Fusion proteins having dual activity of glp and gip and uses thereof

Abstract

The application relates to the field of medicine, in particular to a GLP and GIP dual-activity fusion protein and application thereof. The GLP and GIP dual-activity protein in the GLP and GIP dual-activity fusion protein is obtained through specific design, and is further fused with the GLP and GIP dual-activity protein at the N end of IgG4 heavy chain constant region and light chain constant region or the N end of Fc. Research results show that the GLP and GIP dual-activity fusion protein has obvious effects of reducing the weight of mice, and the application can be applied to drugs or products for slimming or treating fatty liver.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to fusion proteins with dual GLP and GIP activities and their applications. Background Technology

[0002] Glucagon-like peptide (GLP) is a polypeptide translated from the proglucagon gene that exerts its biological effects by binding to its corresponding G protein-coupled receptor. GLP can significantly increase phase I and II insulin secretion, decrease glucagon levels, and delay gastric emptying. Due to its glucose-lowering effect being glucose-dependent and its weight-reducing properties, GLP and its analogues are widely used in research on the treatment of type 2 diabetes and in alleviating insulin resistance caused by obesity.

[0003] Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic β cells in the presence of glucose and protecting pancreatic β cells.

[0004] Although GLP-1 / GIP dual-activity analogs (Tirzepatide) are currently used for weight loss, their effectiveness remains unsatisfactory. Therefore, there is an urgent need in this field to develop a drug that can significantly control patients' weight. Summary of the Invention

[0005] Based on the needs of existing technologies, this application provides a fusion protein with dual GLP and GIP activities and its applications.

[0006] Firstly, this application discloses a protein with dual GLP and GIP activities, which is a self-designed GLP and GIP targeting polypeptide, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] Furthermore, the present invention provides a fusion protein with dual GLP and GIP activities, which further contains Fc or immunoglobulin IgG4 in addition to the above-mentioned protein; wherein the amino acid sequence of Fc is shown in SEQ ID NO.2.

[0008] Specifically, the amino acid sequence of the heavy chain constant region of IgG4 is shown in SEQ ID NO.3.

[0009] The amino acid sequence of the constant region of the light chain of IgG4 is shown in SEQ ID NO.4.

[0010] Specifically, the N-terminus of the GLP-GIP targeting peptide is fused with a signal peptide, the sequence of which is shown in SEQ ID NO.5.

[0011] The present invention provides a fusion protein with dual GLP and GIP activity, which is obtained by fusing the aforementioned GLP and GIP dual-activity protein with IgG4 protein or Fc protein;

[0012] More specifically, it is obtained by fusing the GLP and GIP dual-activity proteins to the N-terminus of Fc, respectively; or by fusing the GLP and GIP dual-activity proteins to the N-terminus of the heavy chain constant region and the light chain constant region of IgG4, respectively.

[0013] More specifically, the amino acid sequence of Fc is shown in SEQ ID NO.2; the amino acid sequence of the constant region of the IgG4 heavy chain is shown in SEQ ID NO.3; and the amino acid sequence of the constant region of the IgG4 light chain is shown in SEQ ID NO.4.

[0014] Furthermore, the N-terminus of the Fc or IgG4 heavy chain constant region and the light chain constant region are respectively fused to proteins with dual GLP and GIP activity via linker peptides. Preferably, the amino acid sequence of the linker peptide is shown in SEQ ID NO.6.

[0015] The amino acid sequence obtained by fusing the protein with dual GLP and GIP activity to the N-terminus of Fc is shown in SEQ ID NO. 7.

[0016] The amino acid sequences of the protein with dual GLP and GIP activity obtained by fusing the N-terminus of the IgG4 heavy chain constant region and the light chain constant region are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0017] Optionally, the N-terminus of the protein with dual GLP and GIP activity is fused with a signal peptide; preferably, the amino acid sequence of the signal peptide is shown in SEQ ID NO.5.

[0018] The present invention also provides the nucleic acid encoding the fusion protein with dual GLP and GIP activity;

[0019] The nucleotide sequence encoding a protein with dual GLP and GIP activities fused to the N-terminus of Fc is shown in SEQ ID NO.10;

[0020] The nucleotide sequence encoding the protein with dual GLP and GIP activity fused to the N-terminus of the constant region of the IgG4 heavy chain is shown in SEQ ID NO. 11, and the nucleotide sequence encoding the protein with dual GLP and GIP activity fused to the N-terminus of the constant region of the IgG4 light chain is shown in SEQ ID NO. 12.

[0021] The present invention also provides a recombinant expression vector encoding the aforementioned nucleic acid.

[0022] The present invention also provides a method for preparing a cell line that produces a fusion protein with dual GLP and GIP activities, which is obtained by transfecting the encoded nucleic acid into mammalian cells, preferably, the mammalian being CHO cells; preferably, it is obtained by constructing a recombinant expression vector containing the encoded nucleic acid, linearizing it, and then transfecting it into mammalian cells.

[0023] The present invention also provides a cell line that produces a fusion protein with dual GLP and GIP activities obtained by the method.

[0024] The present invention ultimately provides the application of the fusion protein with dual GLP and GIP activity, and the cell line thereof, in the preparation of weight loss products or in drugs for the treatment of fatty liver.

[0025] The beneficial effects of this invention: Based on sequence analysis of GIP, GIP-1, and the GLP-1 / GIP dual-activity analog (Tirzepatide, prepared by chemical synthesis), and considering the characteristics of the GLP-1 and GIP sequences, this invention innovatively designs a novel polypeptide sequence co-targeting GLP-1 and GIP (dual-target polypeptide) by selecting amino acids suitable for cell expression. Furthermore, it forms a fusion protein with Fc or IgG4 possessing dual GLP and GIP activities. Research results show that, compared with the control, the fusion protein with dual GLP and GIP activities of this application significantly reduces mouse body weight, with a decrease in both liver volume and weight, and a significant decrease in serum total cholesterol. Unexpectedly, the fusion of the GLP-GIP targeting polypeptide with Fc showed a more significant effect. The fusion protein with dual GLP and GIP activities of this application can be applied to the development of drugs or products for weight loss, treatment of fatty liver, etc. Attached Figure Description

[0026] Figure 1 The present invention describes the changes in body weight in mice treated with the fusion protein.

[0027] Figure 2 Changes in liver volume in mice treated with the fusion protein. * P < 0.01, ** P < 0.05 compared to the control group.

[0028] Figure 3 Changes in liver weight in mice treated with the fusion protein. Compared with the control group, * P < 0.01, ** P < 0.05, *** P < 0.001, and **** P < 0.0001. Detailed Implementation

[0029] The present invention will be described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0030] 1. Construct an expression vector for the GLP-GIP-lgG4 fusion protein.

[0031] (1) Design of GLP-GIP targeting peptide (dual-target peptide): Based on the sequence analysis of GIP, GIP-1, GLP-1 / GIP dual-activity analog (Tirzepatide), this invention has designed a novel GLP-GIP targeting peptide (dual-target peptide), whose amino acid sequence is shown in SEQ ID NO.1: HGEGTFTSDYSILLDKIAQKAFVNWLIAGGPSSGAPPPS.

[0032] (2) The GLP-GIP-Fc fusion protein of this application:

[0033] The GLP-GIP-Fc fusion protein consists of a signal peptide, a GLP-GIP targeting peptide, a transfer peptide, and Fc. The amino acid sequence of Fc is shown in SEQ ID NO: 2: AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0034] The amino acid sequence of the signal peptide is shown in SEQ ID NO.5: METDTLLLWVLLLWVPGSTG;

[0035] The amino acid sequence of the linker peptide is shown in SEQ ID NO.6: GGGGSGGGGSGGGGS.

[0036] The amino acid sequence of the GLP-GIP-Fc fusion protein is shown in SEQ ID NO.7 below:

[0037] METDTLLLWVLLLWVPGSTGHGEGTFTSDYSILLDKIAQKAFVNWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0038] The nucleotide sequence of the GLP-GIP-Fc fusion protein is as shown in SEQ ID NO.10 below:

[0039] ATGGAAACCGATACCCTCTTGCTTTGGGTACTGCTCCTGTGGGTACCTGGCAGCACCGGTCACGGTGAGGGAACATTCACCTCTGATTACTCCATCTTGCTGGATAAGATCGCTCAGAAGGCCTTCGTGAATTGGTTGATCGCAGGTGGACCTAGTTCTGGAGCTCCACCACCAAGTGGGGGCGGTGGATCTGGGGGTGGAGGTTCTGGCGGTGGGGGGTCCGCTGAAAGCAAGTATGGACCTCCTTGCCCACCCTGTCCTGCACCTGAGGCTGCCGGAGGGCCCAGTGTGTTCTTGTTCCCACCAAAACCCAAGGATACCCTCATGATCAGCCGGACCCCAGAGGTAACCTGCGTGGTCGTGGACGTGAGCCAAGAGGACCCAGAAGTACAATTCAATTGGTATGTGGATGGAGTGGAGGTTCATAACGCAAAAACAAAGCCCCGCGAGGAACAGTTCAACTCCACATACCGCGTGGTCAGCGTTCTGACTGTACTGCACCAGGACTGGCTCAACGGAAAGGAGTACAAATGCAAAGTTTCTAACAAGGGTCTGCCATCCAGCATCGAAAAAACTATCTCCAAAGCAAAGGGGCAGCCTCGTGAACCTCAGGTATACACTCTCCCACCCAGCCAGGAGGAAATGACCAAAAATCAGGTATCTCTCACATGTCTGGTCAAGGGATTCTACCCATCCGACATTGCCGTGGAGTGGGAGTCAAATGGCCAGCCCGAAAATAACTATAAGACCACCCCACCCGTCTTGGACAGCGATGGCAGCTTTTTCCTGTACTCACGCCTGACAGTGGATAAGAGCCGTTGGCAGGAGGGTAACGTATTTTCTTGCAGCGTCATGCATGAAGCCCTGCATAATCATTACACCCAGAAGTCTCTGAGCCTGTCCCTGGGC。

[0040] (3) Design different GLP-GIP-lgG4 fusion proteins.

[0041] (a) GLP-GIP-IgG4 fusion protein:

[0042] The heavy chain sequence GLP-GIP-Hc consists of a signal peptide, a GLP-GIP targeting peptide, a transfer peptide, and IgG4 protein Hc.

[0043] Among them, the amino acid sequence of the heavy chain constant region of IgG4 is as SEQ ID Shown in NO.3: PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0044] The amino acid sequence of the heavy chain sequence GLP-GIP-Hc is shown in SEQ ID NO. 8:

[0045] METDTLLLWVLLLWVPGSTGHGEGTFTSDYSILLDKIAQKAFVNWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0046] The nucleotide sequence of the heavy chain sequence GLP-GIP-Hc was optimized using CHO cell-preferred codons, as shown in SEQ ID NO. 11:

[0047]

[0048] The light chain sequence GLP-GIP-Lc consists of a signal peptide, a GLP-GIP targeting peptide and a transfer peptide, and IgG4 protein Lc.

[0049] The amino acid sequence of the light chain constant region of IgG4 is shown in SEQ ID NO.4: APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0050] The amino acid sequence of the light chain GLP-GIP-Lc is shown in SEQ ID NO.9:

[0051] METDTLLLWVLLLWVPGSTGHGEGTFTSDYSILLDKIAQKAFVNWLIAGGPSSGAPPPSGGGGSGGGGSGGGGSAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0052] The nucleotide sequence of the light chain GLP-GIP-Lc was derived from artificially optimized CHO cell-preferred codons, as shown in SEQ ID NO. 12:

[0053] .

[0054] (b) Construction method of GLP-lgG4 (refer to CN202410464079.7)

[0055] The genes encoding the aforementioned fusion proteins were all obtained through chemical synthesis using codon-preferred sequences from artificially optimized CHO cells. The synthesized GLP-GIP-Hc was cloned into the HindIII / EcoRI site of pKS001 to obtain the vector pKS001-GLP-GIP-Hc; the synthesized GLP-GIP-Lc was cloned into the XbaI / NotI site of pKS001-GLP-GIP-Hc to obtain the IgG4 expression vector pKS-GLP-GIP-IgG4. The synthesized GLP-GIP-Fc was cloned into the HindIII / EcoRI site of pKS001 to obtain the vector pKS001-GLP-GIP-Fc. The construction method of GLP-IgG4 is described in patent CN202410464079.7. Standard molecular biology techniques were used in all experiments.

[0056] 2. Constructing a cell line stably expressing the fusion protein

[0057] The recombinant expression plasmid was linearized with PvuI and then transfected into CHO-K1 cells. The specific transfection procedure was described in the Lipofectamine 2000 (Invitrogen) manual. Forty-eight hours after transfection, cells were cultured in selection medium, specifically Hycell CHO medium containing 500 μg / ml G418 and 6 mM Glutamine, which was changed every 3 days. Cell viability initially decreased and then increased after transfection under drug influence. Once cell viability reached over 90%, the transfected cells were seeded into 96-well plates using a limiting dilution method for single-clone selection, with a cell density of 0.5 cells / well. After 14 days of static incubation, the expression supernatant of single-clone cells was detected using ELISA. Based on the test results, the 10 monoclonal cell lines with the highest expression levels were selected from each transfected cell line for gradual expansion culture. After culturing in 6-well plates for 3 days, the cells were counted and the protein expression level was detected. The 3 cell lines with the highest protein expression levels were selected for expansion culture in T125 shake flasks. After culturing for 3 days, the cells were counted and the protein expression level was detected. The cells with the highest expression levels were selected as stable cell lines.

[0058] 3. Expression and purification of fusion proteins

[0059] Cells expressing high levels of the fusion protein were cultured in serum-free CD OptiCHO medium, and the culture supernatant was collected after a certain period of time. The fusion protein was purified using the following Protein A affinity chromatography method: chromatographic column: AT Protein A Diamond affinity chromatography medium, column volume CV: 5 ml, flow rate: 5 ml / min, pressure limit: ≤0.3 MPa.

[0060] Pretreatment: Rinse with purified water for at least 5 CVs.

[0061] Equilibration: First, rinse one CV with elution buffer, then fully equilibrate at least 10 CVs with binding buffer before loading.

[0062] Sample loading: Load the filtered CHO suspension cell culture onto a well-equilibrated chromatography column, collect the sample flow, and take 100 μl of the flow for electrophoresis detection.

[0063] Washing: After loading the sample, increase the flow rate to 5 ml / min and wash with binding buffer for at least 6 CVs until the UV baseline is flat.

[0064] Elution: Elute with 100% elution buffer until the UV baseline is level. Collect each eluted fraction in separate tubes. At the same time, add an appropriate amount of neutralization buffer to each collection tube according to the collection volume and mix well.

[0065] 4. Therapeutic efficacy trials of fusion proteins

[0066] This experiment used male C57BL / 6JNifdc-DIO mice induced with a high-fat diet for 10 weeks. The mice were administered the drug via subcutaneous injection for 5 weeks at a frequency of 2 times per week.

[0067] The fusion proteins used in this experiment included the GLP-GIP-IgG4 fusion protein (GLP-GIP-IgG4) and GLP-GIP-Fc fusion protein (GLP-GIP-Fc) proposed in this application, as well as the GLP-IgG4 fusion protein from a prior patent. The fusion proteins were diluted to the desired concentrations using diluent (30mM PB, 0.04% Tween 80, 3% mannitol, pH 7.0) to obtain the respective fusion protein solutions. Twenty-five SPF-grade male C57BL / 6JNifdc-DIO mice, weighing 30-40g and induced for 10 weeks with Research Diet-D12492 feed, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., and were raised by Beijing Huilin Zegu Biotechnology Co., Ltd. Thirty-five mice were randomly divided into six experimental groups and one control group. Mice in the six experimental groups were injected with 75 μg of GLP-GIP-IgG4, 225 μg of GLP-GIP-IgG4, 75 μg of GLP-GIP-Fc, 225 μg of GLP-GIP-Fc, 75 μg of GLP-IgG4, and 225 μg of GLP-IgG4, respectively. The fusion protein solution for each of the six experimental groups was 100 μl. The control group was injected with 100 μl of physiological saline. The injection procedure was twice weekly for five consecutive weeks. Mice were weighed before each injection. After five weeks, the mice were sacrificed, and their livers were removed. The volume and weight of the livers were measured. The results are shown below. Figure 1-3 As shown.

[0068] Depend on Figure 1 The results showed that, compared with the blank control group, after five weeks, the GLP-GIP-Fc 75μg and GLP-GIP-Fc 225μg groups experienced weight loss of approximately 20% and 23.9%, respectively; GLP-GIP-IgG4 had a slightly better weight loss effect on mice than GLP-IgG4. Figures 2-3 The results showed that, compared with the blank control group, the liver volume and weight of the GLP-GIP-IgG4 225μg, GLP-GIP-Fc 75μg, GLP-GIP-Fc 225μg, and GLP-IgG4 225μg groups all decreased, with the GLP-GIP-Fc 75μg and GLP-GIP-Fc 225μg groups showing the most significant decreases in liver volume and weight.

[0069] In the above experiment, blood was collected from mice 16 hours after their last weighing and fasting. The total cholesterol (TC) assay kit (oxidase method) and triglyceride (TG) assay kit (oxidase method) (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.) were used to detect the total cholesterol and triglyceride levels in the serum of mice in the experimental group and the blank control group. The results of the total cholesterol and triglyceride detection are shown in Table 1.

[0070] Table 1

[0071]

[0072] As shown in Table 1, compared with the blank control group, the total cholesterol content in the experimental groups of this application, namely GLP-GIP-Fc 75μg and GLP-GIP-Fc 225μg, decreased significantly, with the former showing a more significant decrease.

[0073] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fusion protein having dual activities of GLP and GIP, characterized in that, It is a GLP and GIP dual-activity protein fused at the N-terminus of an Fc protein; the amino acid sequence of the GLP and GIP dual-activity protein is shown in SEQ ID No: 1; the amino acid sequence of the Fc is shown in SEQ ID NO. 2; and the amino acid sequence of the GLP and GIP dual-activity fusion protein is shown in SEQ ID NO.

7.

2. The GLP and GIP dual active fusion protein according to claim 1, wherein, The GLP and GIP dual-activity protein is fused at the N-terminus with a signal peptide.

3. The GLP and GIP dual active fusion protein according to claim 2, wherein, The amino acid sequence of the signal peptide is shown in SEQ ID NO.

5.

4. A nucleic acid encoding the GLP and GIP dual-activity fusion protein according to any one of claims 1 to 3.

5. The nucleic acid of claim 4, wherein The nucleotide sequence is shown in SEQ ID NO.

10.

6. A recombinant expression vector encoding the nucleic acid according to claim 5.

7. A method of preparing a cell line producing a fusion protein having dual activities of GLP and GIP, characterized by, It comprises the step of transfecting a mammalian cell with the nucleic acid encoding according to claim 4 or 5 to obtain a cell strain.

8. The method of claim 7, wherein, The mammal is a CHO cell; and the cell strain is obtained by constructing a recombinant expression vector containing the nucleic acid encoding according to claim 6, linearizing and transfecting a mammalian cell.

9. A cell strain producing the GLP and GIP dual-activity fusion protein obtained by the method according to claim 7 or 8.

10. Use of the GLP and GIP dual-activity fusion protein according to any one of claims 1 to 3 or the cell strain according to claim 9 in the preparation of a weight loss product or a medicament for treating fatty liver.

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