A conjugate of a polypeptide and an antibody Fc fragment, a pharmaceutical composition and its uses

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a conjugate of a polypeptide and an antibody Fc fragment, a pharmaceutical composition, and its uses. Addressing the problems of metabolic regulation imbalance, insufficient half-life, and high immunogenicity associated with existing triple agonists, this invention achieves a breakthrough in extending the half-life of diabetes treatment drugs by fusing a GLP-1 receptor / GCGR / GIPR triple agonist with an engineered Fc fragment. The drug described in this invention can simultaneously improve blood glucose, weight, and hepatic lipid metabolism, and is suitable for the combined treatment of diabetes, obesity, and non-alcoholic fatty liver disease. Its clinical advantages include precise metabolic regulation, long-term dosing, and controllable immunogenicity risks.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a conjugate of a polypeptide and an antibody Fc fragment, a pharmaceutical composition and its uses. 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 Technology

[0002] The highly complex pathogenesis of metabolic diseases has spurred advancements in treatment strategies from single-target to multi-target approaches. While drugs, such as GLP-1 single-target agonists, can improve blood glucose and weight, their efficacy is limited by the biological mechanisms of a single pathway—single-target activation cannot overcome metabolic compensation effects, for example, it cannot simultaneously resolve the contradiction between insufficient insulin secretion and energy expenditure imbalance. Dual-target agonists (such as GLP-1 / GIP dual-target drugs) enhance efficacy by introducing incretin effects, but the imbalance in the target activation ratio leads to new metabolic side effects: overactivation of the GIP pathway may promote adipose tissue hyperplasia, partially offsetting weight loss benefits. Currently under development, GLP-1R / GCGR / GIPR triple agonists aim to integrate the regulation of insulin secretion, energy expenditure, and nutrient absorption to achieve more comprehensive control of metabolic disease-related signaling pathways and further achieve more comprehensive therapeutic effects; however, the metabolism of these drugs still faces challenges, primarily due to their rapid clearance from the body, leading to poor patient compliance and adversely affecting disease control.

[0003] Fc fragment conjugation technology offers a novel pathway for the metabolic regulation and long-acting of peptide drugs. Compared to traditional chemical modifications (such as fatty acid chain or PEGylation), Fc fragments, through an FcRn-mediated circulation mechanism, can significantly prolong the drug's half-life, enabling monthly dosing cycles while avoiding the disruption of receptor-binding conformation caused by chemical modifications. Compared to traditional long-acting strategies (such as fatty acid modification or PEGylation), Fc conjugation not only reduces renal clearance by increasing molecular weight but also achieves exponential extension of the metabolic cycle through an FcRn-mediated recovery mechanism. Furthermore, Fc-mediated targeted uptake by hepatic endothelial cells enhances drug accumulation in key metabolic organs, improving treatment specificity, rather than relying on the off-target risks associated with systemic exposure.

[0004] Introducing Fc conjugation technology into GLP-1R / GCGR / GIPR triple agonists holds promise for providing novel approaches to the treatment of metabolic diseases and the structural optimization of peptide drugs. In terms of efficacy, the synergistic effect of Fc fragments can overcome the technical bottleneck of multi-target activation—the flexible design of its structure can maintain the native conformation of the triple agonist, ensuring receptor binding efficiency; while its long-acting characteristics enable continuous and stable metabolic regulation, avoiding efficacy fluctuations caused by fluctuations in blood drug concentrations. Compared with PEGylation or chemical modification, Fc fragment conjugation extends the half-life while minimizing conformational interference with the peptide's active domain. In terms of safety, the engineering modification of Fc fragments (such as deimmunogenic mutations) can significantly reduce the risk of complement activation or antibody response induced by traditional peptide modifications, providing safety assurance for long-term treatment. More importantly, this technology, through long-acting methods and multi-target regulation strategies, holds promise for solving the comorbid management challenges of obesity, diabetes, and non-alcoholic fatty liver disease, opening up new directions for the integrated treatment of metabolic diseases. Summary of the Invention

[0005] One objective of this invention is to provide a peptide-antibody Fc fragment conjugate, wherein the peptide is a GLP-1R / GCGR / GIPR triple agonist, and its amino acid sequence is as follows:

[0006] YAibQGTFTSDYSIaMelLDKKAQAibAFIEYLL d E d GGPSSGAPPPS.

[0007] Preferably, the triple agonist is modified with a fatty acid chain C20.

[0008] Preferably, the modified site is K at position 17, which is connected to C20 via the connector AEEA-γEEYEKEYE.

[0009] Preferably, the amino acid sequence of the Fc fragment is as shown in SEQ ID NO: 1.

[0010] Preferably, the coupling site between the Fc fragment and the triple agonist is K at position 16.

[0011] Preferably, the polypeptide-antibody Fc fragment conjugate is as follows:

[0012] The amino acid sequence of Fc is L[ *C]YPWVYASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDPDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL, [*C] is the connecting arm coupling site.

[0013] In another preferred embodiment of the present invention, a pharmaceutical composition comprising the polypeptide and antibody Fc fragment conjugate described herein is also provided.

[0014] In another preferred embodiment of the present invention, the present invention also provides a method for preparing a peptide-antibody Fc fragment conjugate, which includes the following steps:

[0015] (1) YAibQGTFTSDYSIaMelLD was prepared by solid-state synthesis. * K]K((AEEA)γ-E-EYEKEYE-C20)AQAibAFIEYLL d E d GGPSSGAPPPS-NH2, where [ * The structural formula of the amino acid used in K is shown below:

[0016] ;

[0017] (2) After the synthesis of the polypeptide is completed, it is reacted with the following compound in a buffer solution to obtain a polypeptide with a linker arm coupled.

[0018] ;

[0019] (3) The peptide coupled to the Fc fragment linker arm is reacted in a buffer solution to obtain a conjugate of the peptide and the Fc fragment.

[0020] In another preferred embodiment of the present invention, the present invention also provides the polypeptide-antibody Fc fragment conjugate and / or pharmaceutical composition described herein for the prevention and / or treatment of obesity, diabetes, and non-alcoholic fatty liver disease.

[0021] In another preferred embodiment of the present invention, the present invention also provides the use of the polypeptide-antibody Fc fragment conjugate and / or pharmaceutical composition described herein in the preparation of a medicament for the prevention and / or treatment of obesity, diabetes or non-alcoholic fatty liver disease.

[0022] Compared with the prior art, the main advantages of this invention are:

[0023] 1) Specific conjugation of peptides to antibody Fc fragments is achieved through sequence recognition-based covalent conjugation technology;

[0024] 2) Achieve precise and controllable site-directed peptide coupling through chemical synthesis methods;

[0025] 3) The FcRn-mediated dual circulation mechanism extends the half-life to 25-30 days, enabling monthly dosing.

[0026] The drug described in this invention can simultaneously improve blood glucose, weight, and liver lipid metabolism, and is suitable for the combined treatment of diabetes, obesity, and non-alcoholic fatty liver disease. Its clinical advantages include precise metabolic regulation, long-term dosing cycle, and controllable immunogenicity risk. Detailed Implementation

[0027] The present invention will be described in detail below with reference to specific embodiments, but should not be construed as limiting 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 commercially unless otherwise specified.

[0028] Example 1: Preparation of peptide-antibody Fc fragment conjugates

[0029] A conjugate of a peptide and an antibody Fc fragment was prepared using a site-directed conjugation method. The peptide portion was prepared via solid-phase synthesis, and its structure is YAibQGTFTSDYSIaMelLD[]. * K]K((AEEA)γ-E-EYEKEYE-C20)AQAibAFIEYLL d E d GGPSSGAPPPS-NH2, where L d E d It is a D-type amino acid, Aib is 2-aminoisobutyric acid, and aMeI is α-methyl-L-leucine. * K] is the coupling site of the antibody Fc fragment. The structural formula of the amino acid used in solid-phase synthesis is:

[0030] .

[0031] After the peptide synthesis was completed, it was reacted with the following compound in a 1:1 molar ratio in a buffer solution to obtain a peptide coupled with a linker arm:

[0032]

[0033] The expression of the antibody Fc fragment was performed using an E. coli system; its sequence is L[ * C]YPWVYASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDPDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL. The Fc fragment and the peptide conjugated to the linker arm were reacted in buffer (molar ratio of Fc fragment: peptide = 1:1), incubated overnight at room temperature, dialyzed using a dialysis bag with a molecular weight cutoff of 25 kDa, and then lyophilized to obtain the peptide-Fc fragment conjugate. In the Fc fragment, [ * [C] is the coupling site of the linker arm. Stable site-specific coupling is achieved by forming a covalent bond between the side chain thiol group and the alkynyl group in the polypeptide linker arm.

[0034] Example 2: Assay of the agonistic activity of peptide-antibody Fc fragment conjugates against GLP-1R and GIPR

[0035] The agonistic activity of the peptide-antibody Fc fragment conjugate against three targets—GLP-1R, GCGR, and GIPR—was tested at the protein level. The results are expressed as EC50 values ​​below:

[0036] target GLP-1R GCGR GIPR EC50(nM) 0.108 5.25 0.166

[0037] Example 3: In vivo activity assay of peptide-antibody Fc fragment conjugate in mice

[0038] The in vivo activity of the peptide-antibody Fc fragment conjugate in a mouse obesity model was tested. With a dose of 8 nmol of the drug administered subcutaneously once weekly, high-fat diet-induced obese mice experienced a 10% weight loss after 24 days, and the weight loss effect was maintained for 96 days.

[0039] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined 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 the conjugate of the polypeptide and the antibody Fc fragment is shown in the following formula: ； The amino acid sequence of Fc is L[ * C]YPWVYASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDPDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLEL, [*C] is the connecting arm coupling site; Its preparation method includes the following steps: (1) YAibQGTFTSDYSIaMelLD was prepared by solid-state synthesis. * K]K(AEEAγ-E-EYEKEYE-C20)AQAibAFIEYLL d E d GGPSSGAPPPS-NH2, where [ * The structural formula of the amino acid used in K is shown below: ； (2) After the synthesis of the polypeptide is completed, it is reacted with the following compound in a buffer solution to obtain a polypeptide with a linker arm coupled. ； (3) React the Fc fragment with the linker arm-coupled polypeptide in a buffer solution to obtain a conjugate of polypeptide and Fc fragment.

2. A pharmaceutical composition comprising a conjugate of the polypeptide of claim 1 and an antibody Fc fragment.

3. The conjugate of the polypeptide and antibody Fc fragment of claim 1 or the pharmaceutical composition of claim 2, characterized in that, Used for the prevention and / or treatment of obesity, diabetes, and non-alcoholic fatty liver disease.

4. The use of the conjugate of the polypeptide and antibody Fc fragment of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for the prevention and / or treatment of obesity, diabetes, and non-alcoholic fatty liver disease.

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