Super long acting platform comprising Fc-higher fatty acid chains

Abstract

The present invention relates to a super long acting platform for improving the half-life of a pharmaceutically active molecule, comprising an immunoglobulin Fc and a higher fatty acid chain. The invention also relates to the preparation of the conjugate platform, to compositions containing said conjugates and to therapeutic applications thereof. The present invention specifically relates to a conjugated molecule of structure "Active Molecule-Fc-Cn" wherein the active molecule is selected from any molecule beneficial to the organism, Fc is an immunoglobulin IgG Fc, Cn is a modified moiety comprising a C14-24 fatty acid chain, to the preparation of the conjugated molecule, to compositions containing said conjugate and to therapeutic applications thereof.

Description

Ultra-long-acting platform containing Fc-higher fatty acid chains Technical Field

[0001] The present invention relates to an ultra-long-acting platform for improving the half-life of active pharmaceutical molecules, comprising immunoglobulin Fc and higher fatty acid chains. The present invention also relates to the preparation of the conjugate platform, a composition containing the conjugate, and its therapeutic application. Background Art

[0002] Because peptide drugs are closer to endogenous substances in the body than chemical drugs, they have advantages such as low toxicity and side effects and stable efficacy. Therefore, they are widely used in various clinical treatments such as cancer, cardiovascular disease, and autoimmune diseases. They have broad application prospects and are also favored by pharmaceutical manufacturers and scientific researchers. However, classic peptide drugs have low tolerance to proteases in the body and poor stability. They will be quickly degraded after entering the body, resulting in a short plasma half-life. Most bioactive peptide substances have poor bioavailability and cannot be taken orally. These problems have greatly hindered the clinical application of peptide drugs. In addition, due to limited exposure to target tissues, many protein peptide drugs require more frequent dosing to maintain clinically effective drug concentrations.

[0003] To address these issues, peptide drugs are currently being modified. Peptide drug modifications can be divided into two categories: one is to modify the peptide chain backbone; the other is to maintain the peptide backbone while introducing other groups for structural optimization and performance modification. These include polyethylene glycol modification, glycosylation, protein fusion strategies, higher fatty acid modification, site-directed mutagenesis, and cholesterol modification.

[0004] Bioactive molecules are fused to the Fc region of immunoglobulins to form Fc fusion proteins, which combine the beneficial pharmacological properties of bioactive molecules with the additional properties of the Fc region, thereby increasing the serum half-life of physiologically active molecules and thus reducing the frequency of drug administration. Currently, the fusion of the Fc region with active peptides as ligands or receptors or with the extracellular domain (ECD) has greatly increased the clinical potential of active protein drugs. Specifically, for products with a molecular weight of less than 60kDa, they can be easily cleared by the kidneys, thus having a short serum half-life. By coupling or fusing with the Fc region, their size is increased, exceeding the threshold of renal filtration, thereby increasing their circulation time. When the Fc fusion protein is taken up by endothelial cells and enters the acidified endosome, the Fc fusion protein is protected from lysosomal degradation by binding to FcRn in the endosome through Fc. When transported to the cell surface by the recycling endosome, under neutral and weakly alkaline conditions, Fc dissociates from FcRn, releasing the Fc-fusion protein back into the blood circulation, thereby extending the half-life of the Fc-fusion protein and allowing the target tissue to be exposed to the pharmacologically active portion of the Fc-fusion protein for a longer time, thereby enhancing the latter's therapeutic potential.

[0005] Fc fusion proteins represent a successful class of biopharmaceutical products, with 13 approved in the EU and US, and three biosimilars of etanercept. Potential bioactive molecules are highly diverse, including the extracellular domain of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding structures that act as cytokine traps. Most Fc fusion proteins are generated by fusing a bioactive molecule to the N-terminus of the Fc domain. The strong interactions of the IgG-CH3 domain create a stable Fc structure and allow for the fusion of more complex structures, such as flexible hinge regions and disulfide bonds. Eli Lilly and Company developed the antidiabetic drug dulaglutide by fusing GLP-1 to IgG4 (Fc). Due to its significantly increased molecular size, this reduces the renal clearance of GLP-1, resulting in a prolonged biological half-life.

[0006] Fatty acids are important components of human fat, lipids, and cell membrane phospholipids. As endogenous components, they have low immunogenicity and are therefore used to modify bioactive molecules such as peptide drugs. When fatty acids are used to modify specific amino acid residues in peptide drugs, they increase the serum half-life of the peptide drug by reversibly binding to serum albumin. However, fatty acid modification also has its own limitations: for example, it is prone to produce products at nonspecific modification sites of fatty acids; and because the binding of fatty acids to serum albumin is reversible, peptide drugs that dissociate from albumin are easily eliminated through the kidneys, thereby affecting or reducing the half-life of fatty acid-modified peptide drugs.

[0007] While retaining the efficacy of peptide drugs, improving the serum half-life of peptide drugs and reducing the immunogenicity of the drugs have always been the needs of the scientific research and pharmaceutical fields. This application meets this need by providing an innovative ultra-long-acting platform for improving drug active molecules.

[0008] Summary of the Invention

[0009] The present invention provides ultra-long-acting platforms with the following structures that can improve the half-life of active pharmaceutical molecules: active molecule-fusion protein-Cn conjugate platforms, active molecule-Fc-Cn conjugate platforms, and antibody-Cn conjugate platforms, where Cn represents a modified portion containing a fatty acid chain with n=14-24, and Fc represents the Fc region of an immunoglobulin molecule. The ultra-long-acting platforms provided herein have the following advantages:

[0010] 1. Both the Fc component and the higher fatty acid chain component of the conjugated molecule can bind directly or indirectly to FcRn. Fc binds directly to FcRn, while higher fatty acids bind indirectly to FcRn via serum albumin. Since Fc and serum albumin bind to different sites on FcRn, they do not interfere with each other, thus providing a longer half-life for the active molecule conjugated to them compared to Fc alone or higher fatty acids alone.

[0011] 2. Fusion proteins, antibodies, Fc and higher fatty acids are all endogenous substances in the body and have low immunogenicity, which can reduce the heterologous nature of the conjugated molecules to the body and reduce the possibility of producing corresponding antibodies;

[0012] 3. Conjugated molecules can increase the size of the active molecules contained and reduce their renal excretion rate, thereby prolonging the circulation time of the active molecules in the body;

[0013] 4. Due to the hydrophobicity of fatty acid chains, it helps to improve the membrane permeability of conjugated molecules, thereby allowing more conjugated molecules to enter the circulatory system;

[0014] 5. The Fc in the conjugated molecule can homologously pair with the cognate Fc fragment through its CH2-3 domain, thereby increasing stability and improving the concentration of the local conjugated molecule / active molecule.

[0015] In the first aspect, the present invention provides a conjugate molecule having a structure of "active molecule-Fc-Cn", a conjugate molecule having a structure of "active molecule-fusion protein-Cn", and a conjugate molecule having a structure of "antibody-Cn", wherein the active molecule is selected from any molecule that is beneficial to the body, Fc is derived from the heavy chain constant region of immunoglobulin IgG, and Cn is a conjugate containing C 14 - 24 A modified portion of a fatty acid chain.

[0016] In some embodiments, the Cn in the conjugate molecule with the structure of "active molecule-Fc-Cn", the conjugate molecule with the structure of "active molecule-fusion protein-Cn", and the conjugate molecule with the structure of "antibody-Cn" disclosed in the present invention has the structure of the following formula (I): -ZY (I),

[0017] in

[0018] Z has the following structure:

[0019] -Z1-Z2-Z3-Z4-,

[0020] wherein Z1 is a sulfur atom, nitrogen atom or oxygen atom in Fc,

[0021] Z2 is -C(=O)- or a 5-10 membered heterocyclic group, preferably containing 1 or 2 heteroatoms selected from N, S and O;

[0022] Z3 is selected from a bond, -C(=O)-, -C1-C 10 Alkylene-C(=O)-, -C3-C 10 Alkynylidene-C(=O)-, -C3-C 10 Alkenylene-C(=O)-, -C1-C 10 heteroalkylene-C(=O)-, -C3-C8 cycloalkylene-C(=O)-, -O-C1-C8 alkylene-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C 1- C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-C3-C8 cycloalkylene-C(=O)-, -C3-C8 cycloalkylene-C1-C 10 Alkylene-C(=O)-, -C 3- C8 heterocyclylene-C(=O)-, -C1-C 10 Alkylene-C3-C8 heterocyclylene-C(=O)-, -C3-C8 heterocyclylene-C1-C 10 Alkylene-C(=O)-, wherein the alkylene, alkynylene, alkenylene, heteroalkylene, cycloalkylene, arylene and heterocyclylene groups may be optionally substituted;

[0023] Z4 is a bond or a PEG unit represented by the formula,

[0024] Wherein, R1 is selected from C 1-4 Alkylene, -NH-, -NH-C 1-4 Alkylene-, -NH-C 1-4 Alkylene-heteroaryl-, wherein heteroaryl is a 5-membered or 6-membered nitrogen-containing heteroaryl; R2 is -C(=O)-, -C 1-4 Alkylene, -C 1-4 Alkylene-C(=O)-, -C 1-4 Alkylene-NH-C(=O)-(CH2OCH2) p -C 1-4 Alkylene-, -C 1-4 Alkylene-C(=O)-NH-(CH2OCH2) p -C 1-4 Alkylene-, wherein m is an integer of 2-6, p is an integer of 1-3,

[0025] Y is

[0026] Where Y is connected to Z4 through X, k is an integer from 10 to 30,

[0027] wherein R independently represents hydrogen, C 1-6 Alkyl, C 1-6 aminoalkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl.

[0028] In some embodiments, Z2 is maleimido, The wavy line on the left indicates the position connected to Z1; the wavy line on the right indicates the position connected to Z3.

[0029] In some embodiments, Z3 is -C1-C 10 Alkylene-C(=O)-, wherein the alkylene is optionally substituted and wherein Z3 is linked to Z4 through -C-(=O)-.

[0030] In some preferred embodiments, Z2 is maleimido, Z3 is -C 1-6 Alkylene-C(=O)-.

[0031] In some embodiments, Z4 is a bond, and Z3 is directly connected to Y in formula (I).

[0032] In some embodiments, Z4 is a PEG unit represented by the formula,

[0033] Wherein, R1 is selected from -NH- and -NH-C 1-4 Alkylene-; R2 is -C 1-4 Alkylene or -C 1-4 Alkylene-NH-C(=O)-(CH2OCH2) p -C 1-4 Alkylene-, wherein m is an integer of 2-6, and p is an integer of 1-3.

[0034] In some embodiments, Z4 is a unit comprising 2-6 PEGs. In some embodiments, Z4 is

[0035] wherein m=1-4, the asterisk on the left indicates the position for connection to Z3; the asterisk on the right indicates the position for connection to Y in formula II.

[0036] In some embodiments, Z in formula (I) of the present invention has the following structure:

[0037] where R E It is hydrogen, C 1-6 Alkyl, C 1-6aminoalkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, wherein y=0-4, m=1-4, wherein the asterisk on the left indicates the position of attachment to Ab, and the asterisk on the right indicates the position of attachment to Y.

[0038] Y is

[0039] wherein Y is linked to Z4 through X, and X is -NH-(C=O)- or -(C=O)-NH-,

[0040] k is an integer between 10 and 30,

[0041] wherein R independently represents hydrogen, C 1-6 Alkyl, C 1-6 aminoalkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl.

[0042] In a specific embodiment, Cn is selected from

[0043] In one embodiment, the Fc region is derived from the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4. In a specific embodiment, the Fc region is derived from the heavy chain constant region of IgG1 or IgG4. In another embodiment, the Fc region may further include a hinge region. In a specific embodiment, the Fc region comprises amino acid modifications. In a specific embodiment, the modifications to the Fc region are modifications to positions 254, 308 and 434 (according to EU numbering). In another specific embodiment, the modifications to the Fc region are substitutions of amino acids 254, 308, and 434 with Thr, Pro, and Ala, respectively. In a specific embodiment, the modifications to the Fc region are modifications to positions 228, 234, 235, and / or 447, such as modifications of S228P, F234A, L235A, or deletions of S228P, F234A, L235A, and 447. In a specific embodiment, the Fc region is selected from the sequence of SEQ ID NO: 10, 15, or 16.

[0044] In one embodiment, the active molecule is a peptide active molecule. In one embodiment, the peptide active molecule is fused directly or via a peptide linker to an antibody, fusion protein, or Fc region. In a specific embodiment, the C-terminus of the peptide active molecule is fused to the N-terminus of the antibody, fusion protein, or Fc region. Alternatively, the N-terminus of the peptide active molecule is fused to the C-terminus of the antibody, fusion protein, or Fc region. In another specific embodiment, the peptide active molecule is attached to the antibody, fusion protein, or Fc region in a monomeric form. In another specific embodiment, the peptide active molecule is attached to the antibody, fusion protein, or Fc region in a multimeric form.

[0045] In one embodiment, the active molecule is selected from an enzyme, an enzyme inhibitor, an antigen, an antibody or antibody fragment, a hormone, glucagon-like peptide-1 (GLP-1), glucagon, an interferon, a cytokine, a growth factor and / or a differentiation factor, a factor involved in cell motility or migration, a factor involved in bone tissue development / resorption, a chemokine, a plasma or interstitial adhesion molecule or an extracellular matrix, a bactericidal or antifungal factor, and the like.

[0046] In one specific embodiment, the active molecule is selected from GLP-1, an antibody Fab fragment, and an antibody F(ab')2 fragment. In another specific embodiment, the active molecule is selected from an anti-PD-1 antibody Fab fragment, an anti-PD-1 antibody F(ab')2 fragment, an anti-VEGF antibody Fab fragment, and an anti-VEGF antibody F(ab')2 fragment.

[0047] In one embodiment, the peptide linker comprises the amino acid sequence (G4S)n, wherein n is an integer equal to or greater than 1. In a specific embodiment, the peptide linker comprises (G4S)3, (G4S)4, (G4S)6, GS(G4S)4, DAAALEAAALDAAAREAAARDAAAL, NVDHLPSNTLVDLA, (G3S)2, (G4S)2, (G3S)3, (G4S)3, (G3S)4, (G4S)4, (G3S)5, (G4S)5, (G3S)6, (G4S)6, GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP, and GSTSGSGK PGSGEGSTKG.

[0048] In one embodiment, the conjugate molecules provided herein with the structure of "active molecule-Fc-Cn" or "antibody-Cn" homodimerize through their Fc regions.

[0049] In a specific embodiment, the present application provides a conjugate molecule having the structure "active molecule-Fc-Cn", wherein Fc is selected from IgG1 or IgG4, and Cn comprises a 16-, 18-, or 20-carbon fatty acid chain. In a preferred embodiment, the Fc comprises a modification, such as the modifications mentioned herein. In a specific embodiment, Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, Cn comprises a 20-carbon fatty acid chain.

[0050] In a specific embodiment, the present application provides a conjugate molecule having the structure "antibody-Cn", wherein the antibody is selected from IgG1 or IgG4, and Cn comprises a 16-, 18-, or 20-carbon fatty acid chain. In a preferred embodiment, the Fc of the antibody comprises a modification, such as the modifications mentioned in the present application. In a specific embodiment, Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, Cn comprises a 20-carbon fatty acid chain.

[0051] In a specific embodiment, the present application provides a conjugate molecule having the structure "active molecule-Fc-Cn", wherein the Fc is selected from IgG1 or IgG4, and the Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is coupled / conjugated to a free sulfhydryl group of the Fc. In a preferred embodiment, the Fc comprises a modification, such as those mentioned herein. In a specific embodiment, the Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, the Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, the Cn comprises a 20-carbon fatty acid chain.

[0052] In a specific embodiment, the present application provides a conjugate molecule having the structure "antibody-Cn", wherein the antibody is selected from IgG1 or IgG4, and Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is coupled / conjugated to the sulfur atom of the free thiol group of the antibody Fc. In a preferred embodiment, the Fc of the antibody comprises a modification, such as the modifications mentioned in the present application. In a specific embodiment, Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, Cn comprises a 20-carbon fatty acid chain.

[0053] In a specific embodiment, the present application provides a conjugate molecule having the structure "active molecule-fusion protein-Cn", wherein Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is coupled / conjugated to the sulfur atom of the free sulfhydryl group of the fusion protein. In one specific embodiment, Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, Cn comprises a 20-carbon fatty acid chain.

[0054] In a specific embodiment, the present application provides a conjugate molecule having the structure "active molecule-IgG4 Fc-Cn," wherein Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is coupled / conjugated to a free sulfhydryl group of IgG4 Fc. In a preferred embodiment, the IgG4 Fc comprises modifications, such as those described herein. In one specific embodiment, Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, Cn comprises a 20-carbon fatty acid chain.

[0055] In a specific embodiment, the present application provides a conjugate molecule having the structure "antibody-Cn", wherein Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is coupled / conjugated to the sulfur atom of a free sulfhydryl group of an IgG4 antibody. In a preferred embodiment, the Fc of the IgG4 antibody comprises a modification, such as the modifications mentioned herein. In a specific embodiment, Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, Cn comprises a 20-carbon fatty acid chain.

[0056] In a specific embodiment, the present application provides a conjugate molecule having the structure "active molecule-fusion protein-Cn", wherein Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is coupled / conjugated to the sulfur atom of the free sulfhydryl group of the fusion protein. In one specific embodiment, Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, Cn comprises a 20-carbon fatty acid chain.

[0057] In a specific embodiment, the present application provides a conjugate molecule having the structure "Active Molecule-IgG1 Fc-Cn," wherein Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is coupled / conjugated to the sulfur atom of a free sulfhydryl group of IgG1 Fc. In a preferred embodiment, the IgG1 Fc comprises a modification, such as those described herein. In one specific embodiment, Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, Cn comprises a 20-carbon fatty acid chain.

[0058] In a specific embodiment, the present application provides a conjugate molecule having the structure "antibody-Cn", wherein Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is coupled / conjugated to the sulfur atom of a free sulfhydryl group of an IgG1 antibody. In a preferred embodiment, the Fc of the IgG1 antibody comprises a modification, such as those described herein. In a specific embodiment, Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, Cn comprises a 20-carbon fatty acid chain.

[0059] In a specific embodiment, the present application provides a conjugate molecule having the structure "active molecule-fusion protein-Cn", wherein Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is coupled / conjugated to the sulfur atom of the free sulfhydryl group of the fusion protein. In one specific embodiment, Cn comprises a 16-carbon fatty acid chain, in another specific embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another specific embodiment, Cn comprises a 20-carbon fatty acid chain.

[0060] In a specific embodiment, the conjugate molecule having the structure "active molecule-Fc-Cn" described herein is a GLP-1-Fc-Cn conjugate molecule, wherein the GLP-1 is any active GLP-1 known in the prior art. In one embodiment, Fc is selected from the Fc of IgG1 or the Fc of IgG4. In a specific embodiment, the Cn in the conjugate molecule GLP-1-Fc-Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is coupled / conjugated to the sulfur atom of the free sulfhydryl group of Fc or the nitrogen atom of Fc. In a specific embodiment, the Cn in the conjugate molecule GLP-1-Fc-Cn is selected from TM1, C18-tert-butyl ester, C16-NHS, or C20-NHS. In a preferred technical solution, the conjugated molecule GLP-1-Fc-Cn is GLP-1-IgG4 Fc-TM1, GLP-1-IgG4 Fc-C18 tert-butyl ester, GLP-1-IgG4 Fc-C16-NHS, GLP1-IgG4 Fc-C20-NHS. In one embodiment, Fc comprises the sequence shown in SEQ ID NO: 10, 15 or 16. In a more preferred embodiment, the GLP-1-IgG4 Fc in the GLP-1-IgG4 Fc-TM1, GLP-1-IgG4 Fc-C18, GLP-1-IgG4 Fc-C16-NHS, GLP1-IgG4 Fc-C20-NHS conjugated molecule is derived from dulaglutide, preferably dulaglutide, for example, having a structure as disclosed in CN1802167, preferably having Gly 8 -Glu 22 -Gly 36 -GLP-1(7-37)-1L-IgG4 (S228P, F234A, L235A) structure. In one embodiment, the amino acid sequence of dulaglutide is as shown in SEQ ID NO: 1. In a specific embodiment, the GLP-1-Fc-Cn conjugate molecule is dulaglutide-Cn, such as dulaglutide-TM1, dulaglutide-C18 tert-butyl ester, dulaglutide-C16-NHS, or dulaglutide-C20-NHS.

[0061] In a specific embodiment, the active molecule in the conjugate molecule of the present application having the structure of "active molecule-Fc-Cn" is an anti-PD-1 antibody or an antigen-binding fragment thereof, which can be any known anti-PD-1 antibody or antigen-binding fragment thereof. In a specific embodiment, the antibody in the conjugate molecule of the present application having the structure of "antibody-Cn" is an anti-PD-1 antibody or an antigen-binding fragment thereof, which can be any known anti-PD-1 antibody or antigen-binding fragment thereof. In a preferred embodiment, Fc is selected from IgG1 Fc or IgG4 Fc. In a preferred embodiment, the antibody is selected from IgG1 or IgG4. In a preferred technical solution, the conjugate molecule is anti-PD-1 antibody antigen-binding fragment-IgG4 Fc-TM1, anti-PD-1 antibody antigen-binding fragment-IgG4 Fc-C18, anti-PD-1 antibody antigen-binding fragment-IgG4 Fc-C16-NHS, or anti-PD-1 antibody antigen-binding fragment-IgG4 Fc-C20-NHS. In a preferred embodiment, the conjugate molecule is an anti-PD-1 antibody antigen-binding fragment-IgG1 Fc-TM1, an anti-PD-1 antibody antigen-binding fragment-IgG1 Fc-C18, an anti-PD-1 antibody antigen-binding fragment-IgG1 Fc-C16-NHS, or an anti-PD-1 antibody antigen-binding fragment-IgG1 Fc-C20-NHS. In a preferred embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO:9 and the light chain variable region set forth in SEQ ID NO:11. In a preferred embodiment, the Fc comprises the sequence set forth in SEQ ID NO:10. In a more preferred embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises the heavy chain set forth in SEQ ID NO:8 and the light chain set forth in SEQ ID NO:12. In a more preferred embodiment, the Cn is selected from TM1, C18-tert-butyl ester, C16-NHS, and C20-NHS. In a more preferred embodiment, the Cn is selected from TM1.

[0062] In a specific embodiment, the active molecule in the conjugate molecule of the present application having the structure of "active molecule-Fc-Cn" is an anti-VEGF antibody or an antigen-binding fragment thereof, which can be any known anti-VEGF antibody or antigen-binding fragment thereof. In a specific embodiment, the antibody in the conjugate molecule of the present application having the structure of "antibody-Cn" is an anti-VEGF antibody or an antigen-binding fragment thereof, which can be any known anti-VEGF antibody or antigen-binding fragment thereof. In a preferred embodiment, the Fc is selected from IgG1 Fc or IgG4 Fc. In a preferred embodiment, the antibody is selected from IgG1 or IgG4. In a preferred technical solution, the conjugate molecule is anti-VEGF antibody antigen-binding fragment-IgG4 Fc-TM1, anti-VEGF antibody antigen-binding fragment-IgG4 Fc-C18, anti-VEGF antibody antigen-binding fragment-IgG4 Fc-C16-NHS, or anti-VEGF antibody antigen-binding fragment-IgG4 Fc-C20-NHS. In a preferred embodiment, the conjugate molecule is an anti-VEGF antibody antigen-binding fragment-IgG1 Fc-TM1, an anti-VEGF antibody antigen-binding fragment-IgG1 Fc-C18-tert-butyl ester, an anti-VEGF antibody antigen-binding fragment-IgG1 Fc-C16-NHS, or an anti-VEGF antibody antigen-binding fragment-IgG1 Fc-C20-NHS. In a preferred embodiment, the anti-VEGF antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs set forth in SEQ ID NOs: 2, 3, and 4 and the three light chain CDRs set forth in SEQ ID NOs: 5, 6, and 7. In a preferred embodiment, the Fc comprises the sequence set forth in SEQ ID NO: 15. In a more preferred embodiment, the anti-VEGF antibody or antigen-binding fragment thereof comprises the heavy chain set forth in SEQ ID NO: 13 and the light chain set forth in SEQ ID NO: 14. In a more preferred embodiment, the Cn is selected from TM1, C18-tert-butyl ester, C16-NHS, and C20-NHS. In a more preferred embodiment, the Cn is selected from TM1.

[0063] In a second aspect, the present application provides a method for preparing a conjugate molecule having a structure of "active molecule-Fc-Cn", comprising (a) linking an active molecule polypeptide to the Fc region of an immunoglobulin to prepare an "active molecule-Fc" fusion; and (b) subjecting the "active molecule-Fc" fusion to a coupling reaction with a Cn containing a fatty acid chain under conditions that allow conjugation of the Fc region to the Cn, to produce an "active molecule-Fc-Cn" conjugate molecule.

[0064] The present application also provides a method for preparing a conjugate molecule having a structure of "antibody-Cn", comprising (a) coupling an antibody with Cn containing a fatty acid chain under conditions that allow conjugation of the antibody and Cn to produce an "antibody-Cn" conjugate molecule.

[0065] The present application also provides a method for preparing a conjugated molecule having a structure of "active molecule-fusion protein-Cn", comprising (a) linking an active molecule polypeptide to a fusion protein to prepare an "active molecule-fusion protein" fusion; and (b) subjecting the "active molecule-fusion protein" fusion to a coupling reaction with Cn containing a fatty acid chain under conditions that allow the fusion protein to conjugate with Cn to produce an "active molecule-fusion protein-Cn" conjugated molecule.

[0066] In an alternative embodiment, the present application provides a method for preparing a conjugated molecule having a structure of "active molecule-Fc-Cn", comprising the steps of (a) linking an antibody Fab fragment to an immunoglobulin Fc region to prepare a "Fab-Fc" fusion, and (b) subjecting the "Fab-Fc" fusion to a coupling reaction under conditions that allow conjugation of the Fc region to a Cn containing a fatty acid chain to produce a "Fab-Fc-Cn" conjugated molecule. In one embodiment, the Fab and Fc are derived from the same or different antibody molecules.

[0067] In an alternative embodiment, the present application provides a method for preparing a conjugated molecule having a structure of "active molecule-Fc-Cn", which includes the step of subjecting a whole antibody to a coupling reaction under conditions that allow conjugation of the Fc region to a Cn containing a fatty acid chain to produce an "active molecule-Fc-Cn" conjugated molecule.

[0068] In an alternative embodiment, the present application provides a method for preparing a conjugated molecule having a structure of "antibody-Cn", which includes the step of subjecting a whole antibody to a coupling reaction under conditions that allow conjugation of the Fc region to a Cn containing a fatty acid chain to produce an "active molecule-Fc-Cn" conjugated molecule.

[0069] In a third aspect, the present application provides a composition, such as a pharmaceutical composition, comprising the conjugate molecule of the first aspect. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0070] In a fourth aspect, the present application provides a method for effectively prolonging the serum half-life of an active molecule, comprising the steps of constructing the active molecule into a conjugated molecule having a structure of "active molecule-Fc-Cn", a conjugated molecule having a structure of "antibody-Cn", or a conjugated molecule having a structure of "active molecule-fusion protein-Cn" according to the method of the second aspect, thereby effectively improving the serum half-life of the active molecule.

[0071] In a fifth aspect, the present application provides use of the conjugated molecule described in the first aspect, or the composition described in the third aspect, in the preparation of a compound for treating human diseases.

[0072] In one embodiment, the present invention provides the conjugate molecule of the first aspect, or the composition of the third aspect for use in therapy.

[0073] In a sixth aspect, the present application provides a method for treating human diseases, comprising administering an effective amount of the conjugated molecule described in the first aspect of the present application, or the composition described in the third aspect to a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 shows the HIC-HPLC results of the GLP1-Fc-TM1 conjugated products. The upper figure shows the HIC-HPLC results of GLP1-Fc without TM1 conjugation, and the lower figure shows the HIC-HPLC results of GLP1-Fc-TM1 after conjugation with TM1. "0" in the lower figure indicates GLP1-Fc without TM1 conjugation, "2" indicates GLP1-Fc conjugated with two TM1s, and "4" indicates GLP1-Fc conjugated with four TM1s.

[0075] Figure 2 shows the HIC-HPLC results of the HX006-TM1-1 coupling product. The upper figure shows the HIC-HPLC results of HX006 without TM1 coupling, and the lower figure shows the HIC-HPLC results of HX006-TM1-1 after TM1 coupling. "0" in the lower figure represents HX006 without TM1 coupling, "2" represents HX006 coupled with two TM1s, "4" represents HX006 coupled with four TM1s, and "6" represents HX006 coupled with six TM1s.

[0076] Figure 3 shows the HIC-HPLC results of the HX006-TM1-2 coupling products, where "0" represents HX006 without TM1 coupling, "2" represents HX006 coupled with two TM1s, "4" represents HX006 coupled with four TM1s, "6" represents HX006 coupled with six TM1s, and "8" represents HX006 coupled with eight TM1s.

[0077] Figure 4 shows the HIC-HPLC results of the HX008-TM1 coupling products. Figure 4A is the HIC-HPLC result of HX008 without TM1 coupling. Figure 4B is the HIC-HPLC result of HX008-TM1-2 after TM1 coupling. "0" in the figure represents HX008 without TM1 coupling, "2" represents HX008 coupled with two TM1s, "4" represents HX008 coupled with four TM1s, and "6" represents HX008 coupled with six TM1s. Figure 4C is the HIC-HPLC result of HX008-TM1-3 after TM1 coupling. "0" in the figure represents HX008 without TM1 coupling, "2" represents HX008 coupled with two TM1s, "4" represents HX008 coupled with four TM1s, and "6" represents HX008 coupled with six TM1s.

[0078] FIG5 shows the ELISA detection results of GLP1-Fc-TM1 binding to HSA, wherein HX042 represents GLP1-Fc.

[0079] FIG6 shows the ELISA detection results of HX006-TM1 binding to HSA.

[0080] Figure 7 shows the ELISA test results of HX008-TM1 binding to HSA.

[0081] FIG8 shows the HIC-HPLC results after GLP1-Fc was coupled to C18-tert-butyl ester. “0” in the figure indicates no coupling to C18-tert-butyl ester, and “2” indicates coupling to two C18-tert-butyl esters.

[0082] FIG9 shows the HIC-HPLC results of a GLP1-Fc sample (upper panel) and a GLP1-Fc-C16-NHS sample (lower panel).

[0083] FIG10 shows the HIC-HPLC results of the GLP1-Fc-C20-NHS sample.

[0084] FIG11 shows the ELISA test results of GLP1-Fc-C16-NHS and GLP1-Fc-C20-NHS binding to HSA, respectively, wherein HX042 represents GLP1-Fc.

[0085] FIG12 shows the ELISA test results of HX006-C16-NHS and HX006-C20-NHS binding to HSA.

[0086] FIG13 shows the binding activity of GLP1-Fc-C18-tert-butyl ester to HSA.

[0087] FIG14 shows the binding activity of HX006-C18-tert-butyl ester to HSA.

[0088] FIG15 shows the HIC-HPLC results after coupling HX006 with C18-tert-butyl alcohol ester.

[0089] FIG16 shows that GLP-1-Fc-TM1 can effectively activate the biological activity of the reporter gene.

[0090] FIG17 shows the plasma concentration-time curve of GLP1-Fc-TM1 after a single administration in rats, with dulaglutide as a positive control.

[0091] FIG18 shows the plasma concentration-time curve of GLP1-Fc-TM1 after a single administration in cynomolgus monkeys, with dulaglutide as a positive control.

[0092] Figure 19 shows the efficacy of GLP-1-Fc-TM1 on type II diabetic db / db mice, wherein Figure 19-1 shows the results of reducing 4-hour fasting blood glucose in db / db mice; Figures 19-2 and 19-3 show the results of random blood glucose after the first and last administration; Figures 19-4 and 19-5 show the blood glucose AUC of the OGTT test after the last administration. 0-180min Figure 19-6 shows the results of reducing the glycated hemoglobin content in db / db mice; Figure 19-7 shows the results of increasing the insulin level in db / db mice; Figure 19-8 shows the results of reducing the average daily food intake in db / db mice.

[0093] Figure 20 shows the efficacy results of GLP-1-Fc-TM1 on DIO model mice, wherein Figure 20-1 shows the results of GLP-1-Fc-TM1 reducing the body weight of DIO mice; Figure 20-2 shows the results of GLP-1-Fc-TM1 reducing the food intake of DIO mice; Figure 20-3 shows the results of GLP-1-Fc-TM1 reducing the body fat content of mice; Figure 20-4 shows the results of GLP-1-Fc-TM1 reducing the fasting blood glucose of mice; Figure 20-5 shows the results of GLP-1-Fc-TM1 reducing the blood lipid content of mice; Figure 20-6 shows the results of ALT and AST levels of serum liver function indicators in mice; Figure 20-7 shows the results of GLP-1-Fc-TM1 improving the ballooning and fatty degeneration of liver tissue in DIO mice.

[0094] Figure 21 shows the pharmacological effects of GLP-1-Fc-TM1 on rats, wherein Figures 21-1 to 21-4 show the results of GLP-1-Fc-TM1 lowering blood glucose in SD rats, and Figures 21-5 to 21-8 show the results of GLP-1-Fc-TM1 increasing serum insulin.

[0095] Detailed Description of the Invention

[0096] I. Definition

[0097] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0098] When a trade name is used herein, unless the context indicates otherwise, the trade name includes the product formulation, generic drug, and active pharmaceutical ingredient of the trade name product.

[0099] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0100] The term "and / or" should be understood to mean any one of the options or a combination of any two or more of the options.

[0101] The term "comprising" or "including" means including the stated elements, integers, or steps, but does not exclude any other elements, integers, or steps. In this document, when the term "comprising" or "including" is used, unless otherwise indicated, it also covers the situation consisting of the stated elements, integers, or steps. For example, when it is mentioned that an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0102] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, or antibody fragments thereof that exhibit the desired antigen-binding activity. A complete antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but may comprise fewer chains in certain circumstances, for example, antibodies naturally occurring in camels may comprise only heavy chains.

[0103] The term "whole antibody" refers to an immunoglobulin molecule comprising at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The heavy chain of an antibody can be divided into five main different types based on the amino acid sequence of its constant region: IgA, IgD, IgE, IgG, and IgM, and several of these types can be further divided into subclasses, such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2.

[0104] The terms "antibody fragment" and "antigen-binding fragment" of an antibody are used interchangeably and refer to molecules that are not complete antibodies, which contain a portion of an intact antibody that is used to bind to the antigen to which the intact antibody binds. As will be appreciated by those skilled in the art, to achieve antigen binding purposes, antibody fragments generally contain amino acid residues from a "complementarity determining region" or "CDR." Antibody fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Examples of antibody fragments include, but are not limited to, Fab, scFab, disulfide-linked scFab, Fab', F(ab')2, Fab'-SH, Fv, scFv, and disulfide-linked scFv. In some embodiments, the antibody fragment contains a cysteine ​​residue introduced into the Fc region to provide an amino acid residue site that can be used for sulfhydryl coupling chemistry.

[0105] Herein, when referring to an antibody as an IgG antibody, it is meant that the antibody is a heterotetrameric protein having an IgG class immunoglobulin structure. In IgG antibodies, typically the VH-CH1 of the heavy chain is paired with the VL-CL of the light chain to form a Fab fragment that specifically binds to the antigen. Therefore, an IgG antibody is essentially composed of two Fab molecules connected by the immunoglobulin hinge region and two dimerized Fc regions. IgG immunoglobulins can be divided into subclasses based on the sequence of the heavy chain constant region, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), and γ4 (IgG4). In some embodiments, the antibody according to the present invention is an IgG antibody, such as IgG1, IgG2, IgG3 or IgG4 antibody.

[0106] The term "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" refers to the region of the antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen-contacting residues ("antigen contact points"). The CDR is primarily responsible for binding to the antigen epitope.

[0107] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to its antigen. The heavy chain variable region (VH) and light chain variable region (VL) can be further divided into hypervariable regions (HVRs, also known as complementarity determining regions (CDRs)), which are interspersed with more conserved regions (i.e., framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0108] The term "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. Affinity can be measured by common methods known in the art. One method for measuring affinity is the ELISA assay, and another method is the surface plasmon resonance (SPR) assay described in the Examples herein.

[0109] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions, such as various Ig subtypes and their allotypic Fc region sequences (Gestur Vidarsson et al., IgG subclasses and allotypes:from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the heavy chain carboxyl terminus. However, the C-terminal terminal lysine (Lys447) in the Fc region may be present or absent. In further embodiments, the human IgG heavy chain Fc region carries a hinge sequence or a partial hinge sequence of a natural immunoglobulin at the N-terminus, such as, according to EU numbering, a sequence from E216 to T225 or a sequence from D221 to T225. In certain embodiments, the Fc region of an immunoglobulin comprises two constant domains, namely, CH2 and CH3. In other embodiments, the Fc region of an immunoglobulin comprises three constant domains, namely, CH2, CH3, and CH4.

[0110] The EU numbering scheme is a widely adopted standard for numbering residues in antibodies in a consistent manner, developed based on sequence alignments. In the context of the present application, unless otherwise indicated, the EU numbering scheme of Kabat is used to number and refer to amino acid residues in antibody or fusion protein regions (as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0111] "Fc region mutant", "mutant Fc region" and "mutated Fc region" and similar terms are used interchangeably to refer to an Fc region that comprises at least one amino acid modification and is different from a native sequence Fc region / wild-type Fc region. For example, amino acids at multiple positions in the Fc region of a wild-type immunoglobulin IgG are modified to reduce half-antibody formation, reduce or eliminate effector function, and increase serum half-life. Various methods disclosed in the prior art can be used to further modify the Fc regions known in the prior art, the Fc regions provided in this application, fusion proteins (such as antibodies) comprising the Fc region, etc., for example, to reduce immunogenicity, improve stability, solubility, function and other modifications for clinical benefits. Such modifications include, but are not limited to, the following modifications of, for example, IgG Fc, such as at positions 214-238, 250-260, 297-299, 307-318, 322 or 327 to 331, 380-390, etc., specifically, for example, modifications to positions 228, 233, 234, 235, 252, 254, 256, 297, 307, 308, 311, 380, 385, 386, 389, 428, 434, and 447. The hinge sequence of native human IgG can be modified to express the Fc region as a homogeneous product. In order to improve the chemical stability of the Fc region, asparagine, which is prone to deamidation, can be modified, for example, by replacing it with glutamine, aspartic acid, or glutamic acid, such as substitutions including N297E, N315Q, and N384Q. To improve the physical stability of the Fc region, the leucine at position 235 of the Fc region can be modified, such as by replacing it with L235K. In some embodiments, the immunoglobulin Fc region can also be modified by phosphorylation, sulfation, glycosylation, methylation, acetylation, amidation, etc. to meet specific needs.

[0112] In one embodiment, the modification of the Fc region is a modification of positions 254, 308 and 434, such as the modifications disclosed in CN108299560A. In another embodiment, the modification of the Fc region is a modification as described in CN1802167. In a specific embodiment, the modification of the Fc region is a modification of positions 228, 234, 235 and / or 447, such as modification S228P, F234A, L235A or modification S228P, F234A, L235A and 447 deletion. In this application, the modification of the Fc region is shown in brackets, such as IgG4 Fc (S228P, F234A, L235A) indicates that the amino acids at positions 228, 234 and 235 of the wild-type IgG4 Fc are substituted accordingly.

[0113] Wild-type immunoglobulin Fc regions can be obtained from humans and animals (e.g., cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs), or recombinant forms or derivatives of Fc regions can be obtained from transformed animal cells or microorganisms. For example, the gene encoding the immunoglobulin can be isolated from a corresponding cDNA library using PCR, or the Fc region can be obtained by protease treatment of intact immunoglobulins. For techniques for preparing Fc region derivatives, see, for example, WO 97 / 34631 and WO 96 / 32478.

[0114] In some embodiments, the Fc region used in the present application is derived from an IgG immunoglobulin, such as an Fc region derived from IgG1, IgG2, IgG3, and IgG4 subclasses, preferably an Fc region derived from IgG1 and IgG4 subclasses. In some embodiments, the Fc region used in the present application is an Fc region derived from human IgG1 and IgG4 to reduce the immunogenicity of the conjugate of the present application.

[0115] In some embodiments, the variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by one or more amino acid substitutions, deletions, or additions. In some embodiments, the variant Fc region has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to a wild-type Fc region and / or a parent Fc region.

[0116] The term "neonatal receptor (FcRn)" refers to an IgG antibody receptor located on the cell membrane. FcRn is responsible for transferring maternal IgG to the fetus and regulating immunoglobulin homeostasis in the body. FcRn binds to the Fc portion of IgG, preventing lysosomal cleavage of IgG molecules. This increases the half-life of IgG in the body and participates in the transport, maintenance, and distribution of IgG in the body.

[0117] The term "receptor-mediated endocytosis" refers to a process in which a ligand / receptor complex is internalized and delivered into the cytosol or translocated to an appropriate intracellular compartment, triggered by the binding of a ligand to a corresponding receptor on the cell surface.

[0118] The term "sequence identity" refers to the degree of sequence identity based on Nucleotide or amino acid sequence in a comparison window. "Sequence identity percentage ratio" can be calculated in the following way: the sequences of two optimal comparisons are compared in a comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residues (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) in two sequences, the number of the position to obtain the number of matching positions, the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and the result is multiplied by 100, to produce the sequence identity percentage ratio. For determining the optimal comparison of the sequence identity percentage ratio, it can be realized in several ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of interest.

[0119] In the present invention, with respect to antibody sequences, the percentage of amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the given antibody sequence, in a preferred embodiment, according to the Kabat numbering convention. In this article, without specifying a comparison window (i.e., the region of the target antibody to be compared), the comparison will be applied to the full length of the given antibody sequence. In some embodiments, with respect to antibodies, the sequence identity can be distributed over the entire heavy chain variable region and / or the entire light chain variable region, or the sequence percentage identity can be limited to the framework region only, while the sequences in the corresponding CDR regions remain 100% identical.

[0120] An "amino acid substitution" refers to the replacement of at least one amino acid residue present in a predetermined amino acid sequence with another different "substitution" amino acid residue.

[0121] The term "conservative substitution" refers to the substitution of one amino acid with another within the same class, such as one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid.

[0122] In the context of the conjugates described in this application, the term "peptide linker" refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T), used alone or in combination, or the hinge region from an immunoglobulin, which connects the active ingredient and the Fc region.

[0123] Peptide linkers that can be used in the present invention can be easily determined by those skilled in the art. For example, a peptide linker comprising the amino acid sequence (G4S) n , wherein n is an integer equal to or greater than 1. In a preferred embodiment, the peptide linker includes (G4S)3, (G4S)4, (G4S)6, GS(G4S)4, DAAALEAAALDAAAREAAARDAAAL, NVDHLPSNTLVDLA. The peptide linker that can be used in the antibody molecule of the present invention can also be, for example but not limited to, the following amino acid sequence: (G3S)2, (G4S)2, (G3S)3, (G4S)3, (G3S)4, (G4S)4, (G3S)5, (G4S)5, (G3S)6, (G4S)6, GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP and GSTSGSGK PGSGEGSTKG.

[0124] The term "alkyl" refers to a straight or branched saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms. Specifically, an alkyl group has 1 to 10 carbon atoms, such as 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 carbon atoms. For example, as used herein, the term "C1-C6 alkyl" refers to a straight or branched saturated hydrocarbon group with 1 to 6 carbon atoms, examples of which include methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl or tert-butyl), pentyl (including n-pentyl, isopentyl, neopentyl), hexyl (including n-hexyl, 2-methylpentyl, 3-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl), etc.

[0125] The term "alkylene" refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight or branched saturated alkane. Specifically, the alkylene group has 1-10 carbon atoms, such as 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. For example, as used herein, the term "C1-C6 alkylene" refers to a straight or branched alkylene group having 1 to 6 carbon atoms, including, but not limited to, methylene, ethylene, propylene, butylene, etc.

[0126] The term "cycloalkyl" refers to a monocyclic, fused polycyclic, bridged polycyclic, or spirocyclic non-aromatic monovalent hydrocarbon ring structure having the specified number of ring atoms, which may be saturated or unsaturated, e.g., containing one or more double bonds. A cycloalkyl group may contain 3 or more, e.g., 3-18, 3-10, or 3-8 carbon atoms in the ring, e.g., C 3-10 Cycloalkyl, C 3-8 Cycloalkyl, C 3-6 Cycloalkyl, C 5-6 Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0127] The term "alkenyl" refers to a straight or branched unsaturated hydrocarbon group containing at least one double bond consisting of carbon atoms and hydrogen atoms. Specifically, the alkenyl group has 2-8, such as 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkenyl" refers to a straight or branched alkenyl group with 2 to 6 carbon atoms, such as vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl etc.

[0128] The term "alkenylene" refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight or branched unsaturated olefin containing at least one double bond. Specifically, the alkenylene group has 2-8, such as 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkenylene" refers to a straight or branched alkenylene group having 2 to 6 carbon atoms, such as vinylene, propenylene, allylene, butenylene, pentenylene, and hexenylene.

[0129] The term "alkynyl" refers to a straight or branched unsaturated hydrocarbon group containing at least one triple bond consisting of carbon atoms and hydrogen atoms. Specifically, the alkynyl group has 2-8, such as 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkynyl" refers to a straight or branched alkynyl group with 2 to 6 carbon atoms, such as ethynyl, propynyl, propargyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-methyl-1-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, etc.

[0130] The term "alkynylene" refers to a divalent group derived from the removal of two hydrogen atoms from the same or two different carbon atoms of a straight or branched unsaturated alkyne containing at least one triple bond. Specifically, the alkynylene group has 2-8, e.g., 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkynylene" refers to a straight or branched alkynylene group having 2 to 6 carbon atoms, such as ethynylene, propynylene, propargylene, butynylene, pentynylene, and hexynylene.

[0131] The term "heterocycle" or "heterocyclyl" refers to an aromatic or non-aromatic monocyclic, bicyclic or polycyclic ring system having 1-4 heteroatom ring members independently selected from N, O or S. One or more N, C or S atoms in the heterocycle can be oxidized. Preferably, the heterocycle is a 5-10 ring system, which is a monocyclic or fused bicyclic ring. Representative examples include but are not limited to pyrrolidine, azetidine, piperidine, morpholine, tetrahydrofuran, tetrahydropyran, benzofuran, benzothiophene, indole, benzopyrazole, pyrrole, thiophene (thiophene), furan, thiazole, imidazole, pyrazole, pyrimidine, pyridine, pyrazine, pyridazine, isothiazole and isoxazole. It should be understood that the term includes heteroaryl as defined herein.

[0132] The term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6 to 20, for example 6 to 12, carbon atoms in the ring portion. Preferably, aryl is (C6-C 10 ) aryl. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which may be optionally substituted with 1 to 4 substituents such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, mercapto, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamido, heterocyclyl, and the like.

[0133] The term "heteroaryl" refers to a 5-20 membered (e.g., 5-14 membered, 5-8 membered, 5-6 membered) aromatic monocyclic or polycyclic ring system containing 1-4 heteroatoms selected from N, O or S, which may be substituted or unsubstituted. Preferably, the heteroaryl is a 5-10 membered ring system, which is a monocyclic or fused bicyclic ring. Representative heteroaryl groups include 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, 2-, 4-, or 5-pyrimidinyl.

[0134] The term "heteroalkyl" refers to a stable straight or branched chain hydrocarbon radical which is fully saturated or contains from 1 to 3 degrees of unsaturation, and consists of the indicated number of carbon atoms and from one to ten, preferably from one to three, heteroatoms selected from O, N, Si and S, wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, Si and S may be placed at any interior position of the heteroalkyl radical or at the position at which the heteroalkyl radical is attached to the remainder of the molecule. Representative examples of heteroalkyl groups include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -NH-CH2-CH2-NH-C(O)-CH2-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=NO-CH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Typically, a C1 to C4 heteroalkyl or heteroalkylene group has 1 to 4 carbon atoms and 1 or 2 heteroatoms, and a C1 to C3 heteroalkyl or heteroalkylene group has 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some aspects, the heteroalkyl and heteroalkylene groups are saturated.

[0135] Unless otherwise indicated, the term "substituted" as used herein when defining various groups means that the corresponding group may be substituted with groups such as, but not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, halogen, cyano, nitro, azido, carboxyl, hydroxyl, thiol, amino, mono- or dialkylamino, mono- or dicycloalkylamino, mono- or diarylamino, mono- or diheterocyclylamino, mono- or diheteroarylamino, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-oxy, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-thio, alkyl- or cycloalkylamino, mono- or diarylamino, mono- or diheterocyclylamino, mono- or diheteroarylamino, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl- or aryl-sulfonyl, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-sulfonyloxy, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-sulfonyloxy, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-sulfonylamino, or the above optionally substituted amino-formyl, and each of which is further substituted by remaining optional substituents, wherein each type of group is as defined herein. Examples of substituents include, but are not limited to, one or more groups independently selected from the group consisting of halogen, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, C(O)-amino, OCOCH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO2, phenyl, piperidinyl, piperazinyl, and pyrimidinyl.

[0136] The term "PEG unit" refers to an organic moiety comprising repeated ethyleneoxy subunits (PEG or PEG subunits), which can be polydisperse, monodisperse or discrete (i.e., having a discrete number of ethyleneoxy subunits). Polydisperse PEGs are heterogeneous mixtures of size and molecular weight, while monodisperse PEGs are typically purified from heterogeneous mixtures and therefore have a single chain length and molecular weight. Preferred PEG units comprise discrete PEGs, which are compounds synthesized in a stepwise manner rather than via a polymerization process. Discrete PEGs provide single molecules with defined and specified chain lengths.

[0137] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0138] The term "DAR" as used herein refers to the ratio of the Cn moiety coupled to the Fc described herein to the Fc in a conjugate, or the ratio of the Cn moiety coupled to the antibody described herein to the antibody, or the ratio of the Cn moiety coupled to the fusion protein described herein to the fusion protein. In some embodiments described herein, the DAR may be 1 to 20, such as 1-18, 4-16, 5-12, 6-10, 1-8, 2-8, 1-6, 2-6, 2-4, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. DAR can also be calculated as the average DAR of a population of molecules in the product, i.e., the total ratio of Cn conjugated to the Fc portion described herein to the Fc portion, the total ratio of Cn conjugated to the antibody described herein to the antibody, or the total ratio of Cn conjugated to the fusion protein described herein to the fusion protein in the product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), which DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugate of the invention is 1 to 20, e.g., 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5. 1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0, or a range having two of these values ​​as endpoints.

[0139] The term "drug" as used herein encompasses any substance that is effective in preventing or treating diseases associated with glucose metabolism disorders, cardiovascular and cerebrovascular diseases, kidney diseases, and retinopathy. Diseases associated with glucose metabolism disorders include, for example, diabetes (e.g., type I diabetes, type II diabetes), obesity, hypertension, dyslipidemia, obesity, glucose intolerance, hyperglycemia, hyperinsulinemia, cardiovascular disease, and the like.

[0140] The term "active ingredient" as used herein refers to a substance that has any physiologically active function beneficial to cells and / or the body (e.g., regulating gene expression and physiological function, correcting abnormal conditions caused by the lack or excessive secretion of components involved in regulating body functions), and is generally an active peptide substance. Examples include, but are not limited to, enzymes, enzyme inhibitors, antigens, antibodies, antibody fragments, hormones, glucagon-like peptide-1 (GLP-1), glucagon, interferon, cytokines, growth factors and / or differentiation factors, factors involved in cell motility or migration, factors involved in bone tissue development / resorption, chemokines, plasma or interstitial adhesion molecules or extracellular matrix, bactericidal or antifungal factors, etc.

[0141] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to a subject to which the composition is administered. In some embodiments, the pharmaceutical composition of the present invention comprises a conjugated molecule of the present invention and a pharmaceutical excipient.

[0142] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, buffer, surfactant, carrier, stabilizer, etc., which is administered together with the active substance.

[0143] The term "pharmaceutical combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit and a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients are administered to a patient as separate entities simultaneously, without specific time restrictions, or sequentially at equal or different time intervals, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient's body. In some embodiments, the molecules of the present invention and other therapeutic agents used in the pharmaceutical combination are administered at levels no greater than when they are used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0144] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0145] As used herein, the terms "individual" or "subject" are used interchangeably to refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the subject is a human.

[0146] As used herein, "treat," ...

[0147] When used herein, " prevention " includes the inhibition of the occurrence or development of the symptom of disease or illness or specific disease or illness.In some embodiments, the subject with a family history of a disorder of glucose metabolism related disease (such as diabetes) is a candidate for a preventive regimen.Usually, in the context of a disorder of glucose metabolism related disease (such as diabetes), the term " prevention " refers to the administration of medicine before the signs or symptoms of a disorder of glucose metabolism related disease (such as diabetes) occur, particularly before the occurrence in a subject with a risk of a disorder of glucose metabolism related disease (such as diabetes).

[0148] As used herein, the term "effective amount" refers to an amount or dosage of the conjugate / adjuvant of the present invention or a composition or combination thereof, which produces the desired effect in a patient in need of treatment or prevention after administration in single or multiple doses to the patient.

[0149] As used herein, the term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the conjugate / conjugate of the present invention or its composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably achieves at least about 30%, even more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100% inhibition or reduction of a measurable parameter (e.g., glucagon secretion, blood glucose concentration) relative to an untreated individual.

[0150] As used herein, the term "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Typically, a prophylactic effective amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0151] II. Pharmaceutical Compositions and Kits

[0152] Pharmaceutically acceptable salt forms of the conjugate molecules of the present invention are within the scope of the present invention. In some embodiments, the present invention provides a composition comprising any of the conjugate molecules described herein and pharmaceutically acceptable salts thereof, preferably the composition is a pharmaceutical composition or pharmaceutical formulation.

[0153] In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, a composition, eg, a pharmaceutical composition, comprises a conjugate molecule of the invention in combination with one or more other therapeutic agents.

[0154] The compositions disclosed herein may further comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0155] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0156] For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0157] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0158] Medicaments comprising the conjugate molecules described herein can be prepared by mixing the conjugate molecules of the present invention having the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0159] The pharmaceutical composition or preparation of the present invention may also include more than one active ingredient, which is required for the specific indication being treated, preferably having those active ingredients of complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists). The active ingredient is suitably present in combination in an amount effective for the purpose.

[0160] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the conjugated molecules, which matrices are in the form of shaped articles, eg films, or microcapsules.

[0161] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising a conjugate molecule of the present invention and one or more other therapeutic agents.

[0162] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0163] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0164] - a first container containing a conjugated molecule of the invention;

[0165] - A second container of the pharmaceutical composition containing the other therapeutic agent.

[0166] III. Uses and Methods of Treatment

[0167] The long-acting platforms provided in the present application, namely the active molecule-fusion protein-Cn conjugate platform, the active molecule-Fc-Cn conjugate platform, and the antibody-Cn conjugate platform, can be used to effectively prolong the serum half-life of the active molecule. Specifically, the active molecule is constructed into a conjugate molecule with a structure of "active molecule-Fc-Cn", a conjugate molecule with a structure of "active molecule-fusion protein-Cn", and a conjugate molecule with a structure of "antibody-Cn" through the method disclosed in the present application, thereby effectively increasing the serum half-life of the active molecule, thereby reducing the frequency of administration, reducing the dosage, and saving costs.

[0168] The present application uses the above-mentioned long-acting platform to extend the serum half-life of the active molecule without affecting the biological function of the active molecule. The conjugated molecules with the structure of "active molecule-Fc-Cn", the conjugated molecules with the structure of "active molecule-fusion protein-Cn", and the conjugated molecules with the structure of "antibody-Cn" have low immunogenicity to the body, effectively avoiding the negative impact of common conjugated molecules that easily cause immune responses in the body.

[0169] The "active molecule-Fc-Cn" conjugate molecules, "active molecule-fusion protein-Cn" conjugate molecules, and "antibody-Cn" conjugate molecules obtained herein can be used to prevent or treat a variety of diseases in subjects. Those skilled in the art can readily determine the diseases that can be treated or prevented by the conjugate molecules based on the biological function of the active molecule. For example, when the active molecule is GLP-1, the conjugate molecules can be used to effectively treat metabolic diseases and / or disorders, such as diabetes, such as type I diabetes, type II diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity, metabolic syndrome, cardiovascular disease, and the like.

[0170] When the active molecule is an anti-PD-1 antibody or an antigen-binding fragment thereof, the conjugated molecule can be used to effectively prevent or treat diseases associated with abnormal PD-1 expression, such as various tumors or cancers, such as melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, Hodgkin's lymphoma, head and neck cancer, ovarian cancer, and brain cancer.

[0171] When the active molecule is an anti-VEGF antibody or an antigen-binding fragment thereof, the conjugated molecule can be used to effectively prevent or treat diseases associated with abnormal VEGF expression, such as various diseases associated with angiogenesis, for example, ophthalmic diseases, such as wet or neovascular age-related macular degeneration (AMD) and diabetic macular edema (DME); most cancers; and cardiovascular diseases.

[0172] The present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the conjugate molecule of the present invention or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, a pharmaceutical combination or a pharmaceutical kit.

[0173] In the treatment methods of the present invention, the administration of the conjugated molecules of the present invention may include 1) therapeutic measures that cure, slow down, alleviate the symptoms of the diagnosed pathological condition or disease and / or stop the progression of the diagnosed pathological condition or disease; or 2) preventive or prophylactic measures that prevent and / or slow the development of the pathological condition or disease. In some embodiments, in the treatment methods of the present invention, the individual will benefit from the therapeutic measures or prophylactic measures and show a reduction or improvement in the occurrence, recurrence or development of the disease, disorder, condition, and / or symptom compared to an individual who has not received the treatment.

[0174] The pharmaceutical compositions of the present invention can be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending on whether the administration is short-term or long-term, the administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection. Various administration schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0175] For the prevention or treatment of disease, the appropriate dosage of the pharmaceutical compositions of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease being treated, the specific type of active molecule in the conjugate molecule, the severity and course of the disease, the purpose of the treatment, previous therapy, the patient's clinical history and response to the drugs, and the judgment of the attending physician.

[0176] In some embodiments, the present invention also provides use of the pharmaceutical composition of the present invention in the preparation of a medicament for the aforementioned treatment and prevention methods.

[0177] These and other aspects and embodiments of the present invention are exemplified in the following examples. Any or all of the features described above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention; however, it should be understood that the examples are for illustrative purposes only and should not be construed as constituting any limitation.

[0178] The abbreviations used in the specification and claims have the following meanings: AUC Area under the curve CV Column volume HSA Human serum albumin PBS Phosphate buffered saline tBu Tert-butyl Pbf 2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl Trt Trityl Mmt 4-Methoxytriphenyl Mtt Methyltrityl Alloc (2-propenyloxy)carbonyl DCM Dichloromethane DCC Dicyclohexylcarbodiimide DMF N,N-Dimethylformamide DMAP 4-dimethylaminopyridine DIPEA N,N-Diisopropylethylamine DIC N,N-Diisopropylcarbodiimide HBTU Benzotriazole-N,N,N",N"-tetramethyluronium hexafluorophosphate HATU 2-(7-Azobenzotriazole)-N,N,N",N"-tetramethyluronium hexafluorophosphate HPLC High performance liquid chromatography TBTU O-Benzotriazole-N,N,N",N"-tetramethyluronium tetrafluoroborate HOBT 1-Hydroxybenzotriazole HOAT 1-Hydroxy-7-azobenzotriazole TFA Trifluoroacetic acid TIS Triisopropylsilane TCEP Tris(2-carboxyethyl)phosphine hydrochloride TSTU O-(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate Example

[0179] Example 1. Preparation of higher fatty acid chains

[0180] In this example, a variety of higher fatty acid chains that can react with different sites on the Fc region (or fusion protein), such as free sulfhydryl groups and amino groups, were constructed.

[0181] 1.1 Preparation of higher fatty acid chain TM1

[0182] The fatty acid chain TM1 was prepared according to the following technical route:

[0183] (1) Compound 1 (5.0 g, 29.40 mmol) was dissolved in anhydrous DCM (50 mL) and stirred. EDCI (5.62 g, 29.4 mmol) was added, followed by compound 1-2 (4.35 g, 29.40 mmol). The mixture was reacted at 25°C for 2 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to give a colorless oil 2 (5.5 g, 62.5%).

[0184] (2) Compound 2 (5.5 g, 18.30 mmol) was dissolved in anhydrous DCM (60 mL) and stirred. EDCI (3.50 g, 18.30 mmol) was added, followed by compound 2-1 (2.98 g, 18.30 mmol). The mixture was reacted at 25°C for 2 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to give a colorless oil 3 (4.8 g, 59.3%).

[0185] (3) Compound 3 (4.8 g, 10.78 mmol) was dissolved in anhydrous DCM (40 mL) and stirred. EDCI (2.06 g, 10.78 mmol) was added, followed by compound 3-1 (2.18 g, 10.78 mmol). The mixture was reacted at 25°C for 12 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to give a colorless oil 4 (4.0 g, 58.9%).

[0186] (4) Compound 4 (4.0 g, 6.34 mmol) was dissolved in anhydrous DCM (30 mL), stirred evenly, and TFA (5 ml) was added. The mixture was reacted at 25°C for 0.5 h. After the reaction, the mixture was concentrated to obtain a brown-black oil 5 (3.2 g, 88%).

[0187] (5) Compound 5 (3.2 g, 5.58 mmol) was dissolved in anhydrous DMF (30 mL) and stirred. DCC (1.14 g, 5.58 mmol) and DMAP (0.14 g, 1.16 mmol) were added, followed by compound 5-1 (1.75 g, 5.58 mmol). The mixture was reacted at 25°C for 2 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to obtain a colorless oil TM1 (2.1 g, 43.7%) with a molecular weight of 870.05.

[0188] TM1 structure analysis:

[0189] Mass Spectrometry:

[0190] MS-ESI (m / z): 870.4 [M+H]+

[0191] H NMR spectrum:

[0192] 1 H NMR(400MHz,DMSO-d6)δ8.02(S,2H,NH)7.88(S,1H,COOH)7.65(S,1H,COOH)6.97(S,2H,CH)4.13(S,1H,CH )3.67~3.69(S,2H,CH2)3.65~3.15(m,22H,CH2)2.33~1.17(m,10H,CH2)1.16(S,4H,CH2)1.21(S,26H,CH2)

[0193] C-NMR spectrum:

[0194] 13 C NMR(100MHz,DMSO-d6)δ174.91,173.91,172.88,171.95,171.13,169.77,134.94,70.63,70.3 8,69.33,51.94,40.45,40.25,39.83,39.20,35.53,34.20,32.15,29.57,27.49,25.70,24.95

[0195] 1.2 Preparation of higher fatty acid chain C18 esters

[0196] Fatty acid chain C18 ester (i.e., C18-tert-butyl ester) was prepared according to the following technical route:

[0197] (1) Compound 1 (5.0 g, 29.40 mmol) was dissolved in anhydrous DCM (50 mL) and stirred. EDCI (5.62 g, 29.4 mmol) was added, followed by compound 1-2 (4.35 g, 29.40 mmol). The mixture was reacted at 25°C for 2 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to give a colorless oil 2 (5.5 g, 62.5%).

[0198] (2) Compound 2 (5.5 g, 18.30 mmol) was dissolved in anhydrous DCM (60 mL) and stirred. EDCI (3.50 g, 18.30 mmol) was added, followed by compound 2-1 (2.98 g, 18.30 mmol). The mixture was reacted at 25°C for 2 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to give a colorless oil 3 (4.8 g, 59.3%).

[0199] (3) Compound 3 (4.8 g, 10.78 mmol) was dissolved in anhydrous DCM (40 mL) and stirred. EDCI (2.06 g, 10.78 mmol) was added, followed by compound 3-1 (2.18 g, 10.78 mmol). The mixture was reacted at 25°C for 12 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to give a colorless oil 4 (4.0 g, 58.9%).

[0200] (4) Compound 4 (4.0 g, 6.35 mmol) was dissolved in anhydrous DMF (40 mL) and stirred. DCC (1.31 g, 6.35 mmol) and DMAP (0.155 g, 1.27 mmol) were added, followed by compound 4-1 (2.35 g, 6.35 mmol). The mixture was reacted at 25°C for 2 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to obtain a colorless oily C18 ester (1.8 g, 28.8%) with a molecular weight of 982.27.

[0201] C18 ester structure analysis:

[0202] Mass Spectrometry:

[0203] MS-ESI (m / z): 982.5 [M+H]+

[0204] H NMR spectrum:

[0205] 1 H NMR(400MHz, DMSO-d6)δ8.11(m,2H,NH)7.91~7.62(m,2H,NH)7.01(S,2H,CH)3.87~3.59(m,1H,CH)3.57(S,2H,CH2)3.56~3.55(m,10H,CH2)3.50~3.28(m ,6H,CH2)3.21~3.16(m,6H,CH2)2.25(S,2H,CH2)2.16~2.10(m,6H,CH2)1.9 2~1.85(m,2H,CH2)1.40~1.35(m,22H,CH2,CH3)1.30~1.23(m,24H,CH2,CH3)

[0206] C-NMR spectrum:

[0207] 13 C NMR (100MHz, DMSO-d6) δ172.78, 171.78, 171.19, 169.95, 169.70, 135.02, 80.75, 79.76, 70.62, 70.42, 6 9.94, 69.81, 69.47, 69.35, 52.77, 38.94, 35.23, 34.53, 29.50, 29.38, 29.09, 28.81, 28.23, 28.09, 25.06

[0208] 1.3 Preparation of higher fatty acid chain C16-NHS

[0209] The fatty acid chain C16-NHS was prepared according to the following technical route:

[0210] (1) Compound 1 (3.0 g, 11.7 mmol) was dissolved in anhydrous DCM (30 mL) and stirred. DCC (2.41 g, 11.7 mmol) and DMAP (0.285 g, 2.34 mmol) were added and reacted at 25°C for 12 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to obtain a colorless oil 2 (2.1 g, 41.2%).

[0211] (2) Compound 2 (2.1 g, 4.76 mmol) was dissolved in anhydrous DCM (20 mL) and stirred. TSTU (1.43 g, 4.76 mmol) and DIPEA (0.614 g, 4.76 mmol) were added, followed by compound 2-1 (1.64 g, 14.28 mmol). The mixture was reacted at 25°C for 1 h. After the reaction, the mixture was concentrated to obtain a brown-black oil 3 (2.0 g, 78.1%).

[0212] (3) Compound 3 (2.0 g, 3.71 mmol) was dissolved in anhydrous DCM (20 mL) and stirred. 2 mL of TFA was added and the mixture was reacted at 25°C for 1 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to obtain a colorless oil, designated as C16 ester or C16-NHS (1.2 g, 70.5%), with a molecular weight of 482.26.

[0213] C16-NHS ester structure analysis:

[0214] Mass Spectrometry:

[0215] MS-ESI (m / z): 483.3 [M+H]+

[0216] H NMR spectrum:

[0217] 1 H NMR(400MHz,DMSO-d6)δ12.71(S,1H,COOH)8.20(S,1H,NH)4.32~4.31(m,1H,CH)2.88~2.71(m,6H ,CH2)2.18~2.17(m,4H,CH2)1.61~1.52(m,2H,CH2)1.30~1.28(m,24H,CH2)0.94~0.90(m,3H,CH3)

[0218] C-NMR spectrum:

[0219] 13C NMR (100MHz, DMSO-d6) δ173.43, 172.95, 170.59, 168.87, 51.21, 35.52, 31.77, 29.52, 29.18, 29.05, 27.60, 26.42, 25.90, 25.64, 22.56, 14.41

[0220] 1.4 Preparation of higher fatty acid chain C20-NHS

[0221] The fatty acid chain C20-NHS was prepared according to the following technical route:

[0222] (1) Compound 1 (5.0 g, 12.56 mmol) was dissolved in anhydrous DCM (50 mL) and stirred. DCC (2.58 g, 12.56 mmol) and DMAP (0.306 g, 2.51 mmol) were added, followed by compound 1-2 (2.54 g, 12.56 mmol). The mixture was reacted at 25°C for 2 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to obtain a colorless oil 2 (6.1 g, 83.56%).

[0223] (2) Compound 2 (6.1 g, 10.4 mmol) was dissolved in anhydrous DCM (60 mL) and stirred. DCC (2.14 g, 10.4 mmol) and DMAP (0.53 g, 2.08 mmol) were added, followed by compound 2-1 (3.2 g, 10.4 mmol). The mixture was reacted at 25°C for 2 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to give a colorless oil 3 (4.5 g, 51%).

[0224] (3) Compound 3 (4.5 g, 5.15 mmol) was dissolved in anhydrous DCM (40 mL), stirred evenly, and then 4 ml of TFA was added. The mixture was reacted at 25°C for 1 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to obtain a brown oil 4 (3.2 g, 82%).

[0225] (4) Compound 4 (3.2 g, 4.20 mmol) was dissolved in anhydrous DMF (40 mL) and stirred. DCC (0.865 g, 4.20 mmol) and DMAP (0.103 g, 0.84 mmol) were added, followed by compound 4-1 (0.483 g, 4.2 mmol). The mixture was reacted at 25°C for 2 h. After the reaction, the mixture was directly separated by preparative HPLC and concentrated to obtain a white solid, designated as C20 ester or C20-NHS (1.5 g, 41.7%), with a molecular weight of 859.02.

[0226] C20-NHS ester structure analysis:

[0227] Mass Spectrometry:

[0228] MS-ESI (m / z): 859.4 [M+H] +

[0229] H NMR spectrum:

[0230] 1 H NMR(400MHz,DMSO-d6)δ12.41(S,2H,COOH)8.11~7.72(m,3H,NH)4.66(S,1H,CH)3.92~3.61(m,4H,CH2)3.49~ 3.40(m,18H,CH2)3.44~3.33(m,4H,CH2)2.88~2.21(m,6H,CH2)2.18~2.10(m,2H,CH2)1.52~1.28(m,30H,CH2)

[0231] C-NMR spectrum:

[0232] 13 C NMR (100MHz, DMSO-d6) δ174.95, 174.00, 172.81, 171.87, 170.50, 169.72, 167.05, 70.84, 70.62, 7 0.41, 69.80, 69.56, 69.33, 66.23, 39.88, 34.12, 29.54, 29.37, 29.30, 29.01, 25.94, 25.69, 24.96

[0233] Example 2. GLP-1-Fc-TM1 sample preparation and DAR value detection

[0234] In this example, a commercial GLP-1-Fc fusion protein, dulaglutide, was used as an example to construct a conjugate based on the Fc-higher fatty acid chain platform of this application. Dulaglutide is composed of two identical long chains, one of which has the following amino acid sequence:

[0235] The amino acid sequence of the Fc contained in dulaglutide is as follows:

[0236] 1.1 Conjugation of GLP-1-Fc fusion protein to TM1

[0237] 7.5 mg of GLP-1-Fc fusion protein (dulaglutide, homemade) was transferred to reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) using a 15 ml 30 kDa ultrafiltration tube, and the volume was replaced four times. The final volume was approximately 1 ml, and the protein concentration was determined. TCEP (3 times the molar ratio) was added to the sample and incubated at 25°C in a water bath for 2 hours. The sample was then transferred to coupling buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) using a 15 ml 30 kDa ultrafiltration tube, and the protein concentration and free thiol group count were determined.

[0238] Based on the protein amount, 5-fold molar amount of TM1 was added to the GLP-1-Fc fusion protein sample, mixed, and reacted at 25°C for 2 h with shaking on a microshaker. Immediately after coupling, purification was performed by cationic chromatography.

[0239] The purified sample was named GLP-1-Fc-TM1 (also known as GLP1-Fc-TM1) and replaced into a temporary buffer (25 mM phosphate, 150 mM sodium chloride, pH 7.0) for the next step of detection.

[0240] 1.2 GLP1-Fc-TM1 sample DAR value detection

[0241] In this example, HIC-HPLC analysis was used to determine the coupling ratio (DAR) of GLP1-Fc and TM1. The analytical column used was a TSKgel Butyl-NPR (4.6 mm x 3.5 cm) column, and the analytical method was known, as described in the literature: Drug-to-Antibody Ratio (DAR) and Drug Load Distribution by Hydrophobic Interaction Chromatography and Reversed Phase High-Performance Liquid Chromatography. The results are shown in Figure 1. The average DAR value for TM1 coupling to the GLP1-Fc protein was 3.3.

[0242] Example 3. Preparation and Detection of GLP-1-Fc-C18

[0243] In this example, taking the commercial GLP-1-Fc fusion protein Dulaglutide as an example, a conjugate based on the Fc-higher fatty acid chain platform of the present application was constructed.

[0244] 8.34 mg of GLP1-Fc fusion protein (dulaglutide, homemade) was transferred to reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) using a 15 ml 30 kDa ultrafiltration tube. The volume was then exchanged four times to a final volume of approximately 3 ml, and the protein concentration was determined. TCEP (3x molar ratio) was added to the antibody and incubated at 25°C in a water bath for 2 hours. The sample was then exchanged four times using a 15 ml 30 kDa ultrafiltration tube into coupling buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5). The protein concentration and number of free thiol groups in the sample were then determined.

[0245] Based on the protein amount, add 3 times the molar amount of C18-tert-butyl ester to the GLP-1-Fc fusion protein sample, mix well, and react at 25°C for 2 hours with shaking on a microshaker. Immediately after coupling, purify by cationic chromatography.

[0246] The purified protein was named GLP-1-Fc-C18-tert-butyl ester (also known as GLP1-Fc-C18 tert-butyl ester) and replaced into a temporary buffer for the next step of detection.

[0247] In this example, the coupling ratio (DAR) of GLP1-Fc and C18-tert-butyl ester was determined by HIC-HPLC analysis according to the method of Example 2. The results are shown in FIG8 , and the average DAR value is 1.26.

[0248] Example 4. Sample preparation and detection of GLP1-Fc-C16-NHS

[0249] In this example, taking the commercial GLP-1-Fc fusion protein Dulaglutide as an example, a conjugate based on the Fc-higher fatty acid chain platform of the present application and linked via the amino group of the lysine residue on Fc was constructed.

[0250] 4.56 mg of GLP-1-Fc fusion protein was transferred to coupling buffer (0.1 M MOPS, 20 mM Tris, pH 7.5) using a 30 kDa ultrafiltration tube to a final volume of 0.9 ml, and the protein concentration was determined. A 6-fold molar amount of C16-NHS was added to the fusion protein sample. After mixing, the reaction was incubated at 25°C for 2 h with agitation on a microshaker. Immediately after coupling, the sample was purified by cationic chromatography. The sample was then transferred to holding buffer using a 30 kDa ultrafiltration tube and the concentration was determined. The purified product was designated GLP1-Fc-C16-NHS or GLP1-Fc-C16.

[0251] In this example, HIC-HPLC analysis was performed according to the method of Example 2. The results are shown in FIG9 , and the coupling rate of C16-NHS to GLP1-Fc was 83.07%.

[0252] Example 5 Sample Preparation and Detection of GLP1-Fc-C20-NHS

[0253] In this example, taking the commercial GLP-1-Fc fusion protein Dulaglutide as an example, a conjugate based on the Fc-higher fatty acid chain platform of the present application and linked via the amino group of the lysine residue on Fc was constructed.

[0254] 4.56 mg of GLP1-Fc fusion protein was transferred to coupling buffer (0.1 M MOPS, 20 mM Tris, pH 7.5) using a 30 kDa ultrafiltration tube to a final volume of 0.9 ml, and the protein concentration was determined. A 6-fold molar amount of C20-NHS was added to the fusion protein sample. After mixing, the reaction was incubated at 25°C for 2 h with agitation on a microshaker. Immediately after coupling, the protein was purified by cationic chromatography. The sample was then transferred to holding buffer using a 30 kDa ultrafiltration tube and the concentration was determined. The purified product was designated GLP1-Fc-C20-NHS or GLP1-Fc-C20.

[0255] In this example, HIC-HPLC analysis was performed according to the method of Example 2. The results are shown in FIG10 , and the coupling rate of C20-NHS to GLP1-Fc was 96.38%.

[0256] Example 6 Binding activity of GLP1-Fc-C16-NHS and GLP1-Fc-C20-NHS to HSA protein

[0257] In this example, the ELISA method was used to detect the activity of samples GLP1-Fc-C16-NHS and GLP1-Fc-C20-NHS obtained by lysine coupling of GLP1-Fc fusion protein with HSA protein.

[0258] Dilute HSA-his to 0.5 μg / ml using coating solution (carbonate buffer) and coat eight ELISA plates overnight at 4°C. Wash the plates three times with PBST (PBS containing 0.05% Tween 20), add 200 μl / well of blocking solution (1% BSA in PBST), and incubate at 37°C for 1 hour. Wash the plates four times with PBST, and dilute GLP1-Fc-C16-NHS and GLP1-Fc-C20-NHS samples to a starting concentration of 1000 nM using diluent (1‰ BSA in PBST). Then, perform a 5-fold serial dilution series (7 dilutions totaling 8 steps) and add 100 μl / well of each well to the ELISA plate. Incubate at 37°C for 1 hour. Wash the plates five times with PBST, and add 100 μl / well of HRP-Goat anti-Huamn 10000X working solution and incubate at 37°C for 1 hour. Wash the plate six times with PBST, add 100 μl / well of freshly prepared color development solution (5 ml substrate solution, 250 μl TMB stock solution, 16 μl 0.75% H2O2), and incubate at 37°C for 10 min. Add 50 μl / well of stop solution (2 M H2SO4), and measure the OD value at 450 nm.

[0259] The results are shown in Figure 11. The results show that the GLP1-Fc-C20-NHS sample coupled with C20-NHS can strongly bind to HSA-his, while the GLP1-Fc-C16-NHS sample coupled with C16-NHS binds weakly to HSA-his.

[0260] Example 7. Sample preparation of HX006 antibody coupled to TM1

[0261] In this example, the anti-VEGF antibody HX006 disclosed in CN 104804088 A was used as an example to construct a conjugate based on the Fc-higher fatty acid chain platform of the present application. The HX006 antibody is of IgG1 type, and its heavy chain sequence and light chain sequence are shown in the following table.

[0262] 6.53 mg of HX006 antibody protein was transferred to reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) using a 15 ml 30 kDa ultrafiltration tube, exchanged four times to a final volume of approximately 2 ml, and the protein concentration was determined. TCEP (2x molar ratio) was added to the antibody and incubated at 25°C in a water bath for 2 h. The protein was then exchanged four times using a 15 ml 30 kDa ultrafiltration tube into coupling buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5). The sample was then assayed for protein concentration and free thiol group count.

[0263] Based on the amount of protein, add 2 times the molar amount of TM1 to the antibody sample, mix well, and react at 25°C for 2 hours with shaking on a microshaker. Immediately after coupling, purify by cationic chromatography.

[0264] The purified protein was named HX006-TM1-1 and replaced into temporary buffer for the next step of detection.

[0265] In this example, the coupling ratio (DAR) of HX006 and TM1 in the HX006-TM1-1 molecule was determined by HIC-HPLC analysis according to the method of Example 2.

[0266] The test results are shown in Figure 2. In the HX006-TM1-1 molecule, the average DAR value of TM1 coupled to the HX006 antibody is 3.0.

[0267] Example 8 Sample Preparation of HX006 Antibody Conjugated to TM1

[0268] In this example, a conjugate of the HX006 antibody and TM1 was further prepared.

[0269] 6.56 mg of HX006 antibody protein was transferred into reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) using a 15 ml 30 kDa ultrafiltration tube. The volume was then exchanged four times to a final volume of approximately 2 ml, and the protein concentration was determined. TCEP (3x molar ratio) was added to the antibody and incubated in a 25°C water bath for 2.5 hours. The protein was then exchanged into coupling buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) using a 15 ml 30 kDa ultrafiltration tube. The protein concentration and free thiol group count of the sample were then determined.

[0270] Based on the amount of protein, add 4 times the molar amount of TM1 to the antibody sample, mix well, and react at 25°C for 2 hours with shaking on a microshaker. Immediately after coupling, purify by cationic chromatography.

[0271] The purified protein was named HX006-TM1-2 and replaced into temporary buffer for the next step of detection.

[0272] In this example, the coupling ratio (DAR) of HX006 and TM1 in the HX006-TM1-2 molecule was determined by HIC-HPLC analysis according to the method of Example 2.

[0273] The test results are shown in Figure 3. In the HX006-TM1-2 molecule, the average DAR value of TM1 coupled to the HX006 antibody is 4.67.

[0274] Example 9 Sample Preparation of HX006 Antibody Conjugated to C18-tert-Butyl Ester

[0275] In this example, a conjugate of the HX006 antibody and C18 tert-butyl alcohol ester was prepared.

[0276] 4.3 mg of HX006 antibody protein was transferred to reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) using a 15 ml 30 kDa ultrafiltration tube. The volume was then exchanged four times to a final volume of approximately 2 ml, and the protein concentration was determined. TCEP (3x molar ratio) was added to the antibody and incubated at 25°C in a water bath for 2 h. The protein was then exchanged four times using a 15 ml 30 kDa ultrafiltration tube into coupling buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5). The protein concentration and free thiol group count of the sample were then determined.

[0277] Based on the amount of protein, add 3 times the molar amount of C18 tert-butyl ester to the antibody sample, mix well, and react at 25°C for 2 hours with shaking on a microshaker. Immediately after coupling, purify by cationic chromatography.

[0278] The purified protein was named HX006-C18-tert-butyl ester, also known as HX006-C18, and replaced into a temporary buffer for the next step of detection.

[0279] In this example, the coupling ratio (DAR) of HX006 and C18-tert-butyl ester was determined by HIC-HPLC analysis according to the method of Example 2. The results are shown in FIG15 , and the average DAR value is 2.95.

[0280] Example 10 Sample Preparation of HX006 Antibody Conjugated to C16-NHS

[0281] 4 mg of HX006 antibody protein was transferred to coupling buffer (0.1 M MOPS, 20 mM Tris, pH 7.5) using a 30 kDa ultrafiltration tube to a final volume of 0.9 ml, and the protein concentration was determined. A 6-fold molar amount of C16-NHS was added to the fusion protein sample. After mixing, the reaction was allowed to proceed at 25°C for 2 h with agitation on a microshaker. Immediately after coupling, the sample was purified by cationic chromatography, and then transferred to holding buffer using a 30 kDa ultrafiltration tube. The protein concentration was determined. The protein was designated HX006-C16-NHS.

[0282] Example 11. Sample preparation of HX006 antibody coupled to C20-NHS

[0283] 4 mg of HX006 antibody protein was transferred to coupling buffer (0.1 M MOPS, 20 mM Tris, pH 7.5) using a 30 kDa ultrafiltration tube to a final volume of 0.9 ml, and the protein concentration was determined. A 6-fold molar amount of C20-NHS was added to the fusion protein sample. After mixing, the reaction was incubated at 25°C for 2 h with agitation on a microshaker. Immediately after coupling, the protein was purified by cationic chromatography, and the sample was transferred to holding buffer using a 30 kDa ultrafiltration tube. The protein concentration was determined. The protein was designated HX006-C20-NHS.

[0284] Example 12 Binding activity of HX006-C16-NHS and HX006-C20-NHS to HSA protein

[0285] In this example, the ELISA method was used to detect the activity of samples HX006-C16-NHS and HX006-C20-NHS obtained by lysine coupling of the HX006 antibody protein Fc with the HSA protein.

[0286] Dilute HSA-his to 0.5 μg / ml using coating buffer and coat 8 ELISA plates overnight at 4°C. Wash the plates three times with PBST (PBS containing 0.05% Tween 20), add 200 μl / well of blocking buffer, and incubate at 37°C for 1 hour. Wash the plates four times with PBST, dilute HX006-C16-NHS and HX006-C20-NHS to a starting concentration of 1000 nM using diluent, then perform a 5-fold serial dilution into 7 dilutions, for a total of 8 steps, and add 100 μl / well of each well to the ELISA plate and incubate at 37°C for 1 hour. Wash the plates five times with PBST, add 100 μl / well of HRP-Goat anti-Huamn 10000X working solution, and incubate at 37°C for 1 hour. Wash the plates six times with PBST, add 100 μl / well of freshly prepared color development solution, and incubate at 37°C for 10 minutes. 50ul / well stop solution (2M H2SO4) was added and the OD value was detected at 450nm.

[0287] The results are shown in Figure 12. The results show that the sample HX006-C20-NHS coupled with C20-NHS can bind strongly to HSA-his, while the sample HX006-C16-NHS coupled with C16-NHS binds weakly to HSA-his.

[0288] Example 13. Sample preparation of HX008 antibody coupled to TM1

[0289] In this example, the anti-PD-1 antibody H8L2 (referred to as HX008 in this application) disclosed in patent CN108299560A was used as an example to construct a conjugate based on the Fc-higher fatty acid chain platform of this application, wherein the HX008 antibody is an IgG4 type, and its sequence is shown below:

[0290] The heavy chain sequence of the HX008 antibody is:

[0291] The heavy chain variable region sequence of the HX008 antibody is:

[0292] The Fc region sequence is:

[0293] The light chain variable region sequence of the HX008 antibody is:

[0294] The light chain sequence of the HX008 antibody is:

[0295] 4 mg of HX008 antibody protein was transferred into reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) four times using a 15 ml 30 kDa ultrafiltration tube. The final volume was approximately 1 ml, and the protein concentration was determined. Eight molar amounts of TCEP were added to the antibody and incubated in a 30°C water bath for 2.5 hours. The protein was then transferred into coupling buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) four times using a 15 ml 30 kDa ultrafiltration tube. The protein concentration and number of free thiol groups in the sample were then determined.

[0296] Based on the amount of protein, add 4 times the molar amount of TM1 to the fusion protein sample, mix well, and react at 25°C for 2 hours with shaking on a microshaker. Immediately after coupling, purify by cationic chromatography.

[0297] The purified protein was named HX008-TM1-2 and replaced into temporary buffer for the next step of detection.

[0298] In this example, the coupling ratio (DAR) of HX008 and TM1 in the HX008-TM1-2 molecule was determined by HIC-HPLC analysis according to the method of Example 2.

[0299] The results are shown in FIG4 . In the HX008-TM1-2 molecule, the average DAR value of TM1 coupled to the HX008 antibody is 1.45.

[0300] Example 14. Sample preparation of HX008 antibody coupled to TM1

[0301] In this example, a conjugate of HX008 antibody and TM1 was further prepared.

[0302] 4 mg of HX008 antibody protein was transferred into reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) using a 15 ml 30 kDa ultrafiltration tube, and the volume was adjusted to approximately 1 ml. The protein concentration was then determined. 10 molar TCEP was added to the antibody and incubated at 30°C in a water bath for 2.5 hours. The protein was then transferred into coupling buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) using a 15 ml 30 kDa ultrafiltration tube, and the volume was adjusted to 4. The protein concentration and free thiol group count of the sample were then determined.

[0303] Based on the amount of protein, add 5 times the molar amount of TM1 to the fusion protein sample, mix well, and react at 25°C for 2 hours with shaking on a microshaker. Immediately after coupling, purify by cationic chromatography.

[0304] The purified protein was named HX008-TM1-3 and replaced into temporary buffer for the next step of detection.

[0305] In this example, the coupling ratio (DAR) of HX008 and TM1 in the HX008-TM1-3 molecule was determined by HIC-HPLC analysis according to the method of Example 2.

[0306] The results are shown in FIG4 . In the HX008-TM1-3 molecule, the average DAR value of TM1 coupled to the HX008 antibody is 2.0.

[0307] Example 15. Binding activity of GLP-1-Fc-TM1 to HSA protein

[0308] The fatty acid chains contained in TM1 can bind to serum albumin (HSA). In this example, the binding of the fusion GLP-1-Fc-TM1 to HSA was detected using an ELISA method. HSA-his was diluted to 0.5 μg / ml using a coating solution (carbonate buffer) and coated onto three ELISA plates overnight at 4°C. The plates were washed three times with PBST (PBS containing 0.05% Tween 20), and 200 μl / well of blocking solution (1% BSA in PBST) was added. The plates were incubated at 37°C for 1 hour. The plates were washed four times with PBST, and the GLP-1-Fc-TM1 sample was diluted to a starting concentration of 1000 nM using a diluent (1‰ BSA in PBST). This was then serially diluted 5-fold into seven dilutions, for a total of eight dilutions. 100 μl / well of the sample was added to each well of the ELISA plate and incubated at 37°C for 1 hour. Wash the plate five times with PBST, add 100 μl / well of HRP-Goat anti-Huamn 10000X working solution, and incubate at 37°C for 1 hour. Wash the plate six times with PBST, add 100 μl / well of freshly prepared color development solution (5 ml substrate solution, 250 μl TMB stock solution, 16 μl 0.75% H₂O₂), and incubate at 37°C for 10 minutes. Add 50 μl / well of stop solution (2 M H₂SO₄), and measure the OD at 450 nm.

[0309] The results are shown in Figure 5. The results show that the sample GLP1-Fc-TM1 after coupling with TM1 can strongly bind to HSA-his.

[0310] Example 16. Binding activity of GLP-1-Fc-C18-tert-butyl ester to HSA protein

[0311] This example uses an ELISA method to detect the binding of the fusion GLP-1-Fc-C18-tert-butyl ester to HSA. HSA-his was diluted to 0.5 μg / ml with coating solution and coated onto three ELISA plates overnight at 4°C. The plates were washed three times with PBST (PBS solution containing 0.05% Tween 20), and 200 μl / well of blocking solution was added. The plates were incubated at 37°C for 1 hour. The plates were washed four times with PBST, and the GLP-1-Fc-C18-tert-butyl ester sample was diluted to 1000 nM as a starting concentration with diluent. The sample was then diluted 5-fold into 7 dilutions, for a total of 8 dilutions. 100 μl / well of the sample was added to each well of the ELISA plate and incubated at 37°C for 1 hour. The plates were washed five times with PBST, and 100 μl / well of HRP-Goat anti-Huamn 10000X working solution was added. The plates were incubated at 37°C for 1 hour. Wash the plate six times with PBST, add 100 μl / well of freshly prepared color development solution, and incubate at 37°C for 10 min. Add 50 μl / well of stop solution (2 M H2SO4), and measure the OD value at 450 nm.

[0312] The results are shown in Figure 13. The results show that the sample GLP1-Fc-C18-tert-butyl ester coupled with C18-tert-butyl ester can strongly bind to HSA-his.

[0313] Example 17. Binding activity of HX006-TM1 to HSA protein

[0314] This example uses an ELISA method to detect the binding of the fusion HX006-TM1 to HSA. HSA-his was diluted to 0.5 μg / ml with coating solution and coated onto 8 ELISA plates overnight at 4°C. The plates were washed three times with PBST (0.05% Tween 20 in PBS), 200 μl / well of blocking solution was added, and the plates were incubated at 37°C for 1 hour. The plates were washed four times with PBST, and the HX006-TM1 sample was diluted to 1000 nM as a starting concentration with diluent. Then, a 5-fold gradient dilution was made into 7 dilutions, for a total of 8 dilutions. 100 μl / well of the sample was added to each well of the ELISA plate and incubated at 37°C for 1 hour. The plates were washed five times with PBST, and 100 μl / well of HRP-Goat anti-Huamn 10000X working solution was added, and the plates were incubated at 37°C for 1 hour. The plates were washed six times with PBST, and 100 μl / well of freshly prepared color development solution was added, and the plates were incubated at 37°C for 10 minutes. 50ul / well stop solution (2M H2SO4) was added and the OD value was detected at 450nm.

[0315] The results are shown in Figure 6. The results show that the sample HX006-TM1 after coupling with TM1 can strongly bind to HSA-his, and the binding activity increases with the increase of DAR value.

[0316] Example 18. Binding activity of HX006-C18-tert-butyl ester to HSA protein

[0317] This example uses an ELISA method to detect the binding of the fusion HX006-C18-tert-butyl ester to HSA. HSA-his was diluted to 0.5 μg / ml using coating solution and coated onto eight ELISA plates overnight at 4°C. The plates were washed three times with PBST (0.05% Tween 20 in PBS), and 200 μl / well of blocking solution was added. The plates were incubated at 37°C for 1 hour. The plates were washed four times with PBST, and the HX006-TM1 sample was diluted to 1000 nM as a starting concentration using diluent. This was then serially diluted 5-fold into 7 dilutions, for a total of 8 dilutions. 100 μl / well of the sample was added to each well of the ELISA plate and incubated at 37°C for 1 hour. The plates were washed five times with PBST, and 100 μl / well of HRP-Goat anti-Huamn 10000X working solution was added. The plates were incubated at 37°C for 1 hour. The plates were washed six times with PBST, and 100 μl / well of freshly prepared color development solution was added. The plates were incubated at 37°C for 10 minutes. 50ul / well stop solution (2M H2SO4) was added and the OD value was detected at 450nm.

[0318] The results are shown in Figure 14. The results show that compared with naked antibody HX006-DS, the sample HX006-C18-tert-butyl ester coupled with C18-tert-butyl ester can bind to HSA-his more strongly.

[0319] Example 19. Binding activity of HX008-TM1 to HSA protein

[0320] This example used an ELISA method to test the binding of the HX008-TM1 fusion to HSA. The specific steps are similar to those in Example 15, except that the test sample was replaced with HX008-TM1-3. The results, shown in Figure 7, show that compared to naked antibody HX008-DS, the TM1-conjugated HX008-TM1 sample exhibited stronger binding to HSA-his, with increased binding activity as the DAR increased.

[0321] Example 20. Effect of GLP-1-Fc-TM1 on the activity of HEK293-CRE-Luc-GLP1R cell line

[0322] In HEK-293 cells stably expressing the human GLP-1 receptor (GLP-1R) and the intracellular reporter gene CRE-luciferase (CRE4-luciferase) (HEK293-CRE-Luc-GLP1R, provided by Nanjing GenScript Biotechnology Co., Ltd.), the biological activity of test compound I (GLP-1-Fc-TM1) was assessed by specifically binding to the GLP-1R to produce cAMP, thereby activating the reporter gene. After resuscitation, the HEK293-CRE-Luc-GLP1R cells were cultured in DMEM (containing 10% FBS, 400 μg / ml G418, and 200 μg / ml Hygromycin B) at 37°C and 5% CO2 in an incubator. Cells in the logarithmic growth phase were harvested, counted, and resuspended in complete culture medium. The cell concentration was adjusted to the appropriate concentration and the cells were plated at 2×10 3Cells / well were seeded in a 384-well plate, and 20 μl of cell suspension was added to each well. The cells were incubated overnight in a 37°C, 100% relative humidity, 5% CO2 incubator. The test compound was diluted with culture medium to the corresponding set action concentration, 30 μl of test solution per well (the final action concentration and dilution gradient of the test compound depend on specific requirements), a total of 9 concentration gradients were set, and 2 replicates were added for each concentration. Semaglutide (commercially available) and Dulaglutide (homemade) were used as positive controls. The cells were placed in a 37°C, 100% relative humidity, 5% CO2 incubator and incubated for 6 hours. The supernatant was discarded, 40 μL / well of One-Glo detection solution was added, the reaction was shaken for 5 minutes, and luminescience (RLU) was measured on an ENVISION 2104 microplate reader.

[0323] The results are shown in FIG16 : the efficacy of activating the reporter gene CRE-luciferase, the EC of test substance I (GLP-1-Fc-TM1) 50 8.4×10 -3 nM, EC of Dulaglutide 50 4.4×10 -3 nM, EC of Semaglutide 50 4.7×10 -3 nM, EC values ​​of Test Substance I (GLP-1-Fc-TM1) for dulaglutide and semaglutide in in vitro reporter gene assay conditions 50 At the same order of magnitude, test substance I (GLP-1-Fc-TM1) has stronger biological activity.

[0324] Example 21. Determination of the interaction between the test substance and human serum albumin by surface plasmon resonance (SPR)

[0325] The running reagent consisted of 10 mM N-(2-hydroxyethyl)piperazine-N-2-sulfonic acid (HEPES), 150 mM sodium chloride (NaCl), 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.005% Tween-20, adjusted to pH 7.4. Mouse anti-His antibody was diluted to 50 μg / mL using a fixation reagent (10 mM sodium acetate, pH 4.5). First, the surface of the CM5 chip was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 420 s. Next, 50 μg / mL of mouse anti-His antibody was injected into the channel at a flow rate of 10 μL / min for approximately 420 s, resulting in an immobilization rate of approximately 7,000 to 15,000 RU. Finally, the chip was blocked with 1 M ethanolamine at 10 μL / min for 420 s. Human serum albumin (HSA) was buffer exchanged using a desalting column and the corresponding running reagent. The sample concentration after exchange was measured using a SPECTROstar Nano. Ligands (test substances GLP-1-Fc, GLP-1-Fc-TM1, HX006-DS, HX006-TM1-1, HX006-TM1-2, HX008-DS, HX008-TM1-2, HX008-TM1-3) were diluted to 5 μg / mL in the running reagent and injected into the experimental channel (Fc2) of the His capture chip at a flow rate of 10 μL / min. Ligand (test substance) capture was not required in the reference channel (Fc1). Human serum albumin (HSA) was serially diluted 2-fold with the running reagent for a total of 7 concentrations. The diluted human serum albumin (HSA) was injected into the experimental channel and the reference channel at a flow rate of 30 μL / min, with corresponding binding (120 s) and dissociation (300 s) times. The binding and dissociation steps were performed in the running reagent. After each concentration analysis, the chip needed to be regenerated with glycine hydrochloride at a pH of 1.5 at a flow rate of 20 μL / min for 30 s to wash off the ligand and undissociated analyte. When performing the next concentration analysis, the experimental channel needed to recapture the same amount of ligand (test substance). The KD value of each sample was calculated using Biacore 8K analysis software Biacore Insight Evaluation Software. The reference channel (Fc1) was used for background subtraction.

[0326] The results showed that the affinity of the test substances to human serum albumin detected by Biacore 8K was: the Kd values ​​of unmodified GLP-1-Fc, HX006-DS, and HX008-DS were 0, i.e., no binding; the Kd values ​​of GLP-1-Fc-TM1, HX006-TM1-1, HX006-TM1-2, HX008-TM1-2, and HX008-TM1-3 were 1.01×10 -2nM、1.14×10 -3 nM, 6.92×10 -4 nM、4.87×10 -3 nM、4.05×10 -3 nM, showing strong affinity to human serum albumin.

[0327] Example 22. Study on the efficacy of multiple administrations of GLP-1-Fc-TM1 in type II diabetic db / db mice

[0328] Twelve m / m mice (normal control) and 60 male db / db mice were purchased and bred for adaptive breeding. After the db / db mice reached normal blood glucose levels (around 8-10 weeks of age), 10 m / m mice and 50 db / db mice were selected and grouped into the following groups: normal control (m / m mice), model group, positive control (dulaglutide), low-dose test substance group, medium-dose test substance group, and high-dose test substance group, with 10 mice in each group. Drug administration began after grouping: the positive control group received 10 nmol / kg / dose of TRULICITY (dulaglutide) subcutaneously twice weekly for 4 weeks; the low-, medium-, and high-dose test substance groups received 3, 10, and 30 nmol / kg / dose of test substance I (GLP-1-Fc-TM1) subcutaneously twice weekly for 4 weeks; and the db / db model group and normal control group received the corresponding vehicle (PBS) subcutaneously twice weekly for 4 weeks. The dosage volume for all groups was 5 ml / kg / dose. Clinical observation was performed once a day, and serum insulin and glycosylated hemoglobin were detected at the end of the administration, followed by an OGTT test. During the administration period, body weight and food intake were monitored twice a week. After the first and last administration, random blood glucose was tested at 0 h (0-60 min before administration), 0.5 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h. Fasting blood glucose was tested once a week (4 h fasting) before administration. Random blood glucose, fasting blood glucose, and serum insulin were tested at the end of the administration period.

[0329] The results showed that compared with the positive control drug (Dulaglutide), the test substance I (GLP-1-Fc-TM1) was more effective in lowering glycated hemoglobin, random blood glucose and fasting blood glucose, stimulating serum insulin secretion, and reducing body weight and food intake. The effects of low, medium, and high doses of the test substance I (GLP-1-Fc-TM1) showed a therapeutic effect relationship, indicating that the test substance I (GLP-1-Fc-TM1) had a significant hypoglycemic effect.

[0330] Specifically, the test substance GLP-1-Fc-TM1 can dose-dependently reduce the 4h fasting blood glucose of db / db mice (Figure 19-1), random blood glucose after the first and last administration (Figures 19-2 and 19-3), and the blood glucose AUC of the OGTT test after the last administration. 0-180min (Figure 19-4 and Figure 19-5), reduced the content of glycated hemoglobin in db / db mice (Figure 19-6), increased the insulin level in db / db mice (Figure 19-7), and reduced the average daily food intake in db / db mice (Figure 19-8); at the same dose, the test substance GLP-1-Fc-TM1 had a better effect on the above indicators than the positive control Dulaglutide.

[0331] Example 23. Pharmacological Study on the Effect of GLP-1-Fc-TM1 on Body Weight in DIO Model Mice

[0332] After purchasing 72 male C57BL / 6J mice, except for 12 normal control mice (conventional feed), the remaining 60 mice were fed a high-fat diet to prepare DIO model mice, with free access to food, and fed continuously for about 10 weeks. During the modeling period, food intake was monitored twice a week, and body weight was monitored twice a week. When the body weight exceeded 20% of that of normal mice, the DIO model standard was met, and then grouping began. 10 qualified normal control mice (conventional feed) and 50 DIO model mice were selected and grouped, namely, normal control group (mice fed with conventional feed), model group, positive control group (dulaglutide), low-dose group of the test substance, medium-dose group of the test substance, and high-dose group of the test substance, with 10 mice in each group. After grouping, dosing began. The positive control group received 10 nmol / kg / dose of TRULICITY (dulaglutide) by subcutaneous injection twice weekly for 4 weeks. The low-, medium-, and high-dose groups received 3, 10, and 30 nmol / kg / dose of test substance I (GLP-1-Fc-TM1) by subcutaneous injection twice weekly for 4 weeks. The obese DIO model group and the normal control group received the corresponding vehicle (PBS) by subcutaneous injection twice weekly for 4 weeks. The dosing volume for all groups was 5 ml / kg / dose. All DIO mice continued to be fed a high-fat diet during the dosing period and for the entire experimental period. Body weight and food intake were monitored twice a week during the dosing period. Fasting blood glucose was measured before dosing (4 hours fasting), and blood was collected to test four blood lipids and liver blood biochemistry. The first day of dosing was recorded as D1. After the 28th day of dosing (D28), a 4-hour fasting blood glucose test was performed after the end of the dosing period. Blood was collected and serum was separated to test four blood lipids, liver blood biochemistry, and serum insulin levels. Finally, abdominal adipose tissue was collected and weighed, and the liver was removed and weighed to calculate its organ coefficient. The formula is organ coefficient = organ weight (g) / mouse body weight (g). Part of the liver was removed and fixed in formalin for pathological histological examination (HE staining, Oil Red-O staining).

[0333] The results showed that compared with the positive control drug (dulaglutide), the test substance I (GLP-1-Fc-TM1) was more effective in lowering fasting blood glucose, improving four blood lipids and liver biochemical indicators and liver pathology, stimulating serum insulin secretion, and reducing body weight and food intake. The effects of low, medium, and high doses of the test substance I (GLP-1-Fc-TM1) showed a therapeutic effect relationship, and the test substance I (GLP-1-Fc-TM1) had obvious blood sugar lowering and weight loss effects.

[0334] Specifically, GLP-1-Fc-TM1 dose-dependently reduced body weight (Figure 20-1), food intake (Figure 20-2), body fat content (Figure 20-3), and fasting blood glucose (Figure 20-4) in DIO mice. Furthermore, GLP-1-Fc-TM1 also dose-dependently reduced blood lipid levels (Figure 20-5) and serum liver function indicators ALT and AST levels (Figure 20-6), and improved ballooning and steatosis in DIO mice's liver tissue (Figure 20-7). At the same dose, GLP-1-Fc-TM1 was more effective than the positive control, dulaglutide, in improving hepatic fatty lesions.

[0335] Example 24. Study of GLP-1-Fc-TM1 on rat glucose tolerance test (IVGTT)

[0336] Fifty-eight male Sprague-Dawley rats (6-8 weeks old, weighing 180-220g) were purchased and acclimated for one week. Fifty animals were then divided into the following groups: vehicle control, positive control (dulaglutide), low-dose test substance group, medium-dose test substance group, and high-dose test substance group, with 10 rats in each group. Dosing began after grouping: the positive control group received a single subcutaneous injection of 10 nmol / kg of TRULICITY (dulaglutide); the low-, medium-, and high-dose test substance groups received a single subcutaneous injection of 3, 10, and 30 nmol / kg of test substance I (GLP-1-Fc-TM1); and the vehicle control group received a single subcutaneous injection of the corresponding vehicle (PBS). The dosing volume for all groups was 5 ml / kg / dose. Rats were fasted for 16 hours (without water deprivation) and then intravenously injected with glucose (0.5 g / kg) for stimulation. Blood samples were collected at 2, 4, 6, 10, 20 and 30 minutes after glucose injection to detect blood glucose concentration and insulin levels, and the AUC was calculated.

[0337] The results showed that compared with the positive control drug (Dulaglutide), the test substance I (GLP-1-Fc-TM1) had equivalent or no significant difference in reducing fasting blood glucose and stimulating serum insulin secretion; and the effects of low, medium and high doses of the test substance I (GLP-1-Fc-TM1) showed a certain dose-response relationship.

[0338] Specifically, single administration of the test substance GLP-1-Fc-TM1 at a dose of 3, 10, and 30 nmol / kg can dose-dependently reduce the blood glucose AUC of SD rats 24h and 72h after administration. 0-30min (Figure 21-1, Figure 21-2, Figure 21-3 and Figure 21-4) and increased serum insulin AUC0-30min (Figure 21-5, Figure 21-6, Figure 21-7 and Figure 21-8).

[0339] Example 25. Pharmacokinetic study of the test substance after a single subcutaneous injection in SD rats

[0340] Twelve Sprague-Dawley (SD) rats (6-8 weeks, weighing 180-220 g), half male and half female, were randomly divided into two groups after acclimation: the test substance I (GLP-1-Fc-TM1) group and the positive control (dulaglutide, homemade). Each group consisted of six rats, half male and half female. The corresponding test substance I (GLP-1-Fc-TM1) and the positive control (dulaglutide, homemade) were administered subcutaneously at a dose of 0.1 mg / kg in a 4 ml / kg volume. The rats were administered before, 24 hours (±10 minutes on day 1), 48 hours (±10 minutes on day 2), 72 hours (±15 minutes on day 3), 96 hours (±15 minutes on day 4), 144 hours (±15 minutes on day 6), 192 hours (±30 minutes on day 8), 240 hours (±30 minutes on day 10), and 336 hours (±30 minutes on day 14). Approximately 0.5 mL of blood was collected via the jugular vein and anticoagulated with EDTA-K2. Plasma concentrations of test substance I (GLP-1-Fc-TM1) and the positive control (dulaglutide) were determined by ELISA at various time points. The standard curves for all three test substances ranged from 3.13 to 200 ng / mL, and pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2.

[0341] The results are shown in Figure 17 and Table 1: After a single subcutaneous injection of 0.1 mg / kg of test substance I (GLP-1-Fc-TM1) and Dulaglutide in SD rats, the average T values ​​of test substance I (GLP-1-Fc-TM1) and Dulaglutide in plasma were max All were 24 hours, average C max 225 and 191 ng / mL, respectively, with an average T 1 / 2 The average AUC was 45 and 28 hours respectively. 0-t were 15700 and 7540 h*ng / mL, respectively, with average AUC 0- ∞ were 16100 and 7840 h*ng / mL, respectively, with an average Vz_ F_obs The average Cl _F_obs 6.42 and 13.3 mL / h / kg, respectively, and MRT (0-t) 70 and 39 hours respectively, MRT (0-∞ ) were 77 and 44 h respectively. The half-life of test substance I (GLP-1-Fc-TM1) was T1 / 2 、C max , AUC last , AUC 0- ∞、Vz_ F 、Cl、MRT (0-t) , MRT (0-∞ ) were 1.61, 1.18, 2.08, 2.05, 0.80, 0.48, 1.79 and 1.75 times that of Dulaglutide, respectively, indicating that test substance I (GLP-1-Fc-TM1) can prolong the half-life by reducing the clearance rate.

[0342] Example 26. Pharmacokinetic study of the test substance after a single subcutaneous injection in cynomolgus monkeys

[0343] Four cynomolgus monkeys (5-6 years old, weighing approximately 6.5 kg), half male and half female, were randomly divided into two groups after acclimation: a reference control (dulaglutide, available as Trulicity) and a test substance I (GLP-1-Fc-TM1), with two monkeys in each group (half male and half female). Each group received a single subcutaneous injection of the corresponding reference control, dulaglutide, or test substance I (GLP-1-Fc-TM1), at a molar dose of 1.676 nmol / kg (i.e., 0.1 mg / kg for dulaglutide and 0.101 mg / kg for GLP-1-Fc-TM1) in a 4 ml / kg dosing volume. Blood was collected via the jugular vein (approximately 0.5 mL) and anticoagulated with EDTA-K2 before, at 4, 8, 24, 48, 96, 144, 240, and 336 hours post-dose. Plasma concentrations of the reference preparation, dulaglutide, and test substance I (GLP-1-Fc-TM1) were measured at various time points using ELISA. The standard curve limit of detection (LLOQ) was 15.625 ng / mL, and pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2.

[0344] The results are shown in Figure 18 and Table 2 and below: After a single subcutaneous injection of 0.676 nmol / kg of test substance I (GLP-1-Fc-TM1) and reference preparation control Dulaglutide in cynomolgus monkeys, the average T values ​​of test substance I (GLP-1-Fc-TM1) and Dulaglutide in plasma were max 16 and 8 hours respectively, with an average C max 760 and 602 ng / mL, respectively, with an average T 1 / 2 The mean AUCs were 97.9 and 48.2 h, respectively. 0-t The mean AUC values ​​were 98546 and 49118 h*ng / mL, respectively. 0-∞ were 140678 and 50947 h*ng / mL, respectively, with an average Vz_ F_obs The average Cl _F_obs 0.74 and 1.97 mL / h / kg, respectively, MRT (0-t) 88 and 68 hours respectively, MRT (0-∞ ) were 163 and 77 h respectively. The half-life of test substance I (GLP-1-Fc-TM1) was T max 、C max 、T 1 / 2 , AUC 0-t , AUC 0- ∞、Vz-F、Cl、MRT (0-t) , MRT (0-∞ ) were 2.00, 1.26, 2.03, 2.01, 2.76, 0.70, 0.38, 1.29 and 2.12 times that of Dulaglutide, respectively, indicating that test substance I (GLP-1-Fc-TM1) can prolong the half-life by reducing the clearance rate.

Claims

1. A conjugate molecule with a structure of "active molecule-Fc-Cn", wherein the active molecule is selected from any molecule that is beneficial to the body, Fc is immunoglobulin IgG Fc, and Cn is a C-terminal 14-24 A modified portion of a fatty acid chain.

2. A conjugate molecule with a structure of "antibody-Cn", wherein Cn is a C 14-24 A modified portion of a fatty acid chain.

3. A conjugate molecule with a structure of "active molecule-fusion protein-Cn", wherein the active molecule is selected from any molecule that is beneficial to the body, and Cn is a molecule containing C 14-24 A modified portion of a fatty acid chain.

4. The conjugate molecule according to any one of claims 1 to 3, wherein the Cn has a structure of the following formula (I): -ZY (I), in Z has the following structure: -Z1-Z2-Z3-Z4-, wherein Z1 is a sulfur atom, nitrogen atom or oxygen atom in Fc, Z2 is -C(=O)- or a 5-10 membered heterocyclic group, preferably containing 1 or 2 heteroatoms selected from N, S and O; Z3 is selected from a bond, -C(=O)-, -C1-C 10 Alkylene-C(=O)-, -C3-C 10 Alkynylidene-C(=O)-, -C3-C 10 Alkenylene-C(=O)-, -C1-C 10 heteroalkylene-C(=O)-, -C3-C8 cycloalkylene-C(=O)-, -O-C1-C8 alkylene-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C 1- C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-C3-C8 cycloalkylene-C(=O)-, -C3-C8 cycloalkylene-C1-C 10 Alkylene-C(=O)-, -C 3- C8 heterocyclylene-C(=O)-, -C1-C 10 Alkylene-C3-C8 heterocyclylene-C(=O)-, -C3-C8 heterocyclylene-C1-C 10 Alkylene-C(=O)-, wherein the alkylene, alkynylene, alkenylene, heteroalkylene, cycloalkylene, arylene and heterocyclylene groups may be optionally substituted; Z4 is a bond or a PEG unit represented by the formula, in, R1 is selected from C 1-4 Alkylene, -NH-, -NH-C 1-4 Alkylene-, -NH-C 1-4 Alkylene-heteroaryl-, wherein heteroaryl is a 5-membered or 6-membered nitrogen-containing heteroaryl; R2 is -C(=O)-, -C 1-4 Alkylene, -C 1-4 Alkylene-C(=O)-, -C 1-4 Alkylene-NH-C(=O)-(CH2OCH2) p -C 1-4 Alkylene-, -C 1-4 Alkylene-C(=O)-NH-(CH2OCH2) p -C 1-4 Alkylene-, wherein m is an integer of 2-6, p is an integer of 1-3, Y is Where Y is connected to Z4 through X, k is an integer from 10 to 30, wherein R independently represents hydrogen, C 1-6 Alkyl, C 1-6 aminoalkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl.

5. The conjugate molecule according to any one of claims 1 to 4, wherein Z2 is a maleimido group, The wavy line on the left indicates the position connected to Z1; the wavy line on the right indicates the position connected to Z3.

6. The conjugate molecule of any one of claims 1 to 5, wherein Z3 is -C1-C 10 Alkylene-C(=O)-, wherein the alkylene is optionally substituted and wherein Z3 is linked to Z4 through -C-(=O)-.

7. The conjugate molecule of any one of claims 1 to 6, wherein Z2 is a maleimido group, Z3 is a -C 1-6 Alkylene-C(=O)-.

8. The conjugate molecule according to any one of claims 1 to 7, wherein Z4 is a bond, and Z3 is directly connected to Y in formula (I).

9. The conjugate molecule of any one of claims 1 to 8, wherein Z4 is a PEG unit represented by the formula, in, R1 is selected from -NH- and -NH-C 1-4 Alkylene-; R2 is -C 1-4 Alkylene or -C 1-4 Alkylene-NH-C(=O)-(CH2OCH2) p -C 1-4 Alkylene-, wherein m is an integer of 2-6, and p is an integer of 1-3.

10. The conjugate molecule of any one of claims 1-9, wherein Z4 is a unit comprising 2-6 PEGs.

11. The conjugate molecule of any one of claims 1 to 10, wherein Z4 is wherein m=1-4, the asterisk on the left indicates the position for connection to Z3; the asterisk on the right indicates the position for connection to Y in formula II.

12. The conjugate molecule of any one of claims 1 to 11, wherein Z in formula (I) has the following structure: where R E It is hydrogen, C 1-6 Alkyl, C 1-6 aminoalkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, wherein y=0-4, m=1-4, wherein the asterisk on the left indicates the position of attachment to Ab, and the asterisk on the right indicates the position of attachment to Y.

13. The conjugate molecule of any one of claims 1 to 12, wherein Y is linked to Z4 through X, and X is -NH-(C=O)- or -(C=O)-NH-.

14. The conjugate molecule of any one of claims 1 to 13, wherein the Cn comprises C 16 Fatty acid chains, C 18 Fatty acid chain or C 20 Fatty acid chains.

15. The conjugate molecule of any one of claims 1 to 14, wherein the Cn comprises C 18 The fatty acid chain is coupled to the sulfur atom of the free sulfhydryl group of Fc.

16. The conjugate molecule of any one of claims 1 to 15, wherein the Cn is selected from The conjugate molecule according to any one of claims 1 to 16 , wherein the Fc is IgG1 Fc or IgG4 Fc, preferably human IgG1 Fc or human IgG4 Fc.

18. The conjugate molecule of any one of claims 1-16, wherein the Fc region further comprises an immunoglobulin IgG hinge region.

19. The conjugate molecule of any one of claims 1-17, wherein the Fc region has a modification at amino acid position selected from the group consisting of amino acids 228, 233, 234, 235, 252, 254, 256, 297, 307, 308, 311, 380, 385, 386, 389, 428, 434, and 447 (according to EU numbering).

20. The conjugate molecule of claim 19, wherein the modification is substitution of amino acids 254, 308, and 434 with Thr, Pro, and Ala, respectively (according to EU numbering).

21. The conjugate molecule of claim 20, wherein the modifications are S228P, F234A and L235A, or S228P, F234A, L235A and deletion 447 (according to EU numbering).

22. The conjugate molecule of any one of claims 1-21, wherein the Fc region comprises or consists of the sequence shown in SEQ ID NO: 10, 15 or 16.

23. The conjugated molecule according to any one of claims 1 to 22, wherein the active molecule is a peptide active molecule, and the peptide active molecule is fused to the Fc region directly or through a peptide linker.

24. The conjugate molecule according to any one of claims 1 to 23, wherein the C-terminus of the peptide active molecule and the N-terminus of the Fc region are fused together, or the N-terminus of the peptide active molecule and the C-terminus of the Fc region are fused together.

25. The conjugate molecule of any one of claims 1 to 24, wherein the active molecule is selected from an enzyme, an enzyme inhibitor, an antigen, an antibody or antibody fragment, a hormone, glucagon-like peptide-1 (GLP-1), glucagon, an interferon, a cytokine, a growth factor and / or a differentiation factor, a factor involved in cell motility or migration, a factor involved in bone tissue development / resorption, a chemokine, a plasma or interstitial adhesion molecule or an extracellular matrix, a bactericidal or antifungal factor.

26. The conjugate molecule of any one of claims 1-25, wherein the active molecule is selected from GLP-1, an antibody, or an antigen-binding fragment thereof.

27. The conjugate molecule according to any one of claims 1 to 26, wherein the active molecule is selected from an anti-PD-1 antibody or an antigen-binding fragment thereof, and an anti-VEGF antibody or an antigen-binding fragment thereof.

28. The conjugate molecule of any one of claims 1 to 27, wherein the peptide linker comprises the amino acid sequence (G4S) n , wherein n is an integer equal to or greater than 1, preferably, the peptide linker includes (G4S)3, (G4S)4, (G4S)6, GS(G4S)4, DAAALEAAALDAAAREAAARDAAAL, NVDHLPSNTLVDLA, (G3S)2, (G4S)2, (G3S)3, (G4S)3, (G3S)4, (G4S)4, (G3S)5, (G4S)5, (G3S)6, (G4S)6, GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP and GSTSGSGK PGSGEGSTKG.

29. The conjugate molecule of any one of claims 1-28, wherein the Fc comprises the sequence shown in SEQ ID NO: 10, 15 or 16.

30. The conjugate molecule of any one of claims 1-29, which is GLP-1-IgG4 Fc-TM1, GLP-1-IgG4 Fc-C18 tert-butyl ester, GLP-1-IgG4 Fc-C16-NHS, or GLP1-IgG4 Fc-C20-NHS. The conjugate molecule of claim 30 , wherein the IgG4 Fc comprises the sequence shown in SEQ ID NO:

16.

32. The conjugate molecule of claim 30 or 31, wherein the GLP-1-IgG4 Fc portion in the GLP-1-IgG4 Fc-TM1, GLP-1-IgG4 Fc-C18 tert-butyl ester, GLP-1-IgG4 Fc-C16-NHS, or GLP1-IgG4 Fc-C20-NHS conjugate molecule comprises the amino acid sequence shown in SEQ ID NO:

1.

33. The conjugate molecule of claim 27, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 9 and a light chain variable region shown in SEQ ID NO:

11.

34. The conjugate molecule of claim 33, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises the heavy chain shown in SEQ ID NO: 8 and the light chain shown in SEQ ID NO:

12.

35. The conjugate molecule of claim 33 or 34, wherein the Fc comprises the sequence shown in SEQ ID NO:

10. The conjugate molecule according to claim 35 , which is an HX008-TM1 conjugate molecule, preferably an HX008-TM1-2 conjugate molecule or an HX008-TM1-3 conjugate molecule.

37. The conjugate molecule of claim 27, wherein the anti-VEGF antibody or antigen-binding fragment thereof comprises three heavy chain CDRs set forth in SEQ ID NOs: 2, 3, and 4 and three light chain CDRs set forth in SEQ ID NOs: 5, 6, and 7.

38. The conjugate molecule of claim 37, wherein the anti-VEGF antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO: 13 and a light chain as set forth in SEQ ID NO:

14.

39. The conjugate molecule according to claim 37 or 38, wherein The Fc comprises the sequence shown in SEQ ID NO:

15.

40. The conjugated molecule of any one of claims 37-39, which is a HX006-TM1 conjugated molecule, a HX006-C18-tert-butyl ester conjugated molecule, a HX006-C16-NHS conjugated molecule, and a HX006-C20-NHS conjugated molecule.

41. A method for preparing a conjugate molecule having a structure of "active molecule-Fc-Cn", comprising (a) linking the active molecule polypeptide to the Fc region of an immunoglobulin to prepare an "active molecule-Fc" fusion; and (b) coupling the "active molecule-Fc" fusion protein with Cn containing a fatty acid chain under conditions that allow conjugation of the Fc region to Cn to produce an "active molecule-Fc-Cn" conjugated molecule.

42. A method for preparing a conjugate molecule having a structure of "active molecule-Fc-Cn", comprising (a) linking an antibody Fab fragment to an immunoglobulin Fc region to produce a "Fab-Fc" fusion, (b) A step of coupling the "Fab-Fc" fusion under conditions that allow conjugation of the Fc region to the Cn containing a fatty acid chain to produce a "Fab-Fc-Cn" conjugated molecule.

43. A method for preparing a conjugate molecule having a structure of "active molecule-Fc-Cn" or "antibody-Cn", comprising the step of subjecting a whole antibody to a coupling reaction under conditions that allow conjugation of the Fc region to a Cn containing a fatty acid chain to produce the "active molecule-Fc-Cn" conjugate molecule.

44. A pharmaceutical composition comprising the conjugate molecule of any one of claims 1-40.

45. A method for effectively extending the serum half-life of an active molecule, comprising the step of constructing the active molecule into a conjugated molecule having a structure of "active molecule-Fc-Cn" or a structure of "antibody-Cn" according to the method of any one of claims 41 to 43, thereby effectively increasing the serum half-life of the active molecule.

46. ​​Use of the conjugated molecule according to any one of claims 1 to 40, or the pharmaceutical composition according to claim 44, in the preparation of a drug for treating a human disease.

47. A method for treating a human disease, comprising administering an effective amount of the conjugated molecule of any one of claims 1-40, or the pharmaceutical composition of claim 44, to a subject.

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