GLP-1 analog fusion protein and its application

Abstract

The present application relates to the field of medicine, and specifically to a GLP-1 analog fusion protein and its application. The GLP-1 analog fusion protein is obtained by fusing GLP-1 to the N-terminus of the IgG4 heavy chain constant region and the light chain constant region. The results of the study showed that the GLP-1 analog fusion protein of the present application has a significant effect on reducing the weight of mice. The volume and weight of the mouse liver decreased, and the NAS score and hepatic steatosis were significantly improved. Therefore, the present invention can be applied to drugs or products for weight loss or the treatment of fatty liver.

Description

Technical Field

[0001] The present application relates to the field of medicine, and in particular to GLP-1 analog fusion proteins and their applications. Background Art

[0002] Glucagon-like peptide (GLP) is a polypeptide expressed by the proglucagon gene. It exerts its biological effects by binding to corresponding G protein-coupled receptors. GLP can significantly increase phase I and II insulin secretion, reduce glucagon levels, and delay gastric emptying. Because its blood sugar-lowering effect is blood sugar-dependent and has the potential to reduce weight, GLP and its analogs are widely used in the treatment of type 2 diabetes and to alleviate insulin resistance caused by obesity.

[0003] Numerous weight-loss drugs have been approved overseas, encompassing various categories, such as mitochondrial uncouplers, sympathomimetics, cannabinoid 1 receptor (CB1) antagonists, lipase inhibitors, serotonin (5-HT) receptor agonists, and GLP-1 receptor agonists. However, most of these drugs were withdrawn from use after approval due to extremely serious adverse reactions, such as severe cardiovascular side effects (sibutramine, fenfluramine, dexfenfluramine hydrochloride, and Rainbow Pills), suicidal tendencies (rimonabant), and the risk of drug dependence and abuse (methamphetamine).

[0004] Among weight loss drugs approved domestically and internationally, GLP-1 receptor agonists have relatively few side effects. Therefore, it is necessary to develop a GLP-1 receptor agonist drug. Summary of the Invention

[0005] Based on the needs of the existing technology, the present application provides a GLP-1 analog fusion protein and its application.

[0006] First, the present application provides a GLP-1 analog fusion protein, which is obtained by fusing glucagon-like peptide GLP-1 to the N-termini of the IgG4 heavy chain constant region and the light chain constant region, respectively.

[0007] Preferably, the amino acid sequence of the glucagon-like peptide GLP-1 is shown as SEQ ID NO.1; the amino acid sequence of the IgG4 heavy chain constant region is shown as SEQ ID NO.4; and the amino acid sequence of the IgG4 light chain constant region is shown as SEQ ID NO.8.

[0008] Specifically, the N-termini of the IgG4 heavy chain constant region and the light chain constant region are fused to the glucagon-like peptide GLP-1 via a connecting peptide, respectively. Preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO.3.

[0009] The amino acid sequences of the glucagon-like peptide GLP-1 fused to the N-termini of the IgG4 heavy chain constant region and the light chain constant region are shown in SEQ ID NO. 5 and SEQ ID NO. 9, respectively;

[0010] Furthermore, a signal peptide is fused to the N-terminus of GLP-1; preferably, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 2. The present invention has determined through research that the signal peptide used can achieve a higher level of GLP-1 expression.

[0011] The present invention also provides a nucleic acid encoding the GLP-1 analog fusion protein.

[0012] Preferably, the nucleotide sequence encoding the glucagon-like peptide GLP-1 fused to the N-terminus of the IgG4 heavy chain constant region is shown as SEQ ID NO.6, and the nucleotide sequence encoding the glucagon-like peptide GLP-1 fused to the N-terminus of the IgG4 light chain constant region is shown as SEQ ID NO.10.

[0013] The present invention provides a recombinant expression vector encoding the nucleic acid.

[0014] The present invention provides a method for preparing a cell line producing a GLP-1 analog fusion protein, which is obtained by transfecting the encoding nucleic acid into mammalian cells, preferably, the mammalian cells are CHO cells; preferably, the cell line is obtained by constructing a recombinant expression vector containing the encoding nucleic acid, linearizing it, and then transfecting it into mammalian cells.

[0015] The present invention provides a cell line producing a GLP-1 analog fusion protein obtained by the method.

[0016] The present invention particularly provides the use of the GLP-1 analog fusion protein and the cell line in preparing weight loss products or drugs for treating fatty liver.

[0017] The present invention's research results show that compared with the control, the GLP-1 analog fusion protein of the present invention significantly reduced the weight of mice, with a decrease in both liver volume and weight, and significant improvements in NAS scores and hepatic steatosis. The present GLP-1 analog fusion protein can be used in drugs or products for weight loss and the treatment of fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , the structure of the GLP-1 analog fusion protein of the present application.

[0019] Figure 2 , Reduced SDS-PAGE electrophoresis diagram of the GLP-1 analog fusion protein of the present application.

[0020] Figure 3, body weight changes in mice treated with GLP-1 analog fusion protein.

[0021] Figure 4 The changes in liver volume of mice treated with GLP-1 analog fusion protein were shown in Table 2. Compared with the control group, "*" P < 0.01, "**" P < 0.05, and "***" P < 0.001.

[0022] Figure 5 , changes in liver weight of mice treated with GLP-1 analog fusion protein.

[0023] Figure 6 , NAS scores of GLP-1 analog fusion protein treatment. DETAILED DESCRIPTION

[0024] The present invention is described below through specific embodiments in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0025] Example 1: Construction of an expression vector for a GLP-1 analog fusion protein

[0026] The GLP-1 analog fusion protein of the present application is designed, wherein the amino acid sequence of the glucagon-like peptide GLP-1 polypeptide used is: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO. 1). The specific design is as follows:

[0027] (1) The GLP-1 analog fusion protein of the present application:

[0028] The amino acid sequence of the heavy chain IgG4-Hc is as follows:

[0029] PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

[0030] LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLFPPK

[0031] PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL

[0032] TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC

[0033] LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV

[0034] MHEALHNHYTQKSLSLSLG (SEQ ID NO. 4).

[0035] The heavy chain fusion protein GLP-Hc is composed of a GLP-1 polypeptide connected to the N-terminus of the heavy chain IgG4-Hc via a linker peptide (GGGGSGGGGSGGGGS, SEQ ID NO. 3). Its amino acid sequence is as follows:

[0036] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSPSVFPLAPCSRS

[0037] TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT

[0038] YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPAALGGPSVFLFPPKPKDTLMISRTPEVTCV

[0039] VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK

[0040] CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE

[0041] SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL

[0042] SLG (SEQ ID NO. 5).

[0043] Through research and comparison, in this example, a signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO. 2) is further connected to the N-terminus of the GLP-1 polypeptide.

[0044] Based on the above amino acid sequence, codon optimization was performed according to the codon preference of CHO cells, which is more conducive to the expression of GLP-Hc fusion protein in CHO cells. The optimized GLP-Hc encoding nucleotide sequence is as follows:

[0045]

[0046] The nucleotide sequence of the signal peptide (after screening) connected to the N-terminus of GLP-Hc is: ATGGGATGGTCATGTAT TATTCTGTTTCTCGTCGCCACAGCCACAGGCGTACACTCA (SEQ ID NO. 7).

[0047] The amino acid sequence of the light chain IgG4-Lc is as follows:

[0048] APSVFIFPPSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSKDSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC (SEQ ID NO. 8).

[0049] The light chain fusion protein GLP-Lc is composed of a GLP-1 polypeptide connected to the N-terminus of the light chain IgG4-Lc through a linker peptide (GGGGSGGGGSGGGGS). Its amino acid sequence is as follows:

[0050] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 9).

[0051] In this example, a signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO. 2) is further connected to the N-terminus of the GLP-1 polypeptide.

[0052] Based on the above amino acid sequence, codon optimization was performed according to the codon preference of CHO cells, which is more conducive to the expression of GLP-Lc fusion protein in CHO cells. The optimized GLP-Lc encoding nucleotide sequence is as follows:

[0053] CACGGTGAGGGAACCTTCACTAGTGACGTGAGTAGTTATCTGGAGGAACAGGCCGCCAAAGAGTTCATCGCCTGGCTCGTCAAAGGAGGCGGCGGAGGTGGAGGGTCAGGAGGAGGGGGATCAGGAGGGGGCGGCTCTGCACCCTCTGTCTTTATCTTTCCCCCTAGCGATGAGCAGCTTAAATCCGGGACCGCAAGCGTCGTCTGTCTGCTGAACAATTTTTAT CCCAGAGAGGCCAAGGTGCAGTGGAAAGTGGACAACGCTCTCCAGAGTGGCAACAGTCAGGAGAGCGTAACAGAGCAGGATAGCAAGGACTCTACTTACTCCCTTAGCTCCACTCTGACCCTGTCAAAAGCTGACTATGAGAAGCACAAAGTGTACGCATGCGAGGTGACACATCAGGGTTTGAGCTCCCCAGTCACAAAGTCTTTTAACCGAGGAGAGTGT(SEQ ID NO.10).

[0054] The nucleotide sequence of the signal peptide (after screening) connected to the N-terminus of GLP-Lc is the same as above (SEQ ID NO. 7).

[0055] The genes encoding the aforementioned fusion proteins were all artificially optimized for CHO cell-preferred codons, and the full-length sequences were obtained by chemical synthesis. The synthesized GLP-Hc nucleotide sequence, including a signal peptide, was cloned into the HindIII / EcoRI sites of pKS001 to generate the vector pKS001-GLP-Hc. The synthesized GLP-Lc nucleotide sequence, including a signal peptide, was cloned into the XbaI / NotI sites of pKS001-GLP-Hc to generate the fusion protein expression vector pKS-GLP-IgG4. The relevant experimental methods used were conventional molecular biology methods.

[0056] (2) Dulaglutide (trade name Eli Lilly) was purchased directly. The amino acid sequence of dulaglutide is:

[0057] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(dlu-Fc)(SEQ IDNO.11).

[0058] Example 2: Construction of a GLP-1 analog fusion protein stable expression cell line

[0059] The recombinant expression plasmid pKS-GLP-IgG4 was linearized with PvuI and transfected into CHO-K1 cells. For the specific transfection procedure, refer to the instructions for Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were cultured in a selection medium containing 25 μM L-methionine sulfoximide (MSX), which was replaced every three days. After transfection, cell viability initially decreased and then increased under the action of the drug. When cell viability rose to over 90%, the transfected cells were plated in 96-well plates using the limiting dilution method for monoclonal screening at a cell density of 0.5 cells per well. After 14 days of static culture in an incubator, the expression of the fusion protein in the culture supernatant of the monoclonal cells was determined by ELISA. According to the test results, the 10 monoclonal cell lines with the highest expression levels were screened for each transfected cell and gradually expanded for culture. After 3 days of culture in 6-well plates, 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 3 days of culture, the cells were counted and the protein expression level was detected. The cells with the highest expression level were selected as stable cell lines.

[0060] Example 3. Expression and purification of GLP-1 analog fusion protein

[0061] High-expressing cells were cultured in serum-free CDOptiCHO 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: chromatography column: ATProteinADiamond affinity chromatography medium, column volume CV: 5 ml, flow rate: 5 ml / min, pressure limit: ≤0.3 MPa.

[0062] Pretreatment: Rinse with purified water for at least 5 CV.

[0063] Equilibration: First elute with elution buffer for 1 CV, then fully equilibrate with binding buffer for at least 10 CV before loading.

[0064] Sampling: Load the filtered culture supernatant onto the equilibrated chromatography column, collect the flow-through, and sample 100 μl for electrophoresis detection.

[0065] After loading, increase the flow rate to 5 ml / min and elute with binding buffer for at least 6 CV until the UV baseline is flat.

[0066] Elution: Use 100% elution buffer to elute until the UV baseline is flat, collect the eluted components in separate tubes, and add an appropriate amount of neutralization buffer to each collection tube according to the collection volume and mix well.

[0067] The GLP-1 analog fusion protein was purified by reducing SDS-PAGE electrophoresis. Figure 2 shown.

[0068] Example 4: Effects of different signal peptides on the expression of GLP-1 analog fusion proteins

[0069] An expression vector for a GLP-1 analog fusion protein containing a signal peptide was constructed according to the method of Example 1 above, wherein the heavy chain sequence GLP-Hc (SEQ ID NO. 6) and the light chain sequence GLP-Lc (SEQ ID NO. 10) in the expression vector for the GLP-1 analog fusion protein were the same as those in Example 1, with the only difference being the sequence of the N-terminal fusion signal peptide.

[0070] A cell line stably expressing a GLP-1 analog fusion protein containing a signal peptide was constructed according to the method of Example 2. Expression of the GLP-1 analog fusion protein containing a signal peptide was performed according to the method of Example 3. High-expressing cells were cultured in serum-free CDOptiCHO medium, and the culture supernatant was collected after 14 days.

[0071] The expression level of GLP-1 analog fusion protein in the culture supernatant was determined by ELISA. The specific steps of the ELISA assay include: diluting anti-human IgG (Fc-specific) goat antibody (I2136, Sigma) at a 1:2000 dilution in PBS, adding 100 μl / well of the ELISA plate. Incubate overnight at 4°C for coating. After washing the plate three times with PBST, add 50 g / L BSA blocking buffer at 200 μl / well and incubate at 37°C for 2 h. After washing the plate three times with PBST, add cell expression supernatant diluted at appropriate concentrations and standard curve protein at 100 μl / well and incubate at 37°C for 1 h. After washing the plate three times with PBST, add secondary antibody, anti-human IgG (Fc-specific) peroxidase goat antibody (A0170, Sigma) at a 1:8000 dilution in PBS, adding 100 μl / well and incubating at 37°C for 1 h. After washing the plate three times with PBST, add 100 μl / well of TMB substrate developer and incubate at room temperature in the dark for 10-15 minutes. Stop the reaction by adding 50 μl / well of stop solution. Read the plate in a microplate reader at 450 nm. Calculate the expression level of the GLP-1 analog fusion protein in the culture supernatant using the standard curve.

[0072] Table 1 Expression levels of GLP-1 analog fusion proteins in culture supernatants obtained with different signal peptides

[0073] signal peptide Expression level of GLP-1 analog fusion protein (g / L) MGWSCIILFLVATATGVHS(SEQ ID NO.2) 2.5 MPLLLLLPLLWAGALA 1.2 MAHIRGLWLPGCLALAALCSLVHS 1.5

[0074] From the results in Table 1, it can be seen that the expression level of the GLP-1 analog fusion protein obtained by the signal peptide (SEQ ID NO. 2) finally screened and used in this application is much greater than the expression levels of the GLP-1 analog fusion proteins obtained by the other two signal peptides.

[0075] Example 5: Therapeutic efficacy test of GLP-1 analog fusion protein

[0076] In this experiment, C57BL / 6JNifdc-DIO male mice induced by high-fat diet for 10 weeks were selected, and the efficacy of the drug was investigated in this model mouse model by subcutaneous injection for 5 weeks with a dosing frequency of 2 times / week.

[0077] The fusion proteins used in this experiment include the GLP-1 analog fusion protein (GLP-lgG4) of this application and Eli Lilly's dulaglutide (dlu-Fc, purchased directly). The fusion proteins used were diluted to the required concentration using diluent (30mMPB, 0.04% Tween80, 3% mannitol, pH 7.0) to obtain each fusion protein solution. 25 SPF-grade C57BL / 6JNifdc-DIO male mice weighing 30-40g were induced for 10 weeks using ResearchDiet-D12492 feed and were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. and raised by Beijing Huilinzegu Biotechnology Co., Ltd. 25 mice were randomly divided into 4 experimental groups and 1 blank control group. The mice in the 4 experimental groups were injected with 40μg dlu-Fc, 75μg dlu-Fc, 40μg GLP-IgG4, and 75μg GLP-IgG4, respectively. The injection volume of the fusion protein solution in the 4 experimental groups was 100μl. The blank control group was injected with 100μl normal saline. Injection procedure: 2 injections per week for 5 consecutive weeks. The mice were weighed before each injection. The results are shown in the table. Figure 3 After 5 weeks, the mice were killed and their livers were removed. The volume and weight of the livers were measured. Figures 4-5 The liver tissues were stained with HE and Sirius red. After taking pictures of each mouse liver section, NAS score was performed (NAS score = fatty degeneration score + inflammation score). The results are shown in Figure 6 .

[0078] Figure 3 The results showed that after 5 weeks, the GLP-IgG4 75μg group had a 14.7% weight loss compared to the blank control group. Figures 4-5 As shown in the results, compared with the blank control group, the liver volume of the dlu-Fc 75μg, GLP-IgG4 40μg and GLP-IgG4 75μg groups decreased, and the liver volume and weight of the GLP-IgG4 75μg group decreased most significantly.

[0079] Figure 6 The results showed that the NAS scores of the GLP-IgG4 40μg and GLP-IgG4 75μg groups were significantly better than those of the blank control group, and the NAS score and hepatic steatosis of the GLP-IgG4 75μg group were significantly improved.

[0080] Finally, it should be noted that the foregoing description is merely a preferred embodiment of the present invention and is 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 will be able to modify the technical solutions described in the foregoing embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A GLP-1 analog fusion protein, characterized in that: It is obtained by fusing glucagon-like peptide GLP-1 to the N-terminus of the IgG4 heavy chain constant region and light chain constant region respectively; The amino acid sequence of the glucagon-like peptide GLP-1 is shown in SEQ ID NO.1; The amino acid sequence of the IgG4 heavy chain constant region is shown in SEQ ID NO.4; the amino acid sequence of the IgG4 light chain constant region is shown in SEQ ID NO.

8.

2. The GLP-1 analog fusion protein according to claim 1, wherein The N-termini of the IgG4 heavy chain constant region and the light chain constant region are fused with glucagon-like peptide GLP-1 via connecting peptides.

3. The GLP-1 analog fusion protein according to claim 2, wherein The amino acid sequence of the connecting peptide is shown in SEQ ID NO.

3.

4. The GLP-1 analog fusion protein according to claim 3, wherein The amino acid sequences obtained by fusing glucagon-like peptide GLP-1 to the N-termini of the IgG4 heavy chain constant region and the light chain constant region are shown in SEQ ID NO. 5 and SEQ ID NO. 9, respectively.

5. The GLP-1 analog fusion protein according to claim 1, wherein The N-terminus of the glucagon-like peptide GLP-1 is fused with a signal peptide.

6. The GLP-1 analog fusion protein according to claim 5, characterized in that The amino acid sequence of the signal peptide is shown in SEQ ID NO.

2.

7. A nucleic acid encoding the GLP-1 analog fusion protein according to any one of claims 1 to 6.

8. The encoding nucleic acid according to claim 7, wherein The nucleotide sequence encoding the glucagon-like peptide GLP-1 fused to the N-terminus of the IgG4 heavy chain constant region is shown in SEQ ID NO.6, and the nucleotide sequence encoding the glucagon-like peptide GLP-1 fused to the N-terminus of the IgG4 light chain constant region is shown in SEQ ID NO.

10.

9. The recombinant expression vector encoding the nucleic acid according to claim 7 or 8.

10. A method for preparing a cell line producing a GLP-1 analog fusion protein, characterized in that: The method is obtained by transfecting the encoding nucleic acid according to claim 7 or 8 into mammalian cells.

11. The method according to claim 10, wherein The mammalian cell is a CHO cell.

12. The method according to claim 10 or 11, wherein: The method is obtained by constructing a recombinant expression vector containing the encoding nucleic acid according to claim 5 or 6, linearizing the vector and then transfecting the vector into mammalian cells.

13. A cell line producing a GLP-1 analog fusion protein obtained by the method according to any one of claims 10 to 12.

14. Use of the GLP-1 analog fusion protein according to any one of claims 1 to 6 and the cell line according to claim 13 in the preparation of a medicament for treating fatty liver.

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