressed by the cell as a single protein. The protein may be engineered to include the complete sequence of both original proteins or polypeptides, or only a portion of either.
When forming the fusion proteins of the invention, linkers or linker peptides may be, but need not be, used. The G-rich polypeptides of the invention may be selected from (G) 3-S (i.e., "GGGS"), (G) 4-S (i.e., "GGGGS"), and (G) 5-S (i.e., "GGGGGS"). In some embodiments, the connecting peptide comprises GGGGSGGGGS (SEQ ID NO: 8), GGGGSGGGGSGGGGS (SEQ ID NO: 6), or GGGGSGGGGSGGGGSA (SEQ ID NO: 7). The linkers described herein are exemplary and the linkers of the invention may be much longer linkers as well as linkers comprising other residues. The linker of the invention may also be a non-peptide linker.
As used herein, the terms "comprising" or "including" generally mean including the recited elements, but not excluding other elements.
EXAMPLE 1 preparation of GLP-1/GCG double receptor agonist polypeptide
Fusion genes are formed by adding a SUMO tagged gene sequence to the 5' end of the gene encoding the polypeptide. The fusion gene is cloned to a prokaryotic expression vector and induced to express in an escherichia coli cell. After centrifugation to collect the thalli, ultrasonic crushing and centrifugation to obtain supernatant, and then purifying by a nickel column to obtain the fusion protein. Finally, carrying out enzyme digestion treatment on SUMO protease, and purifying in reverse phase to obtain the target polypeptide. The specific process is as follows:
1. vector construction and primer Synthesis
The specific steps for preparing the polypeptide by taking pET-28a as an expression vector and BL21 (DE 3) as a host are as follows:
1) Designing primers, wherein the primers are mutually used as templates, and amplifying polypeptide gene fragments. And amplifying the fusion gene fragment by using the polypeptide gene fragment and the sumo gene fragment as templates through a fusion PCR method.
2) Constructing a recombinant expression vector: the fusion gene fragment is cloned into a prokaryotic expression vector pET-28a, escherichia coli BL21 (DE 3) is transformed, recombinants are selected, and a recombinant expression plasmid sample is sent to Guangzhou Ai Ji biotechnology Co-Ltd for sequencing verification.
2. Expression purification
The BL21 (DE 3) strain thus constructed was cultured in LB medium, and kanamycin (kanamycin) was added at a final concentration of 50. Mu.g/mL, and after the culture, expression was induced with IPTG for 5 hours. The centrifuged cells were dissolved in an equilibration buffer (20 mM Tris-HCl, pH8.0, 150mM NaCl), and the cells were disrupted by sonication, and the supernatant was used for purification of the fusion protein after centrifugation. Purification by Ni-NTA affinity column (GE HEALTHCARE) and elution with elution buffer (20 mM Tris-HCl, pH8.0, 150mM NaCl,200mM imidazole) gave fusion protein.
3. Fusion protease cleavage
And diluting the protein solution by using an equilibrium buffer solution, adding the SUMO protease according to the ratio of the protein amount to the SUMO protease of 50:1, and performing enzyme digestion for 1.5 hours at room temperature.
4. Purification of polypeptides
The polypeptide solution obtained after the enzyme digestion is added with acetonitrile with the final concentration of 20 percent. Through a 4.6 x 250mm C8 packed column (nano-UniSil 8-120C8 Ultra Plus 8um 4.6*250mm, nano-micro technologies inc. In su state) with a particle size of 8 μm. The preparation and purification were performed in the AKTA purification system. The peptide-containing fraction was collected starting with 20% acetonitrile/H 2 O (20 mM Tris-HCl, pH 8.0) and increasing the ratio of acetonitrile at a gradient (1%/min) at a flow rate of 1mL/min for 30 min. The isolated product was analyzed by liquid chromatography. The amino acid sequences of the obtained GLP-1/GCG double receptor agonist polypeptides and the sample names of the corresponding polypeptide samples are shown in Table 1.
TABLE 1
Example 2 determination of in vitro Activity of Polypeptides
The method comprises the steps of applying the polypeptide prepared by expression, human GLP-1 (SEQ ID NO:17, TOCRIS Co., ltd., product No. 5374 (BATH: 2A) and human GCG (SEQ ID NO:18, norand Nord Co., norand Living) to HEK293 cells expressing GLP-1R or GCGR, respectively, and specifically comprises the following steps:
1 optimizing and conventionally synthesizing genes GCGR and GLP-1R in a gold-only company, cloning the genes to a vector pUC57-Amp, and preparing mini-scale recombinant plasmid DNA and puncture bacteria containing the recombinant plasmid;
2, taking pUC57-GCGR recombinant plasmid DNA, carrying out double enzyme digestion on the pUC57-GLP-1R by using HindIII and EcoRI, carrying out double enzyme digestion on the digested product by using HindIII and XhoI, carrying out electrophoresis on the digested product by using a 1% agarose gel, cutting a target band by using a clean blade, and recycling a target fragment by using a gel recycling kit;
3, the target fragment enzyme digestion recovery product is connected with a vector plasmid pcDNA3.1 fragment through T4 ligase, DH5 alpha competent cells are transformed, a flat plate is coated for separating single colony, and transformant expansion culture is selected for enzyme digestion verification and sequencing verification.
4, 200ML of correct bacterial liquid is inoculated for plasmid large extraction, and the kit is used as follows: pureLink HiPure Plasmid Maxiprep Kit, operate in accordance with instructions. After PCR verification and enzyme digestion verification, the plasmid is linearized by pvuI restriction enzymes. Finally, recovering the plasmid by adopting an ethanol precipitation method.
The 5 host cells were HEK293, and the day before transfection, the cells were plated in 6 well plates at a density of 2X 10-6 cells/well, 1 mL/well. And (3) transfecting the recovered linearized plasmid into HEK293 cells by using a Lipofectamine 3000 liposome transfection method, adding G418 to screen to obtain a mixed strain, and carrying out limiting dilution separation to obtain a monoclonal antibody, and carrying out activity detection verification.
6 CAMP produced by the recipient cells is detected using a cAMP detection kit (Cisbio, 62AM6 PEC) according to the procedures described in the protocol, the steps being as follows:
1) Preparing an Assay buffer: taking a complete culture solution (DMEM culture medium plus 10% FBS), adding 4/1000 of 500mM IBMX mother liquor, and preparing a cAMP-d2 working solution and an anti-cAMP-crytate working solution according to a kit instruction;
2) Diluting the human GLP-1 and GCG of the sample to be detected and the control sample to mother solution with initial concentration of 500nM, and then adding 20 mu L of the mother solution into a gradient of 80 mu L of Assay buffer (diluted 5 times) for dilution, wherein the gradient comprises 8 compound gradients in total of the mother solution;
Human GLP-1:HAEGT FTSDV SSYLE GQAAK EFIAW LVKGRG (SEQ ID NO: 17)
Human GCG HSQGT FTSDY SKYLD SRRAQ DFVQW LMNT (SEQ ID NO: 18)
3) Preparation of cell suspension: taking out cells HEK293-GLP-1R and HEK293-GCGR in a liquid nitrogen tank, immediately carrying out water bath at 37 ℃ for 1.5min, dropwise adding the cells into a 15mL centrifuge tube filled with 8mL warm culture medium in an ultra clean bench, centrifuging at 900rpm for 5min, discarding the supernatant, re-suspending the cells by 1mL of complete culture solution (blowing 15 times), immediately taking 20 mu L of suspension, mixing with an equal volume of trypan blue, taking 20 mu L of suspension, calculating the number of living cells, and diluting the cells to 4x10 pattern 5cells/mL;
4) Dividing a 384-well plate into GLP-1R cells and GCGR cell areas, adding cell suspension into the wells of the corresponding areas according to 5 mu L of each well by using a 12-channel variable-flow-rate dispenser, and adding a sample to be tested and positive control gradient diluent into the 384-well plate of the corresponding cells by using the 12-channel variable-flow-rate dispenser, wherein 5 mu L of each well (2 parallel compound wells of samples with the same concentration) are obtained; negative control: 10 mu L of assay buffer/each well, 3 wells are arranged on each 384-well plate, a white sealing plate film is covered, and the plates are put into a constant temperature incubator at 37 ℃ for half an hour and then taken out;
5) The cAMP-d2 working solution and anti-cAMP-crytate are mixed by using lysis buffer in Hi-range kit before use
The working solution is diluted by 20 times and then mixed evenly in a ratio of 1:1 to prepare a cAMP detection reagent mixed solution, 10 mu LcAMP detection reagent mixed solution is added into each hole of a sample group, 5 mu L of lysis buffer and 5 mu L of diluted anti-cAMP-crytate working solution are added into each hole of a negative control group, a white cover is covered, and the mixture is placed for 1h at room temperature in a dark place;
6) Detecting fluorescence values of 665nm and 620nm in a multifunctional enzyme-labeled instrument;
7) In this way, a dose-response curve was established, EC50 values were calculated and compared with each other, and specific results are shown in table 2.
Table 2 determination of in vitro Activity of polypeptides
As shown in the experimental results of Table 2, the GLP-1/GCG dual receptor agonist polypeptide obtained by the invention can obviously activate GLP-1 receptor cells and GCG receptor cells respectively.
EXAMPLE 3 construction of fusion protein vectors
The GLP-1/GCG double receptor agonist polypeptide and IgG4-Fc (SEQ ID NO: 9) with connecting peptide (SEQ ID NO: 6) are fused by adopting a molecular cloning method, the obtained sequence is inserted between the same enzyme cutting sites of a mammalian cell expression vector after double enzyme cutting, a series of mutant vectors are constructed, and after sequencing verification, plasmid vectors are extracted by adopting an endotoxin-removing plasmid extraction kit (OMEGA), and the plasmid vectors are preserved at-20 ℃. Wherein the amino acid sequence of the GLP-1/GCG dual receptor agonist polypeptide in the vector and the corresponding polypeptide sample name are shown in Table 1. The sample name of the fusion protein comprising the GLP-1/GCG dual receptor agonist polypeptide constructed in example 2 is identical to the sample name of the GLP-1/GCG dual receptor agonist polypeptide obtained in example 1.
Example 4 vector transfection and expression in cells
Chinese hamster ovary Cells (CHO) were resuscitated and subcultured to a density of about 6 x 10 6 cells/mL and transfected with ExpiCHOFectamineTM CHO Transfection Kit (ThermoFisher Scientific) at a final vector concentration of 1 μg/mL as constructed in example 2. The following day of transfection, agents such as enhancers are added to maintain the growth of the transfected cells. Cell culture broth was harvested when cell viability was reduced to about 80%.
Example 5 purification and identification of fusion proteins
The cell culture broth was centrifuged to collect the supernatant and filtered through a 0.22 μm filter to remove residual cell debris. The collected cell culture fluid is purified by using a Protein A chromatographic column, a target peak is collected, the target peak is further purified by using anion exchange chromatography, and finally, the Protein is eluted and collected by using 0.02M PBS. The samples were quantified using a micro nucleic acid protein meter (NanoDrop 2000/2000 cSpectrophotometer). The samples were detected by 12% SDS-PAGE electrophoresis, and the electropherograms showed single bands. The exact molecular weight of the fusion protein was determined by mass spectrometry and was substantially consistent with the theoretical molecular weight.
Example 6 determination of in vitro Activity of fusion proteins
HEK293 cells expressing GLP-1R or GCGR were stimulated by fusion proteins, cAMP produced by receptor cells was detected using cAMP detection kit (Cisbio, 62AM6 PEC), a dose-response curve was established, and EC 50 was calculated and the results are shown in Table 3 and compared with each other.
TABLE 3 determination of in vitro Activity of fusion proteins
As shown in the experimental results of Table 3, the GLP-1/GCG dual receptor agonist sample obtained by the invention can obviously activate GLP-1 receptor cells and GCG receptor cells respectively.
Example 7 Effect of candidate on glucose tolerance in Normal C57BL/6 mice
The experimental method comprises the following steps: normal C57BL/6 mice were divided into 6 groups (Control、Dulaglutide-7.5nmol/kg、HEC-C80-7.5nmol/kg、HEC-C85-7.5nmol/kg、HEC-C86-7.5nmol/kg、HEC-C87-7.5nmol/kg), each of 6 according to blood glucose and body weight, the corresponding vehicle or candidate was subcutaneously administered, animals were fasted 56h after administration, 2g/kg glucose was intraperitoneally administered to each group of animals 72h after administration, and blood glucose tests were performed 15, 30, 60, 90min before and after administration. The results are shown in Table 4. The blood glucose concentration-time curve is plotted from the blood glucose values measured at different time points as shown in fig. 1, and the blood glucose lowering rates at the time of the AUC 0～90min Glu and the blood glucose peak of each dose group are calculated.
Experimental results:
Table 4: effect of fusion proteins on blood glucose in sugar-loaded mice
Conclusion of experiment: HEC-C80, HEC-C85, HEC-C86 and HEC-C87 can significantly reduce blood glucose levels in sugar-loaded mice.
Example 8 Effect of candidate on glucose tolerance in Normal C57BL/6 mice
The experimental method comprises the following steps: normal C57BL/6 mice were divided into 3 groups (Control, dulaglutide-7.5nmol/kg, HEC-C70-7.5 nmol/kg) according to blood glucose and body weight, 8 animals per group were subcutaneously administered with the corresponding vehicle or candidate, 56h after administration, and 72h after administration, each group was intraperitoneally injected with 2g/kg of glucose, and blood glucose tests were performed before and 15, 30, 60, 90min after administration. The results are shown in Table 5. The blood glucose concentration-time curve is plotted from the blood glucose values measured at different time points as shown in fig. 2, and the blood glucose lowering rates at the time of the AUC 0～90min Glu and the blood glucose peak of each dose group are calculated.
Experimental results:
Table 5: effect of HEC-C70 on blood glucose in sugar-loaded mice
Conclusion of experiment: HEC-C70 can significantly reduce blood glucose levels in sugar-loaded mice.
Example 9 Effect of candidates on sugar tolerance and body weight of DIO mice
The experimental method comprises the following steps: the animals were weighed before each administration for 4 weeks, and the IPGTT experiment was performed at week 4, after the high-fat diet of 8-week-old C57BL/6 mice was fed for 15 weeks and then divided into 7 groups (Model、semaglutide-5nmol/kg、HEC-C70-5nmol/kg、HEC-C80-5nmol/kg、HEC-C85-5nmol/kg、HEC-C86-5nmol/kg、HEC-C87-5nmol/kg). at week 16 (corresponding vehicle was administered to the model group), semaglutide was administered once a day, and the other groups were administered twice a week.
Experimental results: HEC-C70-5nmol/kg has an effect of improving glucose tolerance of approximately semaglutide-5nmol/kg. HEC-C70-5nmol/kg, HEC-C80-5nmol/kg, HEC-C85-5nmol/kg, HEC-C86-5nmol/kg and HEC-C87-5nmol/kg all have the effect of significantly reducing DIO mouse weight, and HEC-C70-5nmol/kg and HEC-C85-5nmol/kg reduce weight effect better than semaglutide-5nmol/kg. Specific data are shown in tables 6, 7, and fig. 3 and 4.
Table 6: effect of long-term administration on blood glucose lowering rate in sugar-loaded DIO mice
Table 7: effect of long-term administration on DIO mice body weight
Conclusion of experiment: prolonged administration of HEC-C70, HEC-C80, HEC-C85, HEC-C86, HEC-C87 significantly improved glucose tolerance and significantly reduced body weight in DIO mice.
Example 10 Effect of candidate on sugar tolerance and body weight of DIO mice
The experimental method comprises the following steps: the dosing was started on week 16 (corresponding vehicle dosing in model group) after feeding 6 weeks of age C57BL/6N mice with high fat diet for 15 weeks, divided into 6 groups (Model、LY3298176-10nmo/kg、semaglutide-10nmol/kg、MEDI0382-10nmol/kg、HEC-C70-2.5nmol/kg、HEC-C70-10nmol/kg). according to body weight, semaglutide and MEDI0382 were dosed once daily, the other groups were dosed twice weekly for 4 weeks, animals were weighed before each dosing, and IPGTT experiments were performed on week 4 of dosing.
Control LY3298176 sequence:
YX 1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS-NH2 (SEQ ID NO: 15) wherein X 1 and X 2 are Aib, and K at position 20 is linked via its side chain ε -amino to ([ 2- (2-amino-ethoxy) -ethoxy ] -acetyl) 2-γGlu-CO-(CH2)18-CO2 H.
Control MEDI0382 sequence: HSQGTFTSDKSEYLDSERARDFVAWLEAGG (SEQ ID NO: 16), wherein K in position 10 is linked to γGlu-CO- (CH 2)14-CO2 H) via its side chain ε -amino group.
Experimental results: HEC-C70-10nmol/kg improved glucose tolerance approximately LY3298176-10nmol/kg, better than semaglutide-10nmol/kg and MEDI0382-10nmol/kg. HEC-C70-10nmol/kg has remarkable effect of reducing DIO mouse weight, and is better than semaglutide-10nmol/kg. Specific data are shown in tables 8 and 9 and fig. 5 and 6.
Table 8: effect of long-term administration on blood glucose lowering rate in sugar-loaded DIO mice
Table 9: effect of long-term administration on DIO mice body weight
Conclusion of experiment: prolonged administration of HEC-C70 significantly improved glucose tolerance and significantly reduced body weight in DIO mice.
Example 11 Effect of candidate on blood glucose and body weight in db/db mice
The experimental method comprises the following steps: the 6-7 week old ob/ob mice were divided into 3 groups (Model, dulaglutide-30 nmol/kg, HEC-C70-30 nmol/kg) based on random blood glucose and body weight. Animals of each group were monitored for random blood glucose and body weight during dosing for 4 weeks (model group dosing with corresponding vehicle), dulaglutide and HEC-C70 were dosed twice a week.
Experimental results: HEC-C70-30nmol/kg has better effect in improving sugar tolerance than Dulaglutide-30nmol/kg. HEC-C70-30nmol/kg has remarkable effect of reducing db/db mouse weight, and is superior to Dulaglutide-30nmol/kg. Specific data are shown in tables 10 and 11 and fig. 7 and 8.
Table 10: effects of prolonged administration on blood glucose in db/db mice
Table 11: effect of long-term administration on db/db mice body weight
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
SEQUENCE LISTING
<110> East yang light pharmaceutical Co., ltd
<120> GLP-1/GCG double receptor agonist polypeptide and fusion protein thereof
<130> 2021-12-2
<160> 18
<170> PatentIn version 3.5
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His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
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Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
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His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
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Gly
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His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
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Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
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Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
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Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
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Gly
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His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu
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Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys
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Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
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Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
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Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
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Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
180 185 190
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
210 215 220
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
225 230 235 240
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
245 250 255
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
260 265 270
Gly
<210> 15
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> Control LY3298176 sequence, xaa is Aib
<400> 15
Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys
1 5 10 15
Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 16
<211> 30
<212> PRT
<213> Natural sequence
<220>
<223> Control MEDI0382 sequence
<400> 16
His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Glu Tyr Leu Asp Ser
1 5 10 15
Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly
20 25 30
<210> 17
<211> 31
<212> PRT
<213> Natural sequence
<220>
<223> Human GLP-1 sequence
<400> 17
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210> 18
<211> 29
<212> PRT
<213> Artificial sequence
<220>
<223> Human GCG sequence
<400> 18
His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser
1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr
20 25

Claims (16)

1. A GLP-1/GCG dual receptor agonist polypeptide, characterized in that said polypeptide consists of the amino acid sequence shown below: SEQ ID NO is 1-5.
2. A fusion protein comprising: the polypeptide, linker peptide, and IgG-Fc fragment of claim 1, wherein the linker peptide is disposed between the polypeptide and the head and tail of the IgG-Fc fragment.
3. The fusion protein of claim 2, wherein the N-terminus of the linker peptide is linked to the C-terminus of the polypeptide and the C-terminus of the linker peptide is linked to the N-terminus of the IgG-Fc fragment.
4. A fusion protein according to any one of claims 2 to 3, wherein the linker peptide consists of the amino acid sequence shown in any one of SEQ ID NOs 6 to 8.
5. The fusion protein of claim 2, wherein the IgG-Fc fragment is an IgG4-Fc variant consisting of the amino acid sequence shown in SEQ ID No. 9.
6. The fusion protein of claim 2, wherein the fusion protein consists of an amino acid sequence shown in any one of SEQ ID NOs 10 to 14.
7. A nucleic acid encoding the polypeptide of claim 1 or the fusion protein of any one of claims 2-6.
8. A construct carrying the nucleic acid of claim 7.
9. The construct of claim 8, wherein the vector of the construct is pxc17.4.
10. A recombinant cell expressing the polypeptide of claim 1 or the fusion protein of any one of claims 2 to 6 or comprising the construct of claim 8 or 9 or the recombinant cell genome having the nucleic acid of claim 7 integrated therein.
11. The recombinant cell of claim 10, wherein the recombinant cell is a CHO cell.
12. A pharmaceutical composition comprising the polypeptide of claim 1 or the fusion protein of any one of claims 2-6.
13. The pharmaceutical composition of claim 12, further comprising a pharmaceutically acceptable carrier.
14. The pharmaceutical composition of claim 12, further comprising an additional antidiabetic agent comprising at least one selected from the group consisting of insulins, biguanides, sulfonylureas, rosiglitazone or pioglitazone, alpha-glucosidase inhibitors, and aminodipeptidyl peptidase IV inhibitors.
15. A protein preparation comprising the polypeptide of claim 1 or the fusion protein of any one of claims 2 to 6.
16. Use of the polypeptide of claim 1 or the fusion protein of any one of claims 2 to 6 or the protein preparation of claim 15 in the manufacture of a medicament for the treatment or prevention of a metabolic disorder comprising at least one selected from diabetes, obesity.
CN202111474768.9A 2020-12-03 2021-12-03 GLP-1/GCG double receptor agonist polypeptide and fusion protein thereof Active CN114591415B (en)

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CN116710462A (en) 2021-01-20 2023-09-05 维京治疗公司 Compositions and methods for treating metabolic disorders and liver diseases

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