Conjugate of polypeptide and antibody Fc fragment, pharmaceutical composition and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a conjugate of polypeptide and an antibody Fc fragment, a pharmaceutical composition and application of the conjugate. Aiming at the problems of unbalanced metabolic regulation, insufficient half-life period, high immunogenicity and the like of the existing triple agonist, the GLP-1 receptor / GCGR / GIPR triple agonist is fused with an engineered Fc fragment, so that the half-life period of the diabetes treatment medicine is prolonged in a breakthrough manner. The medicine disclosed by the invention can synchronously improve blood sugar, body weight and liver lipid metabolism, is suitable for combined treatment of diabetes, obesity and non-alcoholic fatty liver, and has the clinical advantages of precise metabolism regulation, long-acting administration period and controllable immunogenicity risk.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a conjugate of a polypeptide and an antibody Fc fragment, a pharmaceutical composition and its use. By fusing a GLP-1 receptor / GCGR / GIPR triple agonist with an antibody Fc fragment, a breakthrough extension of the half-life of a diabetes treatment drug is achieved. Background Art

[0002] The high complexity of the pathogenesis of metabolic diseases has promoted the progress of treatment strategies from single-target to multi-target. Drugs represented by GLP-1 single-target agonists can improve blood glucose and body weight, but their efficacy is limited by the biological mechanism of a single pathway - single-target activation cannot break through the metabolic compensation effect. For example, the contradiction between insufficient insulin secretion and energy consumption imbalance cannot be solved simultaneously. Dual-target agonists (such as GLP-1 / GIP dual-target drugs) have improved efficacy by introducing the incretin effect, but new metabolic side effects are caused by the imbalance of target activation ratios: overactivation of the GIP pathway may promote adipose tissue hyperplasia, partially offsetting the weight loss benefits. The currently developing GLP-1R / GCGR / GIPR triple agonists hope to simultaneously integrate the regulation of insulin secretion, energy consumption and nutrient absorption to achieve a more comprehensive regulation of the signal pathways related to metabolic diseases and further achieve a more comprehensive treatment effect; however, the metabolism of such drugs still has problems, mainly due to their rapid clearance in the body, resulting in poor patient compliance and having an adverse impact on the control of the disease.

[0003] The Fc fragment conjugation technology provides a new path for the metabolic regulation and long-acting of polypeptide drugs. Compared with traditional chemical modifications (such as fatty acid chains or polyethylene glycolylation), the Fc fragment can significantly extend the drug half-life through the recycling mechanism mediated by FcRn, achieve a monthly dosing cycle, and at the same time avoid the destruction of the receptor binding conformation by chemical modifications. Compared with traditional long-acting strategies (such as fatty acid modification or PEGylation), Fc conjugation not only reduces the renal clearance rate by increasing the molecular weight, but also achieves an exponential extension of the metabolic cycle through the recycling mechanism mediated by FcRn. In addition, the Fc-mediated directed uptake by liver endothelial cells can enhance the enrichment of the drug in key metabolic organs, improve the treatment specificity, rather than relying on the off-target risk brought by systemic exposure.

[0004] Introducing Fc conjugation technology into GLP-1R / GCGR / GIPR triple agonists is expected to provide novel ideas for the treatment of metabolic diseases and the structural optimization of polypeptide drugs. At the efficacy level, the synergistic effect of the Fc fragment can break through the technical bottleneck of multi-target activation - the flexible design of its structure can maintain the native conformation of the triple agonist and ensure receptor binding efficiency; while the long-acting property enables continuous and stable metabolic regulation, avoiding efficacy fluctuations caused by blood drug concentration fluctuations. Compared with PEGylation or chemical modification, Fc fragment conjugation minimizes conformational interference with the polypeptide active domain while prolonging the half-life. At the safety level, engineering modification of the Fc fragment (such as deimmunogenic mutation) can significantly reduce the risk of complement activation or antibody response caused by traditional polypeptide modification, providing safety guarantees for long-term treatment. More importantly, through long-acting methods and multi-target regulation strategies, this technology is expected to solve the co-disease management problem of obesity, diabetes, and non-alcoholic fatty liver, opening up a new direction for the integrated treatment of metabolic diseases. Summary of the Invention

[0005] One object of the present invention is to provide a polypeptide conjugated with an antibody Fc fragment, wherein the polypeptide is a GLP-1R / GCGR / GIPR triple agonist, and its amino acid sequence is: YAibQGTFTSDYSIaMelLDKKAQAibAFIEYLL d E d GGPSSGAPPPS.

[0006] Preferably, the triple agonist is modified with a fatty acid chain C20.

[0007] Preferably, the modification site is K at the 17th position, which is connected to C20 through the linker AEEA-γEEYEKEYE.

[0008] Preferably, the amino acid sequence of the Fc fragment is as shown in SEQ ID NO: 1.

[0009] Preferably, the conjugation site of the Fc fragment and the triple agonist is K at the 16th position.

[0010] Preferably, the polypeptide conjugated with the antibody Fc fragment is as shown in the following formula: , where the amino acid sequence of Fc is L *C]YPWVYASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDPDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL, where [*C] is the site conjugated with the linker.

[0011] In another preferred embodiment of the present invention, the present invention also provides a pharmaceutical composition comprising the polypeptide - antibody Fc fragment conjugate of the present invention.

[0012] In yet another preferred embodiment of the present invention, the present invention also provides a method for preparing a polypeptide - antibody Fc fragment conjugate, which comprises the following steps: (1) Prepare YAibQGTFTSDYSIaMelLD * K]K((AEEA)γ - E - EYEKEYE - C20)AQAibAFIEYLL d E d GGPSSGAPPPS - NH2, where * K]the structural formula of the amino acids used is shown as follows: ; (2) After the synthesis of the polypeptide is completed, react it with the following compound in a buffer solution to obtain the linker - conjugated polypeptide ; (3) React the linker - conjugated polypeptide of the Fc fragment in a buffer solution to obtain the conjugate of the polypeptide and the Fc fragment.

[0013] In yet another preferred embodiment of the present invention, the present invention also provides the polypeptide - antibody Fc fragment conjugate and / or the pharmaceutical composition of the present invention for preventing and / or treating obesity, diabetes and non - alcoholic fatty liver.

[0014] In yet another preferred embodiment of the present invention, the present invention also provides the use of the conjugate of the polypeptide and the antibody Fc fragment and / or the pharmaceutical composition according to the present invention in the preparation of a medicament for preventing and / or treating obesity, diabetes or non-alcoholic fatty liver.

[0015] Compared with the prior art, the main advantages of the present invention are as follows: 1) Specific conjugation of the polypeptide and the antibody Fc fragment is achieved through a covalent conjugation technique based on sequence recognition; 2) Precise and controllable site-specific conjugation of the polypeptide is achieved through a chemical synthesis method; 3) The half-life is extended to 25 - 30 days through the double-cycle mechanism mediated by FcRn, enabling monthly administration.

[0016] The medicament according to the present invention can simultaneously improve blood glucose, body weight and liver lipid metabolism, and is applicable to the combined treatment of diabetes, obesity and non-alcoholic fatty liver. The clinical advantages are precise metabolic regulation, long-acting administration cycle and controllable immunogenicity risk. Specific Embodiments

[0017] The present invention will be described in detail below with reference to specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0018] Example 1: Preparation of the conjugate of the polypeptide and the antibody Fc fragment

[0019] A conjugate of the polypeptide and the antibody Fc fragment was prepared by a site-specific conjugation method. Among them, the polypeptide part was prepared by solid-phase synthesis, and its structure was YAibQGTFTSDYSIaMelLD * K]K((AEEA)γ-E-EYEKEYE-C20)AQAibAFIEYLL d E d GGPSSGAPPPS-NH2, where L d 、E d are D-amino acids, Aib is 2-aminoisobutyric acid, aMeI is α-methyl-L-leucine, * K] is the conjugation site of the antibody Fc fragment, and the structural formula of the amino acid used in solid-phase synthesis is: .

[0020] After the synthesis of the polypeptide was completed, it was reacted with the following compound in a buffer solution at a molar ratio of 1:1 to obtain the polypeptide conjugated with the linker:

[0021] The expression of the antibody Fc fragment was carried out using the E. coli system, and its sequence is L * C]YPWVYASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDPDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL. React the Fc fragment with the polypeptide conjugated with the linker in a buffer (molar ratio of Fc fragment: polypeptide = 1:1), dialyze with a dialysis bag with a molecular weight cut-off of 25 kD at room temperature overnight and lyophilize to obtain the conjugate of the polypeptide and the Fc fragment. In the Fc fragment, * C] is the site conjugated with the linker, and a covalent bond is formed between the side-chain thiol group and the alkyne group in the polypeptide linker to achieve stable site-specific conjugation.

[0022] Example 2: Testing the agonistic activity of the polypeptide-antibody Fc fragment conjugate against GLP-1R and GIPR

[0023] The agonistic activity of the polypeptide-antibody Fc fragment conjugate against three targets, GLP-1R, GCGR, and GIPR, was tested at the protein level. The test results are expressed as EC50 values as follows: Target GLP-1R GCGR GIPR EC50 (nM) 0.108 5.25 0.166

[0024] Example 3: Testing the in vivo activity of the polypeptide-antibody Fc fragment conjugate in mice

[0025] The in vivo activity of the polypeptide-antibody Fc fragment conjugate was tested in a mouse obesity model. At a dose of 8 nmol of the drug injected subcutaneously once a week, the body weight of the obese mice induced by a high-fat diet decreased by 10% after 24 days, and the weight loss effect could be maintained for 96 days.

[0026] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in this technical field based on the concept of the present invention through logical analysis, reasoning, or limited experiments on the basis of the prior art shall fall within the protection scope determined by the claims.

Claims

1. A conjugate of a polypeptide and an antibody Fc fragment, wherein the polypeptide is a GLP-1R / GCGR / GIPR triple agonist, and its amino acid sequence is: YAibQGTFTSDYSIaMelLDKKAQAibAFIEYLL d E d GGPSSGAPPPS。 2. The conjugate of the polypeptide and the antibody Fc fragment according to claim 1, wherein The triple agonist is modified with a fatty acid chain C20.

3. The conjugate of the polypeptide and the antibody Fc fragment according to claim 2, characterized in that, The modification site is K at the 17th position, and it is connected to C20 through the linker AEEA-γEEYEKEYE.

4. The conjugate of the polypeptide and the antibody Fc fragment according to claim 1, characterized in that, The amino acid sequence of the Fc fragment is as shown in SEQ ID NO:

1.

5. The conjugate of the polypeptide and the antibody Fc fragment according to claim 4, wherein, The conjugation site of the Fc fragment and the triple agonist is K at the 16th position.

6. The conjugate of the polypeptide and the antibody Fc fragment according to claim 5, characterized in that, The conjugate of the polypeptide and the antibody Fc fragment is shown as follows: , wherein the amino acid sequence of Fc is L * C]YPWVYASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDPDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL, and [*C] is the site coupled by the linker.

7. A pharmaceutical composition comprising the conjugate of a polypeptide and an antibody Fc fragment according to any one of claims 1-6.

8. A method for preparing the conjugate of a polypeptide and an antibody Fc fragment according to claim 6, which comprises the following steps: (1)Prepare YAibQGTFTSDYSIaMelLD by solid-phase synthesis * K]K((AEEA)γ-E-EYEKEYE-C20)AQAibAFIEYLL d E d GGPSSGAPPPS-NH2, where * The structural formula of the amino acids used by K] is as shown in the following formula: ； (2) After the synthesis of the polypeptide is completed, it is reacted with the following compound in a buffer solution to obtain the linker-conjugated polypeptide ； (3) React the linker-conjugated polypeptide of the Fc fragment in a buffer solution to obtain the conjugate of the polypeptide and the Fc fragment.

9. The conjugate of the polypeptide according to any one of claims 1-6 and the antibody Fc fragment and / or the pharmaceutical composition according to claim 7, characterized in that, For preventing and / or treating obesity, diabetes and non-alcoholic fatty liver.

10. Use of the conjugate of a polypeptide and an antibody Fc fragment according to any one of claims 1-6 and / or the pharmaceutical composition according to claim 7 in the preparation of a drug for preventing and / or treating obesity, diabetes or non-alcoholic fatty liver.

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