A fusion protein for treating diabetes and its application

Abstract

The present invention relates to the field of fusion proteins, and in particular to a fusion protein for treating diabetes and its application. The fusion protein containing Exendin-4 provided by the present invention is formed by fusion of Exendin-4-C1, a specific mutated Exendin-4-C2 and a specific mutated Fc fragment mutant of human immunoglobulin IgG1. The fusion protein has higher biological activity of Exendin-4 and therapeutic effects for type I, type II diabetes and obesity, and has stronger glucose tolerance activity and longer half-life. The freeze-dried powder preparation of the fusion protein containing Exendin-4 provided by the present invention is directly administered to the lungs, and its hypoglycemic effect is significantly better than that of the E4F4 fusion protein.

Description

Technical Field

[0001] The present invention relates to the field of fusion proteins, and in particular to a fusion protein for treating diabetes and application thereof. Background Art

[0002] Diabetes is a chronic disease with multiple causes, involving genetics, metabolism, nutrition and lifestyle, etc. Its onset is familial, and genetics is an important factor in the development of diabetes. At the same time, factors other than genetics are controlled by diet, lifestyle, physical activity and weight.

[0003] Exendin-4 is a short incretin peptide and a long-acting agonist of the glucagon-like peptide-1 (GLP-1) receptor. In recent years, studies have found that Exendin-4 can act on pancreatic β cells through multiple pathways to treat diabetes. On the one hand, Exendin-4 can regulate insulin secretion by specifically binding to the GLP-1 receptor on pancreatic β cells; on the other hand, Exendin-4 can also promote the proliferation of pancreatic β cells, regulate cell autophagy and inhibit cell apoptosis, thereby protecting pancreatic function. Exendin-4 can stimulate insulin secretion when blood sugar is high, thereby lowering blood sugar; it has no such effect when blood sugar is low, so it will not cause hypoglycemia. Exendin-4 can significantly reduce fasting and postprandial blood sugar and HbA1c in patients with T2DM, significantly reduce body weight and improve lipid metabolism. Animal studies have shown that exendin-4 increases the number of β cells and has a good therapeutic effect on type 2 diabetes.

[0004] With the rapid development of biotechnology, extending the half-life of drugs in the body and preventing them from being quickly cleared has become a research hotspot in recent years. Established technologies for improving drug half-life include chemical modification, microencapsulation, glycosylation, anti-protease mutants, protein fusion, etc. Many long-acting protein drugs have been successfully developed and applied in clinical treatment. At the same time, the Fc fusion proteins currently on the market often use a target protein fused with the Fc fragment of IgG, which will reduce the in vitro activity of the target protein, especially polypeptides with a small molecular weight. In particular, in the field of exendin-4 and Fc fusion proteins, fusion proteins containing exendin-4 have poor stability, low biological activity, unclear therapeutic effects, and insufficient safety, which cannot meet market needs. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the existing fusion protein containing Exendin-4 polypeptide has no obvious effect on diabetes and obesity, poor glucose tolerance and short half-life.

[0006] The solution to the technical problem of the present invention is to provide a new fusion protein containing Exendin-4 polypeptide, characterized in that the structure of the fusion protein is: Exendin-4-C1, the first connecting peptide, Exendin-4-C2, the second connecting peptide, and the Fc segment mutant of IgM in sequence from the nitrogen end. That is, the structure of the fusion protein containing Exendin-4 polypeptide of the present invention is Ex4-C1-first connecting peptide-Ex4-C2-second connecting peptide-IgG1-Fc Fc segment mutant from the nitrogen end.

[0007] Among them, the amino acid sequence of the Fc region mutant of human IgG1 is shown in SEQ ID No.5;

[0008] Further, the amino acid sequence of Exendin-4-C1 is shown in SEQ ID No. 2;

[0009] Ex4-C1:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(SEQ ID NO:2);

[0010] Further, the amino acid sequence of Exendin-4-C2 is shown in SEQ ID No. 3;

[0011] Ex4-C2:HGEGTATSDLSKQMEREAVRLFIEWLKNGGPSTGAPPPS(SEQ ID NO:3);

[0012] The above fusion protein is recombined from Exendin-4-C1, Exendin-4-C2 and human IgG1Fc mutant. The connecting peptide between the fusion proteins needs to be of sufficient length and good flexibility to ensure that the two connected molecules can fold correctly and ensure their biological activity. Generally, a flexible peptide segment of 5-25 amino acids is used.

[0013] The amino acid sequence of the connecting peptide may be shown as SEQ ID No.6;

[0014] Linker:GGGGSGGGGSGGGGS(SEQ ID NO:6);

[0015] Furthermore, the amino acid sequence of the above fusion protein is shown in SEQ ID No.9.

[0016] Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSHGEGTATSDLSKQMEREAVRLFIEWLKNGGPSTGAPPPSGGGGSGGGGSGGGGSPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSQEDPDVKFNWYVNGAEVHRAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCSVSNKALPAPIQKTISKDKGQPREPQKYTLPPSRE ELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDGSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK(SEQ ID NO:9)

[0017] Furthermore, the above fusion protein also contains a signal peptide at the N-terminus;

[0018] ssmIg:MGWSCIILFLVATATGVHS(SEQ ID NO:7);

[0019] Furthermore, the nucleotide sequence of the above fusion protein is shown in SEQ ID No. 10 or its degenerate sequence;

[0020] The present invention also provides a gene vector containing the nucleotide sequence as described in SEQ ID No.10;

[0021] The present invention also provides a host cell containing the above gene vector;

[0022] In addition, the present invention also provides a drug for treating diabetes or obesity, wherein the drug is prepared using the above-mentioned fusion protein as a main active ingredient, and further the fusion protein is a lyophilized powder preparation;

[0023] The present invention also provides the use of the above-mentioned drug in the preparation of a drug for treating diabetes or obesity. Preferably, the above-mentioned diabetes is type 1 diabetes or type 2 diabetes.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] The fusion protein containing Exendin-4 provided by the present invention is formed by fusing Exendin-4-C1, Exendin-4-C2 and a mutant of the Fc fragment of a human immunoglobulin IgG1 with a specific mutation. The fusion protein has higher biological activity of Exendin-4 and therapeutic effects for type I and type II diabetes and obesity, and has stronger glucose tolerance activity and a longer half-life. Even if a freeze-dried powder preparation of the fusion protein is used for pulmonary administration, the short-term hypoglycemic effect of the fusion protein containing Exendin-4 provided by the present invention is significantly better than the prior art level. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Gel electrophoresis results of pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant gene fragment

[0027] Figure 2 Elution profile of G-25 gel filtration column, SDS-PAGE and Western Blot results of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein

[0028] Figure 3 Changes in body weight and food intake of mice in the NS group, Ex4C1-Ex4C2-F1 group, E4F4 group, and Control group

[0029] Figure 4 Glucose tolerance of mice shown by blood glucose concentration-time curve based on blood glucose values ​​measured at different time points

[0030] Figure 5 Half-life of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein in vivo DETAILED DESCRIPTION

[0031] The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0032] Example 1: Gene synthesis and vector construction of ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein expression

[0033] The fusion protein sequence was designed by fusing the mutant human IgG1-Fc fragment to the C-terminus of Ex4-C1 and Ex4-C2. At the same time, in order to achieve secretory expression of the fusion protein, a mouse signal peptide sequence was added to the N-terminus to obtain the final fusion protein: ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant.

[0034] Exendin-4 parent peptide: HEGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 1);

[0035] Ex4-C1:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(SEQ ID NO:2);

[0036] Among them, the amino acid sequence of Ex4-C1 is exactly the same as that of the Exendin-4 parent peptide;

[0037] Ex4-C2:HGEGTATSDLSKQMEREAVRLFIEWLKNGGPSTGAPPPS(SEQ ID NO:3);

[0038] IgG1-Fc: PTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHHAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCKVSNKALPAPIQ KTISKDKGQPREPQVYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK(SEQ ID NO:4);

[0039] IgG1-Fc-Mutant:PTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHRAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCSVSNKALPAPIQKTISKDKGQPREPQKYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDGSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK(SEQ ID NO:5);

[0040] Linker:GGGGSGGGGSGGGGS(SEQ ID NO:6);

[0041] ssmIg:MGWSCIILFLVATATGVHS(SEQ ID NO:7);

[0042] ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant:MGWSCIILFLVATATGVHSHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSHGEGTATSDLSKQMEREAVRLFIEWLKNGGPSTGAPPPSGGGGSGGGGSGGGGSPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHRAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCSVSNKALPAPIQKTISKDKGQPREPQKYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDGSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK(SEQ ID NO:8);

[0043] Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGSGGGGSGGGGSHGEGTATSDLSKQMEREAVRLFIEWLKNGGPSTGAPPPSGGGGSGGGGSGGGGSPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHRAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCSVSNKALPAPIQKTISKDKGQPREPQKYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDGSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK (SEQ ID NO:9)

[0044] The coding nucleotide sequence corresponding to the ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein is shown below:

[0045]

[0046] The DNA fragment of the ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein containing a mouse signal peptide (SEQ ID No. 10) was obtained by a total gene synthesis method, and the synthesized fragment was directly inserted into the expression vector pcDNA3.1 to obtain the recombinant expression plasmid pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant; the PCR amplified plasmid product was analyzed by 2% agarose gel electrophoresis, and a pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant gene fragment of about 6480 bp was observed, and the size was consistent with the expectation; after double restriction digestion with NotI and EcoRI, a vector fragment of about 5427 bp and a target gene fragment of about 1053 bp were observed ( Figure 1 ), Lane 1: pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant; Lane 2: pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant was double-digested with NotI and EcoRI; the resulting sizes of Lane 1 and Lane 2 were consistent with the theoretical values, respectively. The recombinant plasmids were sent to Shanghai Sangon Biotechnology for sequencing, and the sequencing results were completely consistent with the ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein gene sequence, indicating that the recombinant expression plasmid pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant was successfully constructed.

[0047] Example 2 Expression and Purification of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant Fusion Protein

[0048] At 37°C and 8% CO 2 Saturated humidity and additional shaking speed is 10 9 r min -1 CHO-S suspension cells were cultured and passaged in ExpiCHO Expression Medium under constant temperature incubator conditions. 10 L -1 The transfected cells were diluted to 6 × 10 10 L -1, in ExpiFectamine TM Under the mediation of CHO Reagent, 25ug of recombinant plasmid pcDNA3.1-ssmIg-Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant was transferred into CHO-S suspension cells. 20h after transfection, 150ul of ExpiCHOTM Enhancer and 6mL of ExpiCHOTM Feed were added respectively and the cells continued to be cultured.

[0049] During the culture process, the culture medium was changed every 3 days until cell clones were formed. Monoclonal cells were isolated and expanded into stable cell lines, and the supernatant of tissue culture of these cell lines grown on 24-well plates was used to detect the fusion protein by SDS-PAGE or Elisa. Cells that can secrete a large amount of fusion protein and are stable in passage were selected for subsequent characteristic analysis. The stable CHO engineering cell line was inoculated into a 25 cm2 T-shaped flask, each containing 5 mL of cell suspension. After shaking culture for 4-5 days, it was expanded into a triangular flask and cultured for 7-10 days. The cell culture fluid containing the fusion protein was separated and purified by Protein A affinity chromatography medium, Sepharose SP XL chromatography column, Butyl Sepharose FF chromatography column, and DEAE Sepharose FF chromatography column, and then replaced into the preparation buffer by G-25 gel filtration column. The elution profile of G-25 gel filtration column, SDS-PAGE and Western Blot results of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein ( Figure 2 );

[0050] Figure 2 The results showed that the elution profile of the filtration column showed that the Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein had fewer impurities, and the target protein was mainly concentrated in peak (1); SDS-PAGE showed that the molecular weight of the fusion protein in lane (1) was consistent with the theoretical value of 35 kDa; Western Blot showed that the fusion protein was secreted and expressed in the culture medium of the CHO engineered cell line, and could be specifically recognized by anti-IgG1-Fc antibodies and anti-Exendin-4 antibodies. The above results showed that the Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein was successfully expressed and purified.

[0051] Example 3 Therapeutic effect of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein on body weight and food intake in obese mice with diabetes model

[0052] A diet-induced obesity model (DIO) was established in mice to study the therapeutic effect of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein on the diabetic mouse model in vivo. Experimental method: C57 / B6 male mice weighing 40-50 g were selected and randomly divided into 4 groups:

[0053] Normal saline group (NS group): fed with high-fat diet for 10 weeks to form obesity;

[0054] The experimental group of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein prepared in Example 2 (Ex4C1-Ex4C2-F1 group) was fed with a high-fat diet for 10 weeks to form obesity;

[0055] The E4F4 fusion protein control group (E4F4 group) prepared according to the method described in the patent document CN106146667B was fed with a high-fat diet for 10 weeks to form obesity;

[0056] Normal group (Control group): No high-fat feed was fed during the same period;

[0057] The medication is administered as follows:

[0058] Normal saline group (NS group): subcutaneous administration, 320 μg kg-1 of normal saline once a week;

[0059] Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein experimental group (Ex4C1-Ex4C2-F1 group): subcutaneous administration, 320 μg kg-1 once a week;

[0060] The E4F4 fusion protein control group (E4F4 group) prepared according to the method described in the patent document CN106146667B was administered subcutaneously once a week at 320 μg kg-1; the body weight and food intake of the four groups were measured every day ( Figure 3 ).

[0061] Figure 3 The results showed that both the E4F4 fusion protein group and the Ex4C1-Ex4C2-F1 fusion protein group could significantly inhibit the weight gain of mice by administering the drug once a week; and the effect of inhibiting weight gain of the Ex4C1-Ex4C2-F1 group was much better than that of the E4F4 group; the food intake of mice was weighed every day, and compared with the NS group, the Ex4C1-Ex4C2-F1 fusion protein group had a certain inhibitory effect on the food intake of mice, and the inhibitory effect of food intake was much better than that of the E4F4 fusion protein group.

[0062] Example 4 Glucose tolerance activity test of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein

[0063] Eighteen normal C57BL / 6 mice were randomly divided into three groups according to body weight, with 6 mice in each group. The mice were numbered one by one and administered the drugs according to the following methods:

[0064] The E4F4 fusion protein group (E4F4 group) prepared according to the method described in CN106146667B was given: the dose was 320 μg·kg-1;

[0065] Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein group prepared according to Example 2 (Ex4C1-Ex4C2-F1 group): dose 320 μg·kg-1;

[0066] Blank control group (Control group): 10 mmol·L-1 phosphate buffer, pH 7.4;

[0067] 12 hours after administration, 0.2 mL of 40% glucose was injected intraperitoneally, and blood was collected from the tail vein before injection and 0, 15, 30, 60, 90, 100, and 120 minutes after injection. The blood glucose level was measured with a blood glucose meter to evaluate the glucose tolerance of mice after administration. The blood glucose concentration-time curve was drawn according to the blood glucose values ​​measured at different time points. Figure 4 shown.

[0068] Figure 4 The results showed that compared with the blank control and semaglutide, the E4F4 fusion protein group and the Ex4C1-Ex4C2-F1 fusion protein group still had a significant hypoglycemic effect when glucose was injected 15 minutes and 30 minutes after 12 hours of administration. The Ex4C1-Ex4C2-F1 fusion protein group could maintain a lower blood glucose concentration than the E4F4 fusion protein group, and the low blood glucose concentration was maintained for a longer time, that is, the glucose tolerance activity of the Ex4C1-Ex4C2-F1 fusion protein group was much better than that of the E4F4 fusion protein group.

[0069] Example 5 In vivo half-life detection of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein

[0070] Four 8-week-old SD male rats were selected, and the Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein group prepared according to Example 2 was injected with 200 μg / kg of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein; the changes in Exendin-4 content within 49 days were detected, and blood was collected at different time points of 0, 1, 2, 4, 6, 14, 21, 28, 35, 42, and 49 days, and the Exendin-4 detection kit was used to detect the Exendin-4 content in the serum. The Exendin-4 content in the serum of the Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein group could be continuously detected for 49 days (such as Figure 5 As shown), it was calculated that the half-life t1 / 2 of Ex4C1-Linker-Ex4C2-Linker-IgG1-Fc-Mutant fusion protein in vivo was about 10 days.

[0071] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A fusion protein for treating diabetes, characterized in that: The structure of the fusion protein is: from the nitrogen end, Exendin-4-C1 polypeptide as shown in SEQ ID NO: 2, a first connecting peptide, Exendin-4-C2 polypeptide as shown in SEQ ID NO: 3, a second connecting peptide, and an Fc segment mutant of IgG1 as shown in SEQ ID NO: 5; optionally, the first connecting peptide and the second connecting peptide can be the same.

2. The fusion protein according to claim 1, characterized in that The N-terminus also contains a signal peptide.

3. The fusion protein according to claim 2, characterized in that The signal peptide is the amino acid sequence shown in SEQ ID NO:

7.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The first connecting peptide and the second connecting peptide are the amino acid sequences shown in SEQ ID NO:

6.

5. The fusion protein according to claim 1, characterized in that The fusion protein has an amino acid sequence as shown in SEQ ID NO:

9.

6. A nucleic acid encoding the fusion protein according to any one of claims 2 to 5, characterized in that: The nucleic acid sequence is SEQ ID No. 10 or a degenerate sequence thereof.

7. A gene vector comprising the nucleic acid according to claim 6.

8. A host cell containing the gene vector according to claim 7.

9. A pharmaceutical composition for treating type 2 diabetes or diet-induced obesity, characterized in that: A freeze-dried powder containing the fusion protein according to claim 1 or 5 and pharmaceutically acceptable excipients.

10. Use of the fusion protein according to any one of claims 1 to 5 and the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating diet-induced obesity.

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