GLP-1R / GIPR fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a GLP-1 / GIP fusion protein, and particularly relates to a preparation method and application of a fusion protein activated by a GLP-1R / GIPR double receptor or relayed by the activation of the GLP-1R / GIPR double receptor and activated by the GLP-1R / GIPR double receptor and inhibited by the GLP-1R / GIPR double receptor. Wherein the GLP-1 is a DPP-IV enzyme digestion resisting active sequence and shows GLP-1R activation activity, and when the GIP sequence is DPP-IV enzyme digestion resisting activity, the molecule shows GLP-1R / GIPR double receptor activation activity; when a GIP sequence has non-resistant DPP-IV enzyme digestion activity, after the molecule enters a human body, GLP-1R / GIPR double receptor activation is firstly realized, and then under the action of DPP-IV, the molecule is converted into GLP-1R activation / GIPR inhibition activity, and a weight reduction effect better than that of GLP-1R / GIPR double activation is generated.

Description

Technical Field

[0001] The present invention relates to a GLP-1 / GIP fusion protein, and in particular to the preparation and use of a fusion protein of GLP-1R / GIPR dual receptor activation or GLP-1R / GIPR dual receptor activation relayed by GLP-1R activation / GIPR inhibition. Background Art

[0002] During normal physiological feeding process, humans or animals can produce intestinal insulins GLP-1 and GIP in intestinal cells. GLP-1 is a polypeptide of 31 amino acid residues, and GIP is a polypeptide of 42 amino acid residues. Incretins bind to receptors and play a role in human satiety, insulin secretion, nutrient metabolism, etc. The half-life of GLP-1 and GIP in humans or animals is very short, generally 2-3 minutes. This is mainly because DPP-IV in humans or animals removes and inactivates the two N-terminal amino acid residues of GLP-1 and GIP.

[0003] The effects of GLP-1 and GIP are achieved through GLP-1R and GIPR. Both GLP-1R and GIPR are type B G protein-coupled receptors, and like all G protein-coupled receptors, they have seven transmembrane helical domains. Activation of GLP-1R leads to activation of adenylate cyclase to form cAMP, which activates the PKA signaling pathway and secretes insulin. Activation of GLP-1R also inhibits β-cell apoptosis; the effects of GLP-1 receptor activation also include inhibition of glucagon secretion and inhibition of gastric emptying, appetite suppression, and cardioprotection. Activation of GIPR leads to activation of adenylate cyclase, which increases intracellular cAMP levels and Ca 2+The level of GLP-1R is increased, and the expression of downstream genes is regulated through signaling pathways such as PI3K, PAK, and PKB. GIPR activation promotes insulin secretion, thereby reducing blood sugar levels. At the same time, GIPR can also affect the storage and metabolism of fat cells. GLP-1R and GIPR are co-expressed in the β cells of the pancreatic islets and share signaling pathways. The two receptors send signals by activating adenylate cyclase, leading to an increase in intracellular cAMP, activating the PKA and EPAC2 pathways, thereby increasing insulin secretion, β cell proliferation and survival or anti-apoptosis. The two GPCRs act directly on beta-arrestins, promote receptor internalization, terminate G protein-mediated signaling, and recycle and block the action of Gαs. However, there are also differences in the signaling pathways of GLP-1R and GIPR: activation of the EGFR pathway, leading to activation of PI3K and increased β-cell survival, is only activated by GLP-1R but not GIPR; GLP-1R can be internalized by acting on β-arrestins or Gαq, and the internalization process can be affected by a variety of ligands, while the internalization process of GIPR is completely dependent on β-arrestins; activation of GIPR in β cells has been shown to lead to MAPK-induced signaling pathways, while this effect does not exist for GLP-1R.

[0004] Studies have shown that in people with type 2 diabetes, when they ingest glucose, the change in GIP concentration is not significantly different from that of normal people, while GLP-1 changes significantly, indicating that insufficient secretion of GLP-1 is the main contradiction of type 2 diabetes. In response to this phenomenon, the research and development of GLP-1 has become a focus. However, the half-life of GLP-1 in the human body is 2 minutes, which is obviously not suitable as a therapeutic drug. Therefore, the development of long-acting analogs of GLP-1 has become a necessity. Liraglutide is a long-acting analog of GLP-1 for clinical use developed by Novo Nordisk of Denmark. It is administered once a day and has a homology of up to 97% with human GLP-1. It is a derivative obtained by mutating the 34th lysine to arginine and modifying the 26th lysine in the natural GLP-1 structure. Semaglutide is another long-acting GLP-1 analogue for clinical use developed by Novo Nordisk of Denmark. This derivative is based on liraglutide, with the 8th alanine mutated to the non-natural amino acid Aib, while increasing the length and hydrophilicity of the side chain, thereby significantly extending its half-life and achieving once-a-week dosing. This molecule uses biological fermentation to produce GLP-1 (9-37), then modifies the fatty acid chain on the side chain, couples the dipeptide at the N-terminus, and finally cleaves and purifies to obtain the target product. Dulaglutide is a long-acting GLP-1 analogue developed by Eli Lilly and Company. It is prepared by replacing the 8th Ala of the natural GLP-1 (7-37) with Gly, the 22nd Gly with Glu, and the 36th Arg with Gly, and then by "-(GGGGS) 3-" coupling bridge is fused to the Fc fragment containing 227 amino acids, thereby significantly extending its half-life. The molecule is produced by biological fermentation and has no racemization problem in chemical solid phase synthesis.

[0005] GLP-1 derivatives used alone have a good effect on reducing body weight. The research results of Gault VA et al. show that the combined use of long-acting derivatives of GLP-1 and long-acting derivatives of GIP has more significant hypoglycemic and weight-reducing activities in diabetic and obese animal models than the use of long-acting derivatives of GLP-1 or GIP alone. The main active functional areas of GLP-1 and GIP are at the N-terminus, and GLP-1 and GIP are highly homologous at the N-terminus. Based on the above facts, patents such as US20190218270A1, US010093713B2, US011008375B2, and US009474780B2 have designed a series of GLP-1R and GIPR single-molecule dual receptor activators based on GLP-1 and GIP. Tipoline is the first single-molecule dual receptor activator of GLP-1R and GIPR to be marketed. It showed better weight loss activity in a head-to-head clinical study compared with semaglutide. The polypeptide molecule contains two non-natural amino acid residues and is synthesized using solid-phase chemistry, which has a complex production process.

[0006] As mentioned above, the combination of GLP-1 and GIP or the single-molecule dual receptor activators of GLP-1R and GIPR have shown better weight loss activity than GLP-1R agonists. The research results of Bin Yang et al. showed that the combination of semaglutide and high-dose GIPR inhibitory peptide can produce better weight loss effect than semaglutide. AMG-133 designed by Amgen is an anti-GIPR antibody that connects GLP-1 at the E384 C mutation site to form a GLP-1R activation / GIPR inhibition protein. Phase II clinical results showed that this molecule has good weight loss activity. Summary of the invention

[0007] Based on the above design ideas and existing problems, the present invention designs a GLP-1 / GIP fusion protein molecule according to the structure of GLP-1 and GIP and the enzymatic cleavage characteristics of DPP-IV. Wherein GLP-1 is a sequence of resistance to DPP-IV enzymatic cleavage activity, showing GLP-1R activation activity. When the GIP sequence is a sequence of resistance to DPP-IV enzymatic cleavage activity, the molecule shows GLP-1R / GIPR dual receptor activation activity; when the GIP sequence is a sequence of non-resistance to DPP-IV enzymatic cleavage activity, after entering the human body, the molecule first realizes GLP-1R / GIPR dual receptor activation, and then under the action of DPP-IV, the molecule is converted into GLP-1R activation / GIPR inhibition activity. More importantly, the above fusion protein molecule uses minimal mutations, which can significantly reduce immunogenicity.

[0008] In order to achieve the above-mentioned object, the first aspect of the present invention provides a GLP-1 / GIP fusion protein sequence, the amino acid sequence of which is as follows: HGEGTFTSDVSSYLEX 1 QAAKEFIAWLVKGX 2 GGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFX 3 STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYX 4 TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLX 5 GGGGSGGGGSGGGGSGGGGSDDDDKYX 6 EX 7 TFISDYSIAMDKIX 8 QQQ 9 X 10 VNWLLAQ 11 GGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFX 12 STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLX 13 SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG in: X 1 is G or E; X 2 is R or G; X 3 is N, Q, A or G; X 4 is K or C; X 5is GGGGS, GGGGP, ASGPNQ, VPRGS, or SRSRR; X 6 A or G; X 7 is G or A; X 8 is R or H; X 9 D or E; X 10 is F or W; X 11 is K or R; X 12 is N, Q, A or G; X 13 It is D or C.

[0009] Furthermore, X 1 G is preferred.

[0010] Furthermore, X 2 G is preferred.

[0011] Furthermore, X 3 N, Q or G is preferred.

[0012] Furthermore, X 3 Q or G is preferred.

[0013] Furthermore, X 4 C is preferred.

[0014] Furthermore, X 5 ASGPNQ or VPRGS is preferred.

[0015] Furthermore, X 6 A is preferred.

[0016] Furthermore, X 7 G or A is preferred.

[0017] Furthermore, X 7 G is preferred.

[0018] Furthermore, when conducting animal experiments on mice, X 8 R is preferred. When conducting human experiments, X 8 H is preferred.

[0019] Furthermore, X 9 D is preferred.

[0020] Furthermore, X 10 F is preferred.

[0021] Furthermore, X 11 K is preferred.

[0022] Furthermore, X 12 N, Q or G is preferred.

[0023] Furthermore, X 12 Q or G is preferred.

[0024] Furthermore, X 13 C is preferred.

[0025] Preferably, the GLP-1 / GIP fusion protein is one of the following sequences: SEQ ID NO.1: .

[0026] SEQ ID NO.2: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLASGPNQGGGGSGGGGSGGGGSGGGGSDDDDKYGEGTFISDYSIAMDKIRQQDFVNWLLAQKGGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0027] SEQ ID NO.3: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLASGPNQGGGGSGGGGSGGGGSGGGGSDDDDKYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0028] SEQ ID NO.4: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGPGGGGSGGGGSGGGGSGGGGSDDDDKYAEGTFISDYSIAMDKIRQQDFVNWLLAQKGGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0029] SEQ ID NO.5: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSGGGGSGGGGSDDDDKYAEGTFISDYSIAMDKIRQQDFVNWLLAQKGGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0030] SEQ ID NO.6: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLASGPNQGGGGSGGGGSGGGGSGGGGSDDDDKYAEGTFISDYSIAMDKIRQQDFVNWLLAQKGGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLCSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0031] SEQ ID NO.7: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLASGPNQGGGGSGGGGSGGGGSGGGGSDDDDKYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLCSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0032] SEQ ID NO.8: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSGGGGSGGGGSDDDDKYAEGTFISDYSIAMDKIRQQEFVNWLLAQKGGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0033] SEQ ID NO.9: 。

[0034] SEQ ID NO.10: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLVPRGSGGGGSGGGGSGGGGSGGGGSDDDDKYAEATFISDYSIAMDKIHQQDFVNWLLAQKGGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLCSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0035] SEQ ID NO.11: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFCSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLSRSRRGGGGSGGGGSGGGGSGGGGSDDDDKYAEATFISDYSIAMDKIHQQDFVNWLLAQKGGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFCSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLCSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0036] SEQ ID NO.12: .

[0037] The second aspect of the present invention provides a method for preparing a GLP-1 / GIP fusion protein, using the following preparation process: (1) Synthesizing a coding gene, wherein the gene coding comprises SEQ ID NO.1-SEQ ID NO.12, and an EK restriction site is added to the N-terminus to construct an expression vector; (2) transforming the recombinant expression vector carrying the coding gene into an Escherichia coli host to obtain a recombinant engineered bacterium; (3) Fermentation of recombinant engineered bacteria to induce the expression of inclusion body proteins in cells; (4) The bacterial cells are broken by high-pressure homogenization, the inclusion bodies are collected by centrifugation, and the inclusion bodies are dissolved and renatured; (5) affinity purification of the renatured protein onto a nickel column; (6) The purified protein was dialyzed with EK enzyme digestion buffer and then digested with EK enzyme; (7) Select FAPα, Furin or Thrombin for enzyme cleavage according to the sequence; (8) Purify and obtain active GLP-1 and GIP fusion protein.

[0038] Furthermore, the GLP-1 / GIP fusion protein provided by the present invention is subjected to prokaryotic expression, renaturation, enzyme digestion and purification to obtain a fusion protein having the following Structure-I structure: .

[0039] Furthermore, in the GLP-1 / GIP fusion protein provided by the present invention, when X6 is glycine, the Structure-I structural protein obtained after prokaryotic expression, renaturation, enzyme cleavage and purification of the fusion protein exhibits GLP-1R / GIPR dual receptor activation activity.

[0040] Furthermore, in the GLP-1 / GIP fusion protein provided by the present invention, when X6 is alanine, the Structure-I protein obtained after prokaryotic expression, renaturation, enzyme cleavage and purification of the fusion protein first exhibits GLP-1R / GIPR dual receptor activation after entering the human or mouse body, and is converted into GLP-1R activation / GIPR inhibition activity under the action of DPP-IV enzyme cleavage.

[0041] Furthermore, in the GLP-1 / GIP fusion protein provided by the present invention, when X6 is alanine, the fusion protein is expressed in prokaryotes, renatured, enzymatically digested and purified to obtain the Structure-I structural protein, which, after entering the human or mouse body, exhibits GLP-1R / GIPR dual-target activation relay GLP-1R activation / GIPR inhibition under the action of DPP-IV.

[0042] In the third aspect of the present invention, GLP-1 / GIP fusion protein has better weight loss activity in DIO mice than semaglutide and dulaglutide, and statistically under the same dosage conditions, there are significant differences.

[0043] In the fourth aspect of the present invention, GLP-1 / GIP is fused with Fc, which can significantly prolong the half-life in human or mouse plasma and enable once-a-week administration.

[0044] The present invention provides a GLP-1 / GIP fusion protein and a composition thereof for treating type 2 diabetes, weight loss or fatty liver lesions.

[0045] Compared with the prior art, the present invention provides the following advantages: The GLP-1 / GIP fusion protein provided by the present invention can be prepared by prokaryotic expression, has a simple preparation process, low production cost, and is suitable for large-scale production.

[0046] The GLP-1 / GIP fusion protein provided by the present invention has as few amino acid residue mutations as possible, has the characteristics of low immunogenicity, and can significantly reduce the side effect of antibodies produced during long-term use of the drug.

[0047] Compared with single-target GLP-1R activators such as dulaglutide, the GLP-1R / GIPR dual-target activator provided by the present invention has a better weight loss effect.

[0048] Compared with GLP-1R single-target activators such as dulaglutide and semaglutide, the GLP-1R / GIPR dual-target activation relay GLP-1R activation / GIPR inhibition fusion protein provided by the present invention has better weight loss effect, better treatment effect of liver fatty lesions, and more significant effect of reducing triglyceride and total cholesterol levels in the liver, as shown by animal experimental results.

[0049] Compared with the GLP-1R / GIPR dual-target activator provided by the present invention, the GLP-1R / GIPR dual-target activation relay GLP-1R activation / GIPR inhibition fusion protein provided by the present invention can produce a more significant weight loss effect in obese mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the SDS-PAGE detection diagram of fusion protein expression.

[0051] Figure 2 This is the SDS-PAGE detection diagram of GLP-1 / GIP fusion protein after renaturation digestion.

[0052] Figure 3 This is a graph showing the results of a glucose tolerance experiment on normal mice with SEQ ID NO.4.

[0053] Figure 4 This is a graph showing the weight loss results of GLP-1R / GIPR dual receptor activation relay GLP-1R activation / GIPR inhibition in DIO mice given once a day, where: GLP-1-Fc is Dulaglutide; SEQ ID NO.4 is GLP-1R / GIPR dual activation relay GLP-1R activation / GIPR inhibition; SEQ ID NO.6 is a GLP-1R / GIPR dual activation relay GLP-1R activation / GIPR inhibition.

[0054] Figure 5 HE staining results of liver sections of GLP-1R / GIPR dual receptor activation relay GLP-1R activation / GIPR inhibition in DIO mice given once a day, where: Figure 5A is the control chart of Saline group; Figure 5 B is the liver section image after treatment with Dulaglutide group; Figure 5 C is a liver section image of the SEQ ID NO.4 group after treatment; Figure 5 D is a liver section image of the SEQ ID NO.6 group after treatment.

[0055] Figure 6 Figure 3. Results of hepatic triglyceride and total cholesterol levels in DIO mice dosed once daily with GLP-1R / GIPR dual receptor activation relay GLP-1R activation / GIPR inhibition.

[0056] Figure 7 Figure 3. Weight loss results of GLP-1R / GIPR dual receptor activation relayed by GLP-1R activation / GIPR inhibition in DIO mice given once a week.

[0057] Figure 8 The results of liver sections of GLP-1R / GIPR dual receptor activation relay GLP-1R activation / GIPR inhibition in DIO mice after weekly administration, where: Figure 8 A is the control chart of Saline group; Figure 8 B is a liver section image after treatment with Semaglutide; Figure 8 C is a liver section image of the SEQ ID NO.4 group after treatment; Figure 8 D is a liver section image of the SEQ ID NO.6 group after treatment.

[0058] Fig. 9 This is a graph showing the weight loss results of GLP-1R / GIPR dual receptor activation and GLP-1R / GIPR dual activation. GLP-1R activation / GIPR inhibition in DIO mice given once a week, where: SEQ ID NO.2 is GLP-1R / GIPR dual receptor activation; SEQ ID NO.4 is a GLP-1R / GIPR dual activation relay GLP-1R activation / GIPR inhibition. DETAILED DESCRIPTION Example 1

[0059] Preparation of GLP-1 and GIP fusion protein (1) Vector construction SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.12 protein sequences were selected, GSDDDDK was added to the N-terminus of all protein sequences, and gene optimization was performed according to the codon preference of Escherichia coli to obtain a DNA sequence. The sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and then digested and inserted into the pET28a(+) expression vector, with the digestion sites being NdeI and HindIII.

[0060] (2) Fusion protein expression The constructed expression vector plasmid was transformed into E. coli BL21 (DE3), and a single clone was selected and cultured in LB at 37°C and 250RPM for 16-24 hours. The clone was inoculated into 1 liter of LB medium at a 2% inoculum and cultured at 37°C until OD600 was about 0.8. IPTG was added for induction for 4 hours. The results are shown in the table. Figure 1 As shown, wherein: M is Marker, lane 1 is negative control, lane 2 is SEQ ID NO.1, lane 3 is SEQ ID NO.2, lane 4 is SEQ ID NO.4, lane 5 is SEQ ID NO.6, and lane 6 is SEQ ID NO.12. From the results, it can be seen that the constructed protein sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQID NO.12 are successfully expressed.

[0061] (3) Renaturation, enzyme digestion and purification SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6 were selected for large-scale induced expression, and the induced expression Escherichia coli cells were collected by centrifugation, washed with 0.05 M PBS, broken by a high-pressure homogenizer, and the inclusion body precipitate was collected by centrifugation. According to 40.0 mL of 1.0 g of inclusion bodies, dissolution buffer (20 mM Tris-HCl, pH 9.0, 8 M urea, 2.5 mM DTT) was added, and the mixture was stirred at room temperature for 2 hours until the inclusion bodies were dissolved, and then the supernatant was collected by centrifugation (7000 × g, 4°C, 30 min, and the supernatant was collected by centrifugation). The inclusion body solution was placed in a dialysis bag and dialyzed to remove DTT (dialysis solution: 20 mM Tris-HCl, pH 9.0, 8 M urea) for 8 hours; the dialysate was removed, and renaturation buffer I (20 mM Tris-HCl, pH 9.0; 2 mM Cysteamine, 2 mM Cystamine, 4 M urea) was added for 4 hours; the renaturation buffer I was removed, and renaturation buffer II (20 mM Tris-HCl, pH 9.0; 2 mM Cysteamine, 2 mM Cystamine, 2 M urea) was added for 16-24 hours. After the renaturation was completed, affinity purification was performed using a nickel column to obtain a well-renatured fusion protein, and the purified fusion protein was dialyzed to remove imidazole. The above-mentioned renatured fusion protein was added with EK enzyme (EK enzyme with His6 tag) at a ratio of 3000:1 for enzyme digestion. The enzyme digestion temperature was 16°C for 16 hours. After the digestion was completed, affinity purification was performed using a nickel column. The flow-through was collected and dialyzed with FAPα digestion buffer. After the dialysis was completed, FAPα was added for digestion. The digestion temperature was 23°C. After the digestion was completed, purification was performed to obtain the target protein. The renatured and digested proteins were detected by SDS-PAGE. The results are as follows Figure 2 As shown, lane 1, lane 3, and lane 5 are for renatured protein detection, and there is a target band at 66kD; lane 2, lane 4, and lane 6 are for protein detection after enzyme cleavage, forming two bands, indicating that the enzyme cleavage is complete, forming the N-terminal free active forms of the two sequences of GLP-1 and GIP, and obtaining GLP-1 and GIP fusion protein. The active proteins of SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6 can be used for subsequent experiments, wherein SEQ ID NO.2 is GLP-1R / GIPR dual receptor activation, and SEQ ID NO.4 and SEQ ID NO.6 are GLP-1R / GIPR dual receptor activation relaying GLP-1R activation / GIPR inhibition. Example 2

[0062] Glucose tolerance test Male ICR mice (20-24 g, n=21) were adaptively raised for 7 days before the experiment, during which the bedding was changed every three days. On the day of the experiment, the mice were weighed and divided into three groups (Saline group, 100 nmol / kg Semaglutide group, 100 nmol / kg SEQ ID NO.4 group) according to a group of 7. The mice were given an intraperitoneal injection. The fasting blood glucose of each group of mice was measured 24 hours after administration, and the mice were injected with 200 μL 2 g / kg glucose. Blood was collected from the tail of the mice at 20 min, 40 min, and 60 min. The blood glucose level of the mice was measured and recorded with a blood glucose meter. After the measurement, the mice were fed and fasted for 12 hours before the next fasting blood glucose measurement, but water was not prohibited. After that, the fasting blood glucose at 48 hours, 72 hours, and 7 days and the blood glucose levels at 20 min, 40 min, and 60 min after the injection of glucose were measured as above. The experimental results are shown in the figure. Figure 3 As shown in the figure, it can be seen that the SEQ ID NO.4 group has a better glucose tolerance effect than Semaglutide. Example 3

[0063] Therapeutic effects of once-daily dosing in DIO mice Male ICR mice (20-26 g, n=28) were fed a 7-day adaptive diet of a mixture of high-fat and basal diets in a 12 / 12 hour light / dark cycle, and then all were fed a high-fat diet. The body weight was measured once a week, and mice weighing more than 45 g were selected and divided into 4 groups (Saline group, 33 nmol / kg Dulaglutide group, 33 nmol / kg SEQID NO.4 group, and 33 nmol / kg SEQ ID NO.6 group) according to body weight matching. The drug was intraperitoneally injected once a day for 7 days. Food intake and body weight were recorded every day. The body weight results are shown in Table 1. Figure 4 As shown, compared with the Saline group, the Dulaglutide group, the SEQ ID NO.4 group and the SEQ ID NO.6 group all had extremely significant statistical differences (p < 0.0001); compared with Dulaglutide, SEQ ID NO.4 had a statistical difference (p < 0.05); compared with Dulaglutide, SEQ ID NO.6 had a significant statistical difference (p < 0.001), indicating that GLP-1R / GIPR dual activation relay GLP-1R activation / GIPR inhibition can produce a significant body weight reduction effect. Further, on the 8th day, after fasting for 12 hours, the livers of the above four groups of mice were sliced ​​and HE stained for observation. The results are as follows Figure 5 As shown, Figure 5A is the Saline group. The livers of DIO mice have a large number of fat particles and severe fatty lesions. Figure 5 B is the Dulaglutide treatment group, which has a small amount of fatty lesions, indicating that Dulaglutide cannot completely reverse the fatty lesions; Figure 5 C is the SEQ ID NO.4 treatment group, Figure 5 D is the SEQ ID NO.6 treatment group, Figure 5 C and Figure 5 D had almost no fatty lesions, indicating that both groups were able to reverse the fatty lesions in DIO mice. On the 8th day, after fasting for 12 hours, the livers of the four groups of mice were taken to measure the content of liver triglycerides and total cholesterol. The results are as follows: Figure 6 As shown in the figure, it can be seen that the triglyceride and total cholesterol levels of the Dulaglutide group were statistically different from those of the Saline group (p < 0.05); the triglyceride and total cholesterol levels of the Saline group were extremely significantly different from those of the SEQ ID NO.4 and SEQ ID NO.6 groups (p < 0.0001); the triglyceride levels of the Dulaglutide group were statistically different from those of the SEQ ID NO.4 and SEQ ID NO.6 groups (p < 0.01 and p < 0.05), and the total cholesterol levels were statistically different (p < 0.05); the results indicate that once-a-day administration of GLP-1R / GIPR dual activation relay GLP-1R activation / GIPR inhibition and GLP-1R activators can produce more significant weight loss and therapeutic effects in liver fatty lesions, and can significantly reduce the triglyceride and total cholesterol levels in the liver of mice. Example 4

[0064] Therapeutic effects of weekly dosing in DIO mice Mice weighing more than 45 g were selected and divided into 4 groups (Saline group, 165 nmol / kg Semaglutide group, 165 nmol / kg SEQ ID NO.4 group and 165 nmol / kg SEQ ID NO.6 group) according to body weight matching. The mice were intraperitoneally injected once a week on days 0, 7 and 14. They were fed and watered normally during the whole experiment. The dosing time point was weighing before dosing. The body weight and food intake were measured regularly every day until day 18. The results are shown in the figure. Figure 7As shown, compared with the Saline group, the Semaglutide group, the SEQ ID NO.4 group and the SEQ ID NO.6 group all had extremely significant differences (p < 0.0001); compared with Semaglutide, the SEQ ID NO.4 group had extremely significant statistical differences (p < 0.0001), and the SEQ ID NO.6 group had statistical differences (p < 0.05), indicating that GLP-1R / GIPR dual activation relay GLP-1R activation / GIPR inhibition can produce a significant weight loss effect compared with GLP-1R single target activation. Further, on the 18th day, after fasting for 12 hours, the livers of the above four groups of mice were sliced ​​and HE stained for observation. The results are as follows Figure 8 As shown, Figure 8 A is the Saline group. The livers of DIO mice have a large number of fat particles and severe fatty lesions. Figure 8 B is the Semaglutide treatment group, which has a small amount of fat lesions, indicating that Semaglutide cannot completely reverse the fat lesions; Figure 8 C is the treatment group of SEQ ID NO.4, Figure 8 D is the treatment group of SEQID NO.6, Figure 8 C and Figure 8 D had almost no fatty lesions, indicating that both groups were able to reverse fatty lesions in DIO mice. The results showed that once-weekly administration of GLP-1R / GIPR dual activation relay GLP-1R activation / GIPR inhibition and GLP-1R activator could produce more significant weight loss and therapeutic effects in liver fatty lesions compared with GLP-1R activator. Example 5

[0065] Comparison of weight loss with dual GLP-1R / GIPR receptor activation and dual GLP-1R / GIPR activation relayed by GLP-1R activation / GIPR inhibition Mice weighing more than 45 g were selected and divided into 4 groups (Saline group, 165 nmol / kg Dulaglutide group, 165 nmol / kg SEQ ID NO.2 group and 165 nmol / kg SEQ ID NO.4 group) according to body weight matching. The mice were intraperitoneally injected once a week on days 0, 7 and 14. They were fed and watered normally during the whole experiment. The dosing time point was weighing before dosing. The body weight and food intake were measured regularly every day until day 16. The results are shown in the figure. Fig. 9As shown, compared with the Saline group, the Dulaglutide group, the SEQ ID NO.2 group and the SEQ ID NO.4 group all had extremely significant differences (p < 0.0001); compared with the Dulaglutide group, the SEQ ID NO.2 group had a significant statistical difference (p < 0.01), indicating that the GLP-1R / GIPR dual receptor activation provided by the present invention has more significant weight loss activity than Dulaglutide; compared with SEQ ID NO.2, the SEQ ID NO.4 group had an extremely significant statistical difference (p < 0.0001), indicating that the GLP-1R / GIPR dual receptor activation relayed by the present invention and the GLP-1R activation / GIPR inhibition provided by the present invention produce a more significant weight loss effect than the GLP-1R / GIPR dual receptor activation provided by the present invention.

[0066] The above content is a specific description in combination with a preferred implementation mode. For technicians in the technical field to which the present invention belongs, they can make several deductions based on the technical concept of the present invention, which should be regarded as the protection scope of the present invention.

Claims

1. A GLP-1 / GIP fusion protein, characterized in that: With the following sequence: HGEGTFTSDVSSYLEX1QAAKEFIAWLVKGX2GGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFX3STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYX4TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLX5GGGGSGGGGSGGGGSGGGGSDDDDKYX6EX7TFISDYSIAMDKIX8QQX9X 10 VNWLLAQX 11 GGGGSGGGGSGGGGSGGGGSAGPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFX 12 STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLX 13 SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG in: X1 is G or E; X2 is R or G; X3 is N, Q, A or G; X4 is K or C; X5 is GGGGS, GGGGP, ASGPNQ, VPRGS or SRSRR; X6 is A or G; X7 is G or A; X8 is R or H; X9 is D or E; X 10 is F or W; X 11 is K or R; X 12 is N, Q, A or G; X 13 It is D or C.

2. A GLP-1 / GIP fusion protein according to claim 1, characterized in that: The MGSDDDDK sequence is added to the N-terminus, and after the fusion protein is expressed and renatured, it is cleaved with EK enzyme to form the free active forms of the N-termini of GLP-1 and GIP polypeptides.

3. A method for preparing a GLP-1 / GIP fusion protein, characterized in that: Use the following process: (1) synthesizing a coding gene, wherein the gene encodes the fusion protein of claim 1 and adds a MGSDDDDK sequence at the N-terminus, and constructing an expression vector; (2) transforming the recombinant expression vector carrying the coding gene into an Escherichia coli host to obtain a recombinant engineered bacterium; (3) Fermentation of recombinant engineered bacteria to induce the expression of inclusion body proteins in cells; (4) The bacterial cells are broken by high-pressure homogenization, the inclusion bodies are collected by centrifugation, and the inclusion bodies are dissolved and renatured; (5) affinity purification of the renatured protein onto a nickel column; (6) The purified renatured protein was dialyzed with EK enzyme digestion buffer and then digested with EK enzyme; (7) Select FAPα, Furin or Thrombin for enzyme cleavage according to the sequence; (8) Purify and obtain the active target protein.

4. The method for preparing a GLP-1 / GIP fusion protein according to claim 3, characterized in that: Step (7) FAPα, Furin or Thrombin are selected according to the sequence for enzymatic cleavage to remove the linker sequence, preferably FAPα, to further reduce immunogenicity.

5. The method for preparing a GLP-1 / GIP fusion protein according to claim 3, characterized in that: Step (8) obtains an active target protein having a Structure-I structure.

6. A GLP-1 / GIP fusion protein according to claim 1, characterized in that: When X6 is glycine, the GLP-1 / GIP fusion protein prepared according to claim 3 exhibits GLP-1R / GIPR dual receptor activation activity in humans or mice.

7. A GLP-1 / GIP fusion protein according to claim 1, characterized in that: When X6 is alanine, the GLP-1 / GIP fusion protein prepared according to claim 3 exhibits GLP-1R / GIPR dual receptor activation relay GLP-1R activation / GIPR inhibition activity under the action of DPP-IV cleavage in humans or mice.

8. The GLP-1 / GIP fusion protein according to claim 1, characterized in that: The GLP-1 / GIP fusion protein prepared according to claim 3 has better glucose-lowering activity, more significant weight-reducing activity and better therapeutic effect on fatty liver lesions in humans or mice than the GLP-1R single-target activator.

9. The GLP-1 / GIP fusion protein according to claim 1, characterized in that: The GLP-1 / GIP fusion protein prepared according to claim 3 has more significant weight loss activity and better therapeutic effect on fatty liver lesions in humans or mice than the GLP-1R single-target activator.

10. The GLP-1 / GIP fusion protein according to claim 1, characterized in that: According to the GLP-1 / GIP fusion protein prepared according to claim 3, GLP-1R / GIPR dual receptor activation relayed by GLP-1R activation / GIPR inhibition has more significant weight loss activity in humans or mice than GLP-1R / GIPR dual receptor activation.

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