Ultra-long acting platform containing Fc-long fatty acid chains

Abstract

The present invention relates to an ultra-long-acting platform for improving the half-life of an active molecule of a drug, the platform comprising an immunoglobulin Fc and a long-chain fatty acid chain. The present invention also relates to the preparation of the conjugate platform, a composition comprising the conjugate, and its therapeutic application. The present invention particularly relates to a conjugate molecule having the structure "active molecule-Fc-Cn", where the active molecule is selected from any molecule beneficial to an organism, Fc is immunoglobulin IgG Fc, and Cn is C. 14-24 The modified moiety comprises a fatty acid chain. The present invention further relates to the preparation of the conjugate molecule, compositions comprising the conjugate, and therapeutic applications thereof.

Description

[Technical Field]

[0001] The present invention relates to an ultra-long-acting platform for improving the half-life of an active molecule of a drug, the platform comprising immunoglobulin Fc and a long-chain fatty acid chain. The present invention also relates to the preparation of the conjugate platform, a composition comprising the conjugate, and its therapeutic application. [Background technology]

[0002] Polypeptide drugs, which are more similar to endogenous substances in the body than chemical drugs, have advantages such as reduced toxicity and side effects and stable therapeutic effects. Therefore, they have been widely used in a variety of clinical treatments, including cancer, cardiovascular disease, and autoimmune diseases, and have attracted the attention of pharmaceutical manufacturers and scientific researchers for their broad applicability. However, traditional polypeptide drugs have low resistance to in vivo proteases and poor stability, leading to rapid degradation upon entry into the body and short plasma half-lives. Many bioactive peptide substances with low bioavailability cannot be administered orally, and these issues have significantly hindered the clinical application of polypeptide drugs. Furthermore, due to limited exposure to target tissues, many protein and polypeptide drugs require frequent administration to maintain clinically effective drug concentrations.

[0003] To solve these problems, polypeptide drug modifications are currently being performed. These modifications can be classified into two types: the first type is modification of the peptide chain framework; the second type is the introduction of other groups while maintaining the polypeptide framework to optimize the structure and modify performance, including polyethylene glycol modification, glycosylation modification, protein fusion strategy, long-chain fatty acid modification, site-directed mutagenesis, and cholesterol modification.

[0004] Fc fusion proteins, formed by fusing a bioactive molecule to the Fc region of an immunoglobulin, combine the beneficial pharmacological properties of the bioactive molecule with the additional properties of the Fc region, thereby extending the serum half-life of the bioactive molecule and thereby reducing dosing frequency. Currently, fusing the Fc region to an active peptide as a ligand or receptor or to an extracellular domain (ECD) significantly enhances the clinical potential of active protein drugs. In particular, products with a molecular weight of less than 60 kDa are easily excreted by the kidney and have a short serum half-life; however, increasing the size of the product by conjugation or fusion to the Fc region can exceed the renal filtration threshold and extend the product's circulation time. After being taken up by endothelial cells and translocated into acidified endosomes, Fc fusion proteins are protected from lysosomal degradation by the binding of Fc to FcRn within the endosome; when transported to the cell surface by recycling endosomes, Fc dissociates from FcRn under neutral to slightly alkaline conditions, returning the Fc fusion protein to the blood circulation, thereby extending the half-life of the Fc fusion protein, which allows target tissues to be exposed to the pharmacologically active moiety of the Fc fusion protein for a longer period of time and improving the therapeutic potential of the Fc fusion protein.

[0005] Fc fusion proteins are a successful biopharmaceutical product class, with 13 drugs approved in Europe and the United States, including three etanercept biosimilars. Potential bioactive molecules are diverse, including extracellular domains of natural receptors, functionally active peptides, recombinases, and recombinant binding constructs as cytokine traps. Most Fc fusion proteins are produced by fusing a bioactive molecule to the N-terminus of the Fc domain. Strong interactions between the IgG and CH3 domains form a stable Fc structure, while the flexible hinge region and disulfide bonds allow for the fusion of more complex structures. Dulaglutide, a hypoglycemic drug developed by Eli Lilly and Company by fusing GLP-1 to IgG4 (Fc), has an extended biological half-life due to a significant increase in molecular size, which reduces renal clearance of GLP-1.

[0006] Fatty acids are important components of human body fat, lipids, and cell membrane phospholipids. Because of their low immunogenicity as endogenous components, they are also used to modify biologically active molecules (e.g., polypeptide drugs). Modification of specific amino acid residues of polypeptide drugs with fatty acids extends the serum half-life of the polypeptide drug through reversible binding of the fatty acids to serum albumin. However, fatty acid modification has its own limitations, including the easy generation of nonspecifically modified fatty acid products and the easy elimination of polypeptide drugs from albumin by the kidney due to the reversible binding of fatty acids to serum albumin, thereby affecting or shortening the half-life of the fatty acid-modified polypeptide drug.

[0007] Extending the serum half-life of polypeptide drugs and reducing drug immunogenicity while maintaining their therapeutic efficacy are constant needs in scientific research and the pharmaceutical field, and this application fulfills these needs by providing an innovative ultra-long-acting platform that improves the active molecule of the drug. Summary of the Invention

[0008] The present invention provides ultra-long-acting platforms for improving the half-life of active molecules of drugs, which have the following structures: active molecule-fusion protein-Cn conjugate platform, active molecule-Fc-Cn conjugate platform, and antibody-Cn conjugate platform, where Cn represents a modified moiety containing n=14-24 fatty acid chains, and Fc is the Fc region of an immunoglobulin molecule. The ultra-long-acting platforms provided by the present application have the following advantages: 1. Both the Fc component and the long-chain fatty acid chain component of the conjugate molecule can bind directly or indirectly to FcRn, with Fc binding directly to FcRn and the long-chain fatty acid binding indirectly to FcRn via serum albumin, where Fc and serum albumin bind to different sites on FcRn and therefore do not interfere with each other, so that the active molecule conjugated thereto has a longer half-life than that of Fc alone or the long-chain fatty acid alone; 2. Fusion proteins, antibodies, Fc, and long-chain fatty acids are all endogenous substances in vivo and have low immunogenicity, which reduces the heterogeneity of the conjugated molecule to the living body and reduces the possibility of generating corresponding antibodies; 3. The conjugate molecule can increase the size of the active molecule contained therein and reduce the renal excretion rate, thereby prolonging the in vivo circulation time of the active molecule; 4. The hydrophobicity of the fatty acid chain contributes to improving the membrane permeability of the conjugated molecule, allowing more conjugated molecules to enter the circulatory system; 5. The Fc in the conjugate molecule can be linked to the cognate Fc fragment via its CH2-3 domain, improving stability and local conjugate / active molecule concentration.

[0009] In a first aspect, the present invention provides a conjugate molecule having the structure of "active molecule-Fc-Cn", a conjugate molecule having the structure of "active molecule-fusion protein-Cn", and a conjugate molecule having the structure of "antibody-Cn", wherein the active molecule is selected from any molecule beneficial to the living body, Fc is derived from the heavy chain constant region of immunoglobulin IgG, and Cn is C 14-24 The modified moiety comprises a fatty acid chain.

[0010] In some embodiments, the present invention provides conjugate molecules having the structure "active molecule-Fc-Cn", conjugate molecules having the structure "active molecule-fusion protein-Cn", and conjugate molecules having the structure "antibody-Cn", wherein Cn has the structure of formula (I): -ZY (I) wherein Z has the following structure: -Z1-Z2-Z3-Z4- where Z1 is a sulfur atom, a nitrogen atom, or an oxygen atom in Fc, Z2 is -C(=O)- or a 5- to 10-membered heterocyclyl containing 1 or 2 heteroatoms preferably selected from N, S, and O; Z3 is a bond, -C(=O)-, -C1-C 10 Alkylene-C(=O)-, -C3-C 10 Alkynylene-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)-, -allylidene-C(=O)-, -C1-C 10 Alkylene-arylidene-C(=O)-, -arylidene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-C3-C8 cycloalkylene-C(=O)-, -C3-C8 cycloalkylene-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclylene-C(=O)-, -C1-C 10 Alkylene-C3-C8 heterocyclylene-C(=O)-, -C3-C8 heterocyclylene-C1-C 10 alkylene-C(=O)-, wherein alkylene, alkynylene, alkenylene, heteroalkylene, cycloalkylene, arylidene, and heterocyclylene are optionally substituted; Z4 is a bond or a PEG unit represented by the formula: [ka] where: R1 is C 1-4 Alkylene, -NH-, -NH-C 1-4 Alkylene-, -NH-C 1-4 alkylene-heteroaryl-, where heteroaryl is a 5- 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-, where m is an integer of 2 to 6 and p is an integer of 1 to 3; Y is [ka] where: Y is bonded to Z4 via X, and k is an integer of 10 to 30. where R is independently hydrogen, C 1-6 Alkyl, C 1-6 Aminoalkyl, C 1-6 Haloalkyl, C 1-6 represents hydroxyalkyl.

[0011] In some embodiments, Z2 is a maleimide group. [ka] where the wavy line on the left indicates the position where Z1 binds, and the wavy line on the right indicates the position where Z3 binds.

[0012] In some embodiments, Z3 is -C1-C 10 It is alkylene-C(=O)-, wherein the alkylene is optionally substituted, and Z3 is bonded to Z4 via -C-(=O)-.

[0013] In some preferred embodiments, Z2 is a maleimide group and Z3 is -C 1-6 It is alkylene -C(=O)-.

[0014] In some embodiments, Z4 is a bond and Z3 is directly bonded to Y in formula (I).

[0015] In some embodiments, Z4 is a PEG unit having the formula: [ka] where: R1 is -NH- and -NH-C 1-4 alkylene-; R2 is selected from the group consisting of -C1-4 Alkylene or -C 1-4 Alkylene-NH-C(=O)-(CH2OCH2) p -C 1-4 alkylene-, where m is an integer of 2 to 6, and p is an integer of 1 to 3.

[0016] In some embodiments, Z4 is a unit comprising 2 to 6 PEG. [ka] where: m=1 to 4, the asterisk on the left indicates the position where Z3 is attached; the asterisk on the right indicates the position where Y is attached in formula II.

[0017] In some embodiments, Z in formula (I) of the present invention has the following structure: [ka] where R E is hydrogen, C 1-6 Alkyl, C 1-6 Aminoalkyl, C 1-6 Haloalkyl, C 1-6 It is hydroxyalkyl, where y=0 to 4 and m=1 to 4, the asterisk on the left indicates the position where Ab binds, and the asterisk on the right indicates the position where Y binds.

[0018] Y is [ka] wherein Y is bonded to Z4 via X, and X is -NH-(C=O)- or -(C=O)-NH-; k is an integer from 10 to 30, where R is independently hydrogen, C 1-6 Alkyl, C 1-6 Aminoalkyl, C 1-6 Haloalkyl, C 1-6 represents hydroxyalkyl.

[0019] In one particular embodiment, Cn is [ka] is selected from.

[0020] In one embodiment, the Fc is derived from the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4. In a specific embodiment, the Fc is derived from the heavy chain constant region of IgG1 or IgG4. In yet another embodiment, the Fc region can further comprise a hinge region. In a specific embodiment, the Fc comprises an amino acid modification. In a specific embodiment, the modification in the Fc region is at positions 254, 308, and 434 (according to EU numbering). In another specific embodiment, the modification in the Fc region is a substitution of the amino acids at positions 254, 308, and 434 with Thr, Pro, and Ala, respectively. In a specific embodiment, the modification in the Fc region is at positions 228, 234, 235, and / or 447, for example, including modifications S228P, F234A, and L235A, or modifications S228P, F234A, and L235A and a deletion at position 447. In one specific embodiment, the Fc region is selected from the sequence of SEQ ID NO: 10, 15, or 16.

[0021] In one embodiment, the active molecule is an active peptide molecule. In one embodiment, the active peptide molecule is fused to an antibody, fusion protein, or Fc region directly or via a peptide linker. In one embodiment, the C-terminus of the active peptide molecule is fused to the N-terminus of the antibody, fusion protein, or Fc region. Alternatively, the N-terminus of the active peptide molecule is fused to the C-terminus of the antibody, fusion protein, or Fc region. In yet another specific embodiment, the active peptide molecule is bound to the antibody, fusion protein, or Fc region in a monomeric form. In yet another specific embodiment, the active peptide molecule is bound to the antibody, fusion protein, or Fc region in a multimeric form.

[0022] In one embodiment, the active molecule is selected from the group consisting of 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 differentiation factor, a factor involved in cell motility or migration, a factor involved in bone formation / resorption, a chemokine, a plasma or stromal adhesion molecule or extracellular matrix, a bactericidal or antifungal factor, etc.

[0023] In one specific embodiment, the active molecule is selected from the group consisting of GLP-1, an antibody Fab fragment, and an antibody F(ab')2 fragment. In another specific embodiment, the active molecule is selected from the group consisting of 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.

[0024] In one embodiment, the peptide linker comprises the amino acid sequence (G4S)n, where n is an integer greater than or equal to 1. In a particular 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, LQRDGERP, LRQKDGGGSERP, and GSTSGSGK PGSGEGSTKG.

[0025] In one embodiment, a conjugate molecule having the structure of "active molecule-Fc-Cn" or "antibody-Cn" provided in the present application is homodimerized via its Fc region.

[0026] In one specific embodiment, the present application provides a conjugate molecule having the structure "active molecule-Fc-Cn," where Fc is selected from IgG1 or IgG4, and Cn comprises a 16-, 18-, or 20-carbon fatty acid chain. In a preferred embodiment, Fc comprises a modification as mentioned 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.

[0027] In one specific embodiment, the present application provides a conjugate molecule having an "antibody-Cn" structure, where 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, e.g., as mentioned 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.

[0028] In one specific embodiment, the present application provides a conjugate molecule having the structure "active molecule-Fc-Cn," where Fc is selected from IgG1 or IgG4, and Cn comprises a 16-, 18-, or 20-carbon fatty acid chain, bound / conjugated to a free thiol group of Fc. In a preferred embodiment, Fc comprises a modification, e.g., as mentioned 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.

[0029] In one specific embodiment, the present application provides a conjugate molecule having an "antibody-Cn" structure, wherein the antibody is selected from IgG1 or IgG4, and Cn comprises a 16-, 18-, or 20-carbon fatty acid chain, which is attached / conjugated to a sulfur atom of a free thiol group of the antibody Fc. In a preferred embodiment, the Fc of the antibody comprises a modification, e.g., as mentioned 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.

[0030] In one particular embodiment, the present application provides a conjugate molecule having the structure "active molecule-fusion protein-Cn," where Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is attached / conjugated to a sulfur atom of a free thiol group of the fusion protein. In one particular embodiment, Cn comprises a 16-carbon fatty acid chain, in another particular embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another particular embodiment, Cn comprises a 20-carbon fatty acid chain.

[0031] In one specific embodiment, the present application provides a conjugate molecule having the structure "active molecule-IgG4 Fc-Cn," where Cn comprises a 16-, 18-, or 20-carbon fatty acid chain, which is bound / conjugated to a free thiol group of IgG4 Fc. In a preferred embodiment, the IgG4 Fc comprises a modification, e.g., as mentioned 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.

[0032] In one specific embodiment, the present application provides a conjugate molecule having an "antibody-Cn" structure, where Cn comprises a 16-, 18-, or 20-carbon fatty acid chain, which is attached / conjugated to the sulfur atom of a free thiol group of an IgG4 antibody. In a preferred embodiment, the Fc of the IgG4 antibody comprises a modification, e.g., as mentioned 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.

[0033] In one particular embodiment, the present application provides a conjugate molecule having the structure "active molecule-fusion protein-Cn," where Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is attached / conjugated to a sulfur atom of a free thiol group of the fusion protein. In one particular embodiment, Cn comprises a 16-carbon fatty acid chain, in another particular embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another particular embodiment, Cn comprises a 20-carbon fatty acid chain.

[0034] In one specific embodiment, the present application provides a conjugate molecule having the structure "active molecule-IgG1 Fc-Cn," where Cn comprises a 16-, 18-, or 20-carbon fatty acid chain, bound / conjugated to the sulfur atom of a free thiol group of IgG1 Fc. In a preferred embodiment, the IgG1 Fc comprises a modification, e.g., as mentioned 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.

[0035] In one specific embodiment, the present application provides a conjugate molecule having an "antibody-Cn" structure, where Cn comprises a 16-, 18-, or 20-carbon fatty acid chain, bound / conjugated to the sulfur atom of a free thiol group of an IgG1 antibody. In a preferred embodiment, the Fc of the IgG1 antibody comprises a modification, e.g., as mentioned 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.

[0036] In one particular embodiment, the present application provides a conjugate molecule having the structure "active molecule-fusion protein-Cn," where Cn comprises a 16-, 18-, or 20-carbon fatty acid chain and is attached / conjugated to a sulfur atom of a free thiol group of the fusion protein. In one particular embodiment, Cn comprises a 16-carbon fatty acid chain, in another particular embodiment, Cn comprises an 18-carbon fatty acid chain, and in yet another particular embodiment, Cn comprises a 20-carbon fatty acid chain.

[0037] In a specific embodiment, the conjugate molecule having the structure "active molecule-Fc-Cn" in the present application is a GLP-1-Fc-Cn conjugate molecule, wherein GLP-1 is any active GLP-1 known in the art. In one embodiment, Fc is selected from IgG1 Fc or IgG4 Fc. In a specific embodiment, Cn in the conjugate molecule GLP-1-Fc-Cn comprises a 16-, 18-, or 20-carbon fatty acid chain, which is bonded / conjugated to the sulfur atom of a free thiol group of Fc or to a nitrogen atom of Fc. In a specific embodiment, Cn in the conjugate molecule GLP-1-Fc-Cn is selected from TM1, C18-tert-butanol ester, C16-NHS, or C20-NHS. In a preferred technical solution, the conjugate molecule GLP-1-Fc-Cn is GLP-1-IgG4 Fc-TM1, GLP-1-IgG4 Fc-C18 tert-butanol ester, GLP-1-IgG4 Fc-C16-NHS, or GLP1-IgG4 Fc-C20-NHS. In one embodiment, Fc comprises the sequence shown in SEQ ID NO: 10, 15, or 16. In a further preferred embodiment, the GLP-1-IgG4 Fc in the conjugate molecules GLP-1-IgG4 Fc-TM1, GLP-1-IgG4 Fc-C18, GLP-1-IgG4 Fc-C16-NHS, and GLP1-IgG4 Fc-C20-NHS is derived from dulaglutide, preferably dulaglutide, and has, for example, the structure disclosed in Chinese Patent No. 1802167, preferably Gly. 8 -Glu 22 -Gly 36 and dulaglutide having the structure -GLP-1(7-37)-1L-IgG4(S228P, F234A, L235A). In one embodiment, the amino acid sequence of dulaglutide is as set forth 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-butanol ester, dulaglutide-C16-NHS, or dulaglutide-C20-NHS.

[0038] In a specific embodiment, the active molecule in the conjugate molecule having the structure "active molecule-Fc-Cn" in the present application is an anti-PD-1 antibody or antigen-binding fragment thereof, which may be any known anti-PD-1 antibody or antigen-binding fragment thereof. In a specific embodiment, the antibody in the conjugate molecule having the structure "antibody-Cn" in the present application is an anti-PD-1 antibody or antigen-binding fragment thereof, which may be any known anti-PD-1 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 an antigen-binding fragment of anti-PD-1 antibody-IgG4 Fc-TM1, an antigen-binding fragment of anti-PD-1 antibody-IgG4 Fc-C18, an antigen-binding fragment of anti-PD-1 antibody-IgG4 Fc-C16-NHS, or an antigen-binding fragment of anti-PD-1 antibody-IgG4 Fc-C20-NHS. In a preferred technical solution, the conjugate molecules are an antigen-binding fragment of anti-PD-1 antibody-IgG1 Fc-TM1, an antigen-binding fragment of anti-PD-1 antibody-IgG1 Fc-C18, an antigen-binding fragment of anti-PD-1 antibody-IgG1 Fc-C16-NHS, and an antigen-binding fragment of anti-PD-1 antibody-IgG1 Fc-C20-NHS. In a preferred embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:9 and a 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 further preferred embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain set forth in SEQ ID NO:8 and a light chain set forth in SEQ ID NO:12. In a further preferred embodiment, Cn is selected from the group consisting of TM1, C18-tert-butanol ester, C16-NHS, and C20-NHS. In a preferred embodiment, Cn is selected from TM1.

[0039] In a specific embodiment, the active molecule in the conjugate molecule having the structure "active molecule-Fc-Cn" in the present application is an anti-VEGF antibody or an antigen-binding fragment thereof, which may be any known anti-VEGF antibody or an antigen-binding fragment thereof. In a specific embodiment, the antibody in the conjugate molecule having the structure "antibody-Cn" in the present application is an anti-VEGF antibody or an antigen-binding fragment thereof, which may be any known anti-VEGF antibody or an 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 an antigen-binding fragment of anti-VEGF antibody-IgG4 Fc-TM1, an antigen-binding fragment of anti-VEGF antibody-IgG4 Fc-C18, an antigen-binding fragment of anti-VEGF antibody-IgG4 Fc-C16-NHS, or an antigen-binding fragment of anti-VEGF antibody-IgG4 Fc-C20-NHS. In a preferred technical solution, the conjugate molecule is an antigen-binding fragment of an anti-VEGF antibody-IgG1 Fc-TM1, an antigen-binding fragment of an anti-VEGF antibody-IgG1 Fc-C18-tert-butanol ester, an antigen-binding fragment of an anti-VEGF antibody-IgG1 Fc-C16-NHS, or an antigen-binding fragment of an anti-VEGF antibody-IgG1 Fc-C20-NHS. In a preferred embodiment, 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. In a preferred embodiment, the Fc comprises the sequence set forth in SEQ ID NO: 15. In a further preferred embodiment, the anti-VEGF antibody or antigen-binding fragment thereof comprises a heavy chain set forth in SEQ ID NO: 13 and a light chain set forth in SEQ ID NO: 14. In a further preferred embodiment, Cn is selected from the group consisting of TM1, C18-tert-butanol ester, C16-NHS, and C20-NHS. In one preferred embodiment, Cn is selected from TM1.

[0040] In a second aspect, the present application provides a method for preparing a conjugate molecule having an "active molecule-Fc-Cn" structure, the method comprising: (a) binding an active molecule polypeptide to an immunoglobulin Fc region to prepare an "active molecule-Fc" fusion; and (b) subjecting the "active molecule-Fc" fusion and a Cn comprising a fatty acid chain to a conjugation reaction under conditions that allow conjugation of the Fc region to the Cn, thereby obtaining an "active molecule-Fc-Cn" conjugate molecule.

[0041] The present application further provides a method for preparing a conjugate molecule having an "antibody-Cn" structure, the method comprising: (a) subjecting an antibody and a Cn comprising a fatty acid chain to a conjugation reaction under conditions that allow conjugation of the antibody to the Cn, thereby obtaining an "antibody-Cn" conjugate molecule.

[0042] The present invention further provides a method for preparing a conjugate molecule having an "active molecule-fusion protein-Cn" structure, the method comprising: (a) conjugating 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 and a Cn comprising a fatty acid chain to a conjugation reaction under conditions that allow conjugation of the fusion protein to the Cn, resulting in an "active molecule-fusion protein-Cn" conjugate molecule.

[0043] In another embodiment, the present application provides a method for preparing a conjugate molecule having the structure "active molecule-Fc-Cn," comprising the steps of: (a) binding an antibody Fab fragment to an immunoglobulin Fc region to prepare an "Fab-Fc" fusion; and (b) subjecting the "Fab-Fc" fusion to a conjugation reaction under conditions that allow conjugation to a Cn comprising a fatty acid chain of the Fc region, thereby obtaining an "Fab-Fc-Cn" conjugate molecule. In one embodiment, the Fab and Fc are derived from the same or different antibody molecules.

[0044] In another embodiment, the present application provides a method for preparing a conjugate molecule having an "active molecule-Fc-Cn" structure, comprising the step of subjecting a whole antibody to a conjugation reaction under conditions that allow conjugation to a Cn comprising a fatty acid chain of the Fc region, thereby obtaining an "active molecule-Fc-Cn" conjugate molecule.

[0045] In another embodiment, the present application provides a method for preparing a conjugate molecule having an "antibody-Cn" structure, comprising the step of subjecting a whole antibody to a conjugation reaction under conditions that allow conjugation to a Cn comprising a fatty acid chain of the Fc region, resulting in an "active molecule-Fc-Cn" conjugate molecule.

[0046] In a third aspect, the present application provides a composition, e.g., a pharmaceutical composition, comprising the conjugate molecule described in the first aspect. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0047] In a fourth aspect, the present application provides a method for effectively extending the serum half-life of an active molecule, the method comprising the step of constructing the active molecule into a conjugate molecule having an "active molecule-Fc-Cn" structure, an "antibody-Cn" structure, or an "active molecule-fusion protein-Cn" structure according to the method of the second aspect, thereby effectively increasing the serum half-life of the active molecule.

[0048] In a fifth aspect, the present application provides the use of a conjugate molecule of the first aspect or a composition of the third aspect in the manufacture of a medicament for the treatment of a human disease.

[0049] In one embodiment, the invention provides a conjugated molecule of the first aspect, or a composition of the third aspect, for use in therapy.

[0050] In a sixth aspect, the present application provides a method of treating a human disease comprising administering to a subject an effective amount of a conjugated molecule of the first aspect, or a composition of the third aspect. [Brief explanation of the drawings]

[0051] [Figure 1] Figure 1 shows the HIC-HPLC results of the GLP1-Fc-TM1 conjugate product, where the top row shows the HIC-HPLC results of GLP1-Fc not conjugated with TM1, and the bottom row shows the HIC-HPLC results of GLP1-Fc-TM1 after conjugation with TM1. In the bottom row, "0" indicates GLP1-Fc not conjugated with TM1, "2" indicates GLP1-Fc conjugated with two TM1s, and "4" indicates GLP1-Fc conjugated with four TM1s. [Figure 2] FIG. 2 shows the HIC-HPLC results of the HX006-TM1-1 conjugation product, where the top row shows the HIC-HPLC results of HX006 not conjugated with TM1, and the bottom row shows the HIC-HPLC results of HX006-TM1-1 after conjugation with TM1. In the bottom row, "0" indicates HX006 not conjugated with TM1, "2" indicates HX006 conjugated with two TM1s, "4" indicates HX006 conjugated with four TM1s, and "6" indicates HX006 conjugated with six TM1s. [Figure 3] FIG. 3 shows the HIC-HPLC results of the HX006-TM1-2 conjugate product, where in the panels, "0" indicates HX006 with no TM1 conjugated, "2" indicates HX006 with two TM1 conjugated, "4" indicates HX006 with four TM1 conjugated, "6" indicates HX006 with six TM1 conjugated, and "8" indicates HX006 with eight TM1 conjugated. [Figure 4]Figure 4 shows the HIC-HPLC results of the HX008-TM1 conjugated product. Figure 4A shows the HIC-HPLC results of HX008 without TM1 conjugation, and Figure 4B shows the HIC-HPLC results of HX008-TM1-2 after TM1 conjugation. Within each panel, "0" represents HX008 without TM1 conjugation, "2" represents HX008 with two TM1 conjugations, and "4" represents HX008 with four TM1 conjugations. Gated HX008, "6" indicates HX008 conjugated with six TM1s; Figure 4C shows the HIC-HPLC results of HX008-TM1-3 after conjugation with TM1, where in the panel "0" indicates HX008 without TM1, "2" indicates HX008 conjugated with two TM1s, "4" indicates HX008 conjugated with four TM1s, and "6" indicates HX008 conjugated with six TM1s. [Figure 5] FIG. 5 shows the results of an ELISA assay for the binding of GLP1-Fc-TM1 to HAS, where HX042 represents GLP1-Fc. [Figure 6] FIG. 6 shows the results of an ELISA assay for binding of HX006-TM1 to HAS. [Figure 7] FIG. 7 shows the results of an ELISA assay for binding of HX008-TM1 to HAS. [Figure 8] Figure 8 shows the results of HIC-HPLC of GLP1-Fc after conjugation with C18-tert-butanol esters. In the panel, "0" indicates GLP1-Fc without C18-tert-butanol ester conjugation, and "2" indicates GLP1-Fc with two C18-tert-butanol esters conjugated. [Figure 9] FIG. 9 shows the results of HIC-HPLC of the GLP1-Fc sample (top row) and the GLP1-Fc-C16-NHS sample (bottom row). [Figure 10] FIG. 10 shows the results of HIC-HPLC of the GLP1-Fc-C20-NHS sample. [Figure 11]FIG. 11 shows the results of ELISA assays for the binding of GLP1-Fc-C16-NHS and GLP1-Fc-C20-NHS to HSA, respectively, where HX042 represents GLP1-Fc. [Figure 12] FIG. 12 shows the results of ELISA assays for the binding of HX006-C16-NHS and HX006-C20-NHS to HAS, respectively. [Figure 13] FIG. 13 shows the binding activity of GLP1-Fc-C18-tert-butanol ester to HSA. [Figure 14] FIG. 14 shows the binding activity of HX006-C18-tert-butanol ester to HSA. [Figure 15] FIG. 15 shows the HIC-HPLC results of HX006 after conjugation with C18-tert-butanol ester. [Figure 16] FIG. 16 shows that GLP-1-Fc-TM1 can effectively activate the biological activity of the reporter gene. [Figure 17] FIG. 17 shows the plasma concentration-time curve of GLP1-Fc-TM1 after a single dose in rats, with dulaglutide as a positive control. [Figure 18] FIG. 18 shows the plasma concentration-time curve of GLP1-Fc-TM1 after a single dose in cynomolgus monkeys, with dulaglutide shown as a positive control. [Figure 19-1] Figure 19 shows the efficacy results of GLP-1-Fc-TM1 on type II diabetic db / db mice, where Figure 19-1 shows the results of reducing 4-hour fasting blood glucose levels in db / db mice; Figures 19-2 and 19-3 show the results of random blood glucose levels after the first administration and the final administration; Figures 19-4 and 19-5 show the results of blood glucose AUC0-180 minutes in the OGTT test after the final administration; Figure 19-6 shows the results of reducing glycated hemoglobin levels in db / db mice; Figure 19-7 shows the results of increasing insulin levels in db / db mice; and Figure 19-8 shows the results of reducing average daily food intake in db / db mice. [Figure 19-2]Figure 19 shows the efficacy results of GLP-1-Fc-TM1 on type II diabetic db / db mice, where Figure 19-1 shows the results of reducing 4-hour fasting blood glucose levels in db / db mice; Figures 19-2 and 19-3 show the results of random blood glucose levels after the first administration and the final administration; Figures 19-4 and 19-5 show the results of blood glucose AUC0-180 minutes in the OGTT test after the final administration; Figure 19-6 shows the results of reducing glycated hemoglobin levels in db / db mice; Figure 19-7 shows the results of increasing insulin levels in db / db mice; and Figure 19-8 shows the results of reducing average daily food intake in db / db mice. [Figure 19-3] Figure 19 shows the efficacy results of GLP-1-Fc-TM1 on type II diabetic db / db mice, where Figure 19-1 shows the results of reducing 4-hour fasting blood glucose levels in db / db mice; Figures 19-2 and 19-3 show the results of random blood glucose levels after the first administration and the final administration; Figures 19-4 and 19-5 show the results of blood glucose AUC0-180 minutes in the OGTT test after the final administration; Figure 19-6 shows the results of reducing glycated hemoglobin levels in db / db mice; Figure 19-7 shows the results of increasing insulin levels in db / db mice; and Figure 19-8 shows the results of reducing average daily food intake in db / db mice. [Figure 19-4] Figure 19 shows the efficacy results of GLP-1-Fc-TM1 on type II diabetic db / db mice, where Figure 19-1 shows the results of reducing 4-hour fasting blood glucose levels in db / db mice; Figures 19-2 and 19-3 show the results of random blood glucose levels after the first administration and the final administration; Figures 19-4 and 19-5 show the results of blood glucose AUC0-180 minutes in the OGTT test after the final administration; Figure 19-6 shows the results of reducing glycated hemoglobin levels in db / db mice; Figure 19-7 shows the results of increasing insulin levels in db / db mice; and Figure 19-8 shows the results of reducing average daily food intake in db / db mice. [Figure 19-5]Figure 19 shows the efficacy results of GLP-1-Fc-TM1 on type II diabetic db / db mice, where Figure 19-1 shows the results of reducing 4-hour fasting blood glucose levels in db / db mice; Figures 19-2 and 19-3 show the results of random blood glucose levels after the first administration and the final administration; Figures 19-4 and 19-5 show the results of blood glucose AUC0-180 minutes in the OGTT test after the final administration; Figure 19-6 shows the results of reducing glycated hemoglobin levels in db / db mice; Figure 19-7 shows the results of increasing insulin levels in db / db mice; and Figure 19-8 shows the results of reducing average daily food intake in db / db mice. [Figure 19-6] Figure 19 shows the efficacy results of GLP-1-Fc-TM1 on type II diabetic db / db mice, where Figure 19-1 shows the results of reducing 4-hour fasting blood glucose levels in db / db mice; Figures 19-2 and 19-3 show the results of random blood glucose levels after the first administration and the final administration; Figures 19-4 and 19-5 show the results of blood glucose AUC0-180 minutes in the OGTT test after the final administration; Figure 19-6 shows the results of reducing glycated hemoglobin levels in db / db mice; Figure 19-7 shows the results of increasing insulin levels in db / db mice; and Figure 19-8 shows the results of reducing average daily food intake in db / db mice. [Figure 19-7] Figure 19 shows the efficacy results of GLP-1-Fc-TM1 on type II diabetic db / db mice, where Figure 19-1 shows the results of reducing 4-hour fasting blood glucose levels in db / db mice; Figures 19-2 and 19-3 show the results of random blood glucose levels after the first administration and the final administration; Figures 19-4 and 19-5 show the results of blood glucose AUC0-180 minutes in the OGTT test after the final administration; Figure 19-6 shows the results of reducing glycated hemoglobin levels in db / db mice; Figure 19-7 shows the results of increasing insulin levels in db / db mice; and Figure 19-8 shows the results of reducing average daily food intake in db / db mice. [Figure 19-8]Figure 19 shows the efficacy results of GLP-1-Fc-TM1 on type II diabetic db / db mice, where Figure 19-1 shows the results of reducing 4-hour fasting blood glucose levels in db / db mice; Figures 19-2 and 19-3 show the results of random blood glucose levels after the first administration and the final administration; Figures 19-4 and 19-5 show the results of blood glucose AUC0-180 minutes in the OGTT test after the final administration; Figure 19-6 shows the results of reducing glycated hemoglobin levels in db / db mice; Figure 19-7 shows the results of increasing insulin levels in db / db mice; and Figure 19-8 shows the results of reducing average daily food intake in db / db mice. [Figure 20-1] Figure 20 shows the efficacy results of GLP-1-Fc-TM1 in DIO model mice. Figure 20-1 shows the weight loss results of DIO mice treated with GLP-1-Fc-TM1; Figure 20-2 shows the reduction in food intake of DIO mice treated with GLP-1-Fc-TM1; Figure 20-3 shows the reduction in body fat content in mice treated with GLP-1-Fc-TM1; Figure 20-4 shows the reduction in fasting blood glucose levels in mice treated with GLP-1-Fc-TM1; Figure 20-5 shows the reduction in blood lipid levels in mice treated with GLP-1-Fc-TM1; Figure 20-6 shows the levels of liver function indicators ALT and AST in mouse serum; and Figure 20-7 shows the improvement in hepatocellular balloon lesions and hepatocellular lipid metabolism abnormalities in DIO mice treated with GLP-1-Fc-TM1. [Figure 20-2]Figure 20 shows the efficacy results of GLP-1-Fc-TM1 in DIO model mice. Figure 20-1 shows the weight loss results of DIO mice treated with GLP-1-Fc-TM1; Figure 20-2 shows the reduction in food intake of DIO mice treated with GLP-1-Fc-TM1; Figure 20-3 shows the reduction in body fat content in mice treated with GLP-1-Fc-TM1; Figure 20-4 shows the reduction in fasting blood glucose levels in mice treated with GLP-1-Fc-TM1; Figure 20-5 shows the reduction in blood lipid levels in mice treated with GLP-1-Fc-TM1; Figure 20-6 shows the levels of liver function indicators ALT and AST in mouse serum; and Figure 20-7 shows the improvement in hepatocellular balloon lesions and hepatocellular lipid metabolism abnormalities in DIO mice treated with GLP-1-Fc-TM1. [Figure 20-3] Figure 20 shows the efficacy results of GLP-1-Fc-TM1 in DIO model mice. Figure 20-1 shows the weight loss results of DIO mice treated with GLP-1-Fc-TM1; Figure 20-2 shows the reduction in food intake of DIO mice treated with GLP-1-Fc-TM1; Figure 20-3 shows the reduction in body fat content in mice treated with GLP-1-Fc-TM1; Figure 20-4 shows the reduction in fasting blood glucose levels in mice treated with GLP-1-Fc-TM1; Figure 20-5 shows the reduction in blood lipid levels in mice treated with GLP-1-Fc-TM1; Figure 20-6 shows the levels of liver function indicators ALT and AST in mouse serum; and Figure 20-7 shows the improvement in hepatocellular balloon lesions and hepatocellular lipid metabolism abnormalities in DIO mice treated with GLP-1-Fc-TM1. [Figure 20-4]Figure 20 shows the efficacy results of GLP-1-Fc-TM1 in DIO model mice. Figure 20-1 shows the weight loss results of DIO mice treated with GLP-1-Fc-TM1; Figure 20-2 shows the reduction in food intake of DIO mice treated with GLP-1-Fc-TM1; Figure 20-3 shows the reduction in body fat content in mice treated with GLP-1-Fc-TM1; Figure 20-4 shows the reduction in fasting blood glucose levels in mice treated with GLP-1-Fc-TM1; Figure 20-5 shows the reduction in blood lipid levels in mice treated with GLP-1-Fc-TM1; Figure 20-6 shows the levels of liver function indicators ALT and AST in mouse serum; and Figure 20-7 shows the improvement in hepatocellular balloon lesions and hepatocellular lipid metabolism abnormalities in DIO mice treated with GLP-1-Fc-TM1. [Figure 20-5] Figure 20 shows the efficacy results of GLP-1-Fc-TM1 in DIO model mice. Figure 20-1 shows the weight loss results of DIO mice treated with GLP-1-Fc-TM1; Figure 20-2 shows the reduction in food intake of DIO mice treated with GLP-1-Fc-TM1; Figure 20-3 shows the reduction in body fat content in mice treated with GLP-1-Fc-TM1; Figure 20-4 shows the reduction in fasting blood glucose levels in mice treated with GLP-1-Fc-TM1; Figure 20-5 shows the reduction in blood lipid levels in mice treated with GLP-1-Fc-TM1; Figure 20-6 shows the levels of liver function indicators ALT and AST in mouse serum; and Figure 20-7 shows the improvement in hepatocellular balloon lesions and hepatocellular lipid metabolism abnormalities in DIO mice treated with GLP-1-Fc-TM1. [Figure 20-6]Figure 20 shows the efficacy results of GLP-1-Fc-TM1 in DIO model mice. Figure 20-1 shows the weight loss results of DIO mice treated with GLP-1-Fc-TM1; Figure 20-2 shows the reduction in food intake of DIO mice treated with GLP-1-Fc-TM1; Figure 20-3 shows the reduction in body fat content in mice treated with GLP-1-Fc-TM1; Figure 20-4 shows the reduction in fasting blood glucose levels in mice treated with GLP-1-Fc-TM1; Figure 20-5 shows the reduction in blood lipid levels in mice treated with GLP-1-Fc-TM1; Figure 20-6 shows the levels of liver function indicators ALT and AST in mouse serum; and Figure 20-7 shows the improvement in hepatocellular balloon lesions and hepatocellular lipid metabolism abnormalities in DIO mice treated with GLP-1-Fc-TM1. [Figure 20-7] Figure 20 shows the efficacy results of GLP-1-Fc-TM1 in DIO model mice. Figure 20-1 shows the weight loss results of DIO mice treated with GLP-1-Fc-TM1; Figure 20-2 shows the reduction in food intake of DIO mice treated with GLP-1-Fc-TM1; Figure 20-3 shows the reduction in body fat content in mice treated with GLP-1-Fc-TM1; Figure 20-4 shows the reduction in fasting blood glucose levels in mice treated with GLP-1-Fc-TM1; Figure 20-5 shows the reduction in blood lipid levels in mice treated with GLP-1-Fc-TM1; Figure 20-6 shows the levels of liver function indicators ALT and AST in mouse serum; and Figure 20-7 shows the improvement in hepatocellular balloon lesions and hepatocellular lipid metabolism abnormalities in DIO mice treated with GLP-1-Fc-TM1. [Figure 21-1] Figure 21 shows the results of the efficacy of GLP-1-Fc-TM1 in rats, Figures 21-1 to 21-4 show the results of blood glucose level reduction in SD rats by GLP-1-Fc-TM1, and Figures 21-5 to 21-8 show the results of serum insulin increase by GLP-1-Fc-TM1. [Figure 21-2]Figure 21 shows the results of the efficacy of GLP-1-Fc-TM1 in rats, Figures 21-1 to 21-4 show the results of blood glucose level reduction in SD rats by GLP-1-Fc-TM1, and Figures 21-5 to 21-8 show the results of serum insulin increase by GLP-1-Fc-TM1. [Figure 21-3] Figure 21 shows the results of the efficacy of GLP-1-Fc-TM1 in rats, Figures 21-1 to 21-4 show the results of blood glucose level reduction in SD rats by GLP-1-Fc-TM1, and Figures 21-5 to 21-8 show the results of serum insulin increase by GLP-1-Fc-TM1. [Figure 21-4] Figure 21 shows the results of the efficacy of GLP-1-Fc-TM1 in rats, Figures 21-1 to 21-4 show the results of blood glucose level reduction in SD rats by GLP-1-Fc-TM1, and Figures 21-5 to 21-8 show the results of serum insulin increase by GLP-1-Fc-TM1. [Figure 21-5] Figure 21 shows the results of the efficacy of GLP-1-Fc-TM1 in rats, Figures 21-1 to 21-4 show the results of blood glucose level reduction in SD rats by GLP-1-Fc-TM1, and Figures 21-5 to 21-8 show the results of serum insulin increase by GLP-1-Fc-TM1. [Figure 21-6] Figure 21 shows the results of the efficacy of GLP-1-Fc-TM1 in rats, Figures 21-1 to 21-4 show the results of blood glucose level reduction in SD rats by GLP-1-Fc-TM1, and Figures 21-5 to 21-8 show the results of serum insulin increase by GLP-1-Fc-TM1. [Figure 21-7] Figure 21 shows the results of the efficacy of GLP-1-Fc-TM1 in rats, Figures 21-1 to 21-4 show the results of blood glucose level reduction in SD rats by GLP-1-Fc-TM1, and Figures 21-5 to 21-8 show the results of serum insulin increase by GLP-1-Fc-TM1. [Figure 21-8] Figure 21 shows the results of the efficacy of GLP-1-Fc-TM1 in rats, Figures 21-1 to 21-4 show the results of blood glucose level reduction in SD rats by GLP-1-Fc-TM1, and Figures 21-5 to 21-8 show the results of serum insulin increase by GLP-1-Fc-TM1. DETAILED DESCRIPTION OF THE INVENTION

[0052] I. Definition

[0023] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For purposes of the present invention, the following terms are defined as follows:

[0053] When trademarks are used herein, they are intended to include product forms, generic versions and active pharmaceutical ingredients of the trademarked products, unless the context clearly indicates otherwise.

[0054] The term "about" used in conjunction with a numerical value is meant to include numerical values ​​within a range having a lower limit of 5% below the specified numerical value and an upper limit of 5% above the specified numerical value.

[0055] The term "and / or" means any alternative or combination of two or more alternatives.

[0056] The term "comprising" or "including" means including the stated elements, integers, or steps, but does not exclude other elements, integers, or steps. When the term "comprising" or "including" is used herein, unless otherwise specified, it also encompasses situations consisting of the stated elements, integers, or steps. For example, reference to an antibody variable region "comprising" a particular sequence is intended to encompass an antibody variable region consisting of that particular sequence.

[0057] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody constructs, 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. Intact antibodies typically contain at least two complete heavy chains and two complete light chains, although in some cases they may contain fewer chains; for example, antibodies naturally occurring in camels may contain only heavy chains.

[0058] The term "whole antibody" refers to an immunoglobulin molecule comprising at least two heavy (H) chains and two light (L) chains. Each heavy chain may consist of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain may consist of a light chain variable region (abbreviated herein as VL) and a light chain constant region. Antibody heavy chains can be classified into five major different classes based on the amino acid sequence of their constant regions: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further classified into subclasses, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0059] The terms "antibody fragment" and "antigen-binding fragment" of an antibody are used interchangeably and refer to a molecule that is not an intact antibody but contains a portion of the intact antibody that is used to bind to the antigen to which the intact antibody binds. As those skilled in the art will appreciate, for purposes of antigen binding, antibody fragments typically contain amino acid residues from the "complementarity-determining regions" or "CDRs." Antibody fragments may be prepared by recombinant DNA techniques 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, antibody fragments contain cysteine ​​residues introduced into the Fc region to provide amino acid residue sites for thiol-linking chemistry.

[0060] When an antibody is referred to herein as an "IgG antibody," this refers to a heterotetrameric protein having an immunoglobulin structure of the IgG class. In IgG antibodies, the VH-CH1 of the heavy chain usually pairs with the VL-CL of the light chain to form a Fab fragment that specifically binds to an antigen. Thus, IgG antibodies essentially consist of two Fab molecules connected by an immunoglobulin hinge region and two dimerized Fc regions. IgG immunoglobulins can be classified into subclasses, e.g., gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), and gamma 4 (IgG4), based on the sequence of the heavy chain constant region. In some embodiments, the antibody of the present invention is an IgG antibody, e.g., an IgG1, IgG2, IgG3, or IgG4 antibody.

[0061] The terms "complementary determining region" or "CDR region" or "CDR" or "hypervariable region" are regions of antibody variable domains that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes.

[0062] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding that antibody to an antigen. The heavy chain variable region (VH) and light chain variable region (VL) can be further subdivided into hypervariable regions (HVRs, also called complementarity-determining regions (CDRs)) interspersed with more conserved regions (i.e., framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0063] 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 an ELISA assay, and another method is a surface plasmon resonance (SPR) assay, as described in the examples herein.

[0064] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain and includes native Fc region sequences and variant Fc regions, e.g., Fc region sequences of various Ig subtypes and their allotypes (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, a human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or Pro230 to the carboxy terminus of the heavy chain, although the C-terminal lysine (Lys447) of the Fc region may or may not be present. In still other embodiments, a human IgG heavy chain Fc region includes a native immunoglobulin hinge sequence or partial hinge sequence at the N-terminus, e.g., the sequence E216-T225 or the sequence D221-T225 according to EU numbering. In certain embodiments, the Fc region of an immunoglobulin comprises two constant regions, namely, CH2 and CH3, while in other embodiments, the Fc region of an immunoglobulin comprises three constant regions, namely, CH2, CH3, and CH4.

[0065] The EU numbering scheme is a widely adopted standard for numbering antibody residues in a consistent manner, developed based on sequence alignments. In the context of this application, unless otherwise specified, amino acid residues of antibodies or fusion protein regions will be numbered and referenced using the Kabat EU numbering scheme (as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0066] Similar terms, such as "Fc region variant," "variant Fc region," and "variant Fc region," are used interchangeably to refer to an Fc region that contains at least one amino acid modification that differs from the native / wild-type Fc region sequence. For example, amino acids at various positions in the Fc region of a wild-type immunoglobulin IgG are modified to reduce hapten formation, reduce or eliminate effector function, or extend serum half-life. Further modifications to Fc regions known in the art, Fc regions provided herein, fusion proteins (e.g., antibodies) comprising Fc regions, and the like, can be made using a variety of methods disclosed in the art, such as modifications that reduce immunogenicity, improve stability, solubility, function, and clinical benefit. Such modifications include, but are not limited to, modifications at the following positions of IgG Fc, such as amino acid residue positions 214-238, 250-260, 297-299, 307-318, 322 or 327-331, 380-390, and particularly, for example, 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 ensure that the Fc region is expressed as a homogeneous product. To improve the chemical stability of the Fc region, asparagine, which is vulnerable to deamidation, can be modified, for example, by substitution with glutamine, aspartic acid, or glutamic acid, modifications including, for example, 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, for example, by an L235K substitution. In some embodiments, immunoglobulin Fc regions may also be modified to meet specific requirements by phosphorylation, sulfation, glycosylation, methylation, acetylation, amidation, etc.

[0067] In one embodiment, the modifications to the Fc region are modifications at positions 254, 308, and 434, such as the modifications disclosed in Chinese Patent Application Publication No. 108299560. In another example, the modifications to the Fc region are modifications described in Chinese Patent Application Publication No. 1802167. In a particular embodiment, the modifications to the Fc region are modifications at positions 228, 234, 235, and / or 447, such as the modifications S228P, F234A, L235A or the modifications S228P, F234A, L235A and a deletion at position 447. In the present application, modifications to the Fc region are indicated in parentheses, for example, IgG4 Fc(S228P, F234A, L235A) indicates that the amino acids at positions 228, 234, and 235 of wild-type IgG4 Fc have been substituted at the corresponding positions.

[0068] Wild-type immunoglobulin Fc regions can be obtained from humans and animals (e.g., cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc.). Alternatively, recombinant forms or derivatives of Fc regions can be obtained from transformed animal cells or microorganisms. For example, Fc regions can be obtained by isolating immunoglobulin-encoding genes from corresponding cDNA libraries using PCR techniques, or by subjecting intact immunoglobulins to protease treatment. Techniques for producing derivatives of Fc regions are described, for example, in WO 97 / 34631 and WO 96 / 32478.

[0069] In some embodiments, the Fc region used in the present application is derived from an IgG immunoglobulin, for example, an Fc region derived from the IgG1, IgG2, IgG3, and IgG4 subclasses, preferably the IgG1 and IgG4 subclasses. In some embodiments, the Fc region used in the present application is derived from human IgG1 and IgG4 in order to reduce the immunogenicity of the conjugates of the present application.

[0070] In some embodiments, the variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native Fc region sequence by one or more amino acid substitutions, deletions, or additions, hi some embodiments, the variant Fc region has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the wild-type and / or parent Fc region.

[0071] The term "neonatal Fc receptor (FcRn)" refers to an IgG antibody receptor localized on the surface of the cell membrane. FcRn is responsible for the transfer of maternal IgG to the fetus and regulates immunoglobulin homeostasis in vivo. FcRn can bind to the Fc portion of IgG and prevent IgG molecules from being degraded by lysosomes, thereby extending the half-life of IgG in vivo and is involved in the metabolic processes of IgG transport, maintenance, and distribution in vivo.

[0072] The term "receptor-mediated endocytosis" refers to the process by which a ligand / receptor complex is internalized and delivered to the cytoplasm or transported to an appropriate intracellular compartment, triggered by binding of the ligand to the corresponding receptor on the cell surface.

[0073] The term "sequence identity" refers to the degree to which sequences are identical on a nucleotide or amino acid basis within a comparison window. "Percentage of sequence identity" can be calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions where the same nucleotide base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occurs in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment for determining percentage of sequence identity can be achieved by various methods 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 sequence alignment, including algorithms needed to achieve maximal alignment over the entire sequence or region of the target sequence being compared.

[0074] In the present invention, with respect to antibody sequences, the percentage of amino acid sequence identity is determined by optimally aligning a candidate antibody sequence with a given antibody sequence, in a preferred embodiment according to the Kabat numbering convention. As used herein, alignment is applicable to the full-length alignment of a given antibody sequence, without specifying a comparison window (i.e., the target antibody region to be compared). In some embodiments, with respect to antibodies, the percentage of sequence identity may be distributed over the heavy and / or light chain variable regions, or the percentage of sequence identity may be limited to framework regions, with the sequences of the corresponding CDR regions remaining 100% identical.

[0075] The term "amino acid substitution" refers to the replacement of at least one amino acid residue in a given amino acid sequence with another, different, "substituted" amino acid residue.

[0076] The term "conservative substitution" refers to the substitution of amino acids within the same class, e.g., an acidic amino acid for another acidic amino acid, a basic amino acid for another basic amino acid, or a neutral amino acid for another neutral amino acid.

[0077] In the context of this application relating to conjugates, the term "peptide linker" refers to a short amino acid sequence that links the active ingredient to the Fc region and consists of amino acids (e.g., glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination), or the hinge region of an immunoglobulin.

[0078] Peptide linkers that can be used in the present invention can be easily determined by those skilled in the art. For example, n (n is an integer greater than or equal to 1). In a preferred embodiment, the peptide linker comprises (G4S)3, (G4S)4, (G4S)6, GS(G4S)4, DAAALEAAALDAAAREAAARDAAAL, and NVDHLPSNTLVDLA. Peptide linkers that can be used in the antibody molecules of the present invention can be, for example, but are not limited to, the following amino acid sequences: (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, LQRDGERP, LRQKDGGGSERP, and GSTSGSGKPGSGEGSTKG.

[0079] The term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group composed of carbon and hydrogen atoms. Specifically, alkyl has 1 to 10 carbon atoms, e.g., 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. For example, the term "C1-C6 alkyl" used herein refers to a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms, examples of which include methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, and tert-butyl), pentyl (including n-pentyl, isopentyl, and neopentyl), and 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, and 2-ethylbutyl).

[0080] The term "alkylene" refers to a divalent group obtained by removing two hydrogen atoms from two of the same or different carbon atoms from a straight- or branched-chain saturated alkane. Specifically, alkylene has 1 to 10 carbon atoms, e.g., 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. For example, the term "C1-C6 alkylene" as used herein refers to a straight- or branched-chain alkylene group having 1 to 6 carbon atoms, including, but not limited to, methylene, ethylidene, propylidene, butylidene, and the like.

[0081] The term "cycloalkyl" refers to a monocyclic, fused polycyclic, bridged polycyclic, or spiro non-aromatic monovalent hydrocarbon ring structure having the specified number of ring atoms, and may be saturated or unsaturated (e.g., containing one or more double bonds). A cycloalkyl group can contain three or more carbon atoms in the ring, e.g., 3 to 18, 3 to 10, or 3 to 8 carbon atoms, such as C 3-10 Cycloalkyl, C 3-8 Cycloalkyl, C 3-6 Cycloalkyl, C 5-6Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

[0082] The term "alkenyl" refers to a straight- or branched-chain unsaturated hydrocarbon group consisting of carbon and hydrogen atoms and containing at least one double bond. In particular, alkenyl has 2 to 8 carbon atoms, e.g., 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, the term "C2-C6 alkenyl" as used herein refers to a straight- or branched-chain alkenyl group having 2 to 6 carbon atoms, such as ethenyl, 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.

[0083] The term "alkenylene" refers to a divalent group obtained by removing two hydrogen atoms from two identical or different carbon atoms from a straight- or branched-chain unsaturated alkene containing at least one double bond. In particular, the alkenylene group has 2 to 8 carbon atoms, e.g., 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, the term "C2-C6 alkenylene" as used herein refers to a straight- or branched-chain alkenylene group having 2 to 6 carbon atoms, such as ethenylene, propenylene, arylidene, butenylene, pentenylene, and hexenylene.

[0084] The term "alkynyl" refers to a straight- or branched-chain unsaturated hydrocarbon group consisting of carbon and hydrogen atoms and containing at least one triple bond. In particular, alkynyl has 2 to 8, e.g., 2 to 6, 2 to 5, 2 to 4, or 2 to 3, carbon atoms. For example, the term "C2-C6 alkynyl" as used herein refers to a straight- or branched-chain alkynyl group having 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, and the like.

[0085] The term "alkynylene" refers to a divalent group obtained by removing two hydrogen atoms from two identical or different carbon atoms from a straight- or branched-chain unsaturated alkyne containing at least one triple bond. In particular, alkynylene has 2 to 8 carbon atoms, e.g., 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, the term "C2-C6 alkynylene" as used herein refers to a straight- or branched-chain alkynylene group having 2 to 6 carbon atoms, such as ethynylene, propynylene, propargylene, butynylene, pentynylene, and hexynylene.

[0086] The term "heterocycle" or "heterocyclyl" refers to a 5- to 20-membered (e.g., 5- to 14-, 5- to 8-, or 5- to 6-membered) aromatic or non-aromatic monocyclic, bicyclic, or polycyclic ring system containing 1 to 4 heteroatom ring members independently selected from N, O, or S. One or more of the N, C, or S atoms in the heterocycle may be oxidized. Preferably, the heterocycle is a 5- to 10-membered ring system, and 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, furan, thiazole, imidazole, pyrazole, pyrimidine, pyridine, pyrazine, pyridazine, isothiazole, and isoxazole. It is understood that this term encompasses heteroaryl as defined herein.

[0087] The term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6 to 20, e.g., 6 to 12, carbon atoms in the ring portion. Preferably, aryl is a C6-C 10 Aryl groups. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which is 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, thiol, alkyl-S—, aryl-S—, nitro, cyano, carboxy, alkyl-OC(O)—, carbamoyl, alkyl-S(O)—, sulfonyl, sulfonamido, heterocyclyl, and the like.

[0088] 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, which may be substituted or unsubstituted, containing 1-4 heteroatoms selected from N, O, or S. Preferably, the heteroaryl is a 5-10 membered ring system, which is monocyclic or fused bicyclic. 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.

[0089] The term "heteroalkyl" refers to a stable straight- or branched-chain hydrocarbon that is fully saturated or has one to three degrees of unsaturation, and consists of the specified number of carbon atoms and one to ten, preferably one to three, heteroatoms selected from O, N, Si, and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, Si, and S can be placed at any position within the heteroalkyl group or at the position at which the heteroalkyl group is attached to the remainder of the molecule. Representative examples of heteroalkyl groups include -CH-CH-O-CH, -CH-CH-NH-CH, -CH-CH-N(CH)-CH, -CH-S-CH-CH, -CH-CH-S(O)-CH, -NH-CH-CH-NH-C(O)-CH-CH, -CH-CH-S(O)-CH, -CH=CH-O-CH, -Si(CH), -CH-CH=NO-CH, and -CH=CH-N(CH)-CH. Up to two heteroatoms can be present consecutively, for example, -CH-NH-OCH and -CH-O-Si(CH). Typically, a C1-C4 heteroalkyl or heteroalkylene group has 1 to 4 carbon atoms and 1 or 2 heteroatoms, and a C1-C3 heteroalkyl or heteroalkylene group has 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some aspects, heteroalkyl and heteroalkylene groups are saturated.

[0090] Unless otherwise specified, the term "substituted" as used in defining groups herein means that the group can be substituted with, for example, but not limited to, the following groups: 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 diheteroylamino, 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 cycloalkyl- or heterocyclyl- or heteroaryl. - or aryl-acyl, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-acylamino, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-acyloxy, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-sulfonyl, 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 groups, and each group can be further substituted by any other substituent, wherein each group is as defined herein.Examples of substituents include, but are not limited to, one or more groups independently selected from the following: halogen, OH, SH, CN, NH, NHCH, N(CH), NO, N, C(O)CH, COOH, C(O)-amino, OCOCH, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO, phenyl, piperidinyl, piperazinyl, and pyrimidinyl.

[0091] The term "PEG unit" refers to an organic moiety containing repeating ethyleneoxy subunits (PEG or PEG subunits), which may be polydisperse, monodisperse, or discrete (i.e., the number of ethyleneoxy subunits is discrete). Polydisperse PEGs are mixtures of PEGs that are heterogeneous in 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 include discrete PEGs, which are compounds synthesized in a stepwise manner rather than by a polymerization process. Discrete PEGs provide single molecules with a defined and specified chain length.

[0092] "Pharmaceutically acceptable" and "for pharmaceutical use" are used interchangeably herein unless the context dictates otherwise.

[0093] The term "DAR" in this application refers to the ratio of a Cn moiety conjugated to an Fc as described herein to the Fc in the conjugate, or the ratio of a Cn moiety conjugated to an antibody as described herein to the antibody in the conjugate, or the ratio of a Cn moiety conjugated to a fusion protein as described herein to the fusion protein in the conjugate. In some embodiments described herein, the DAR can be 1 to 20, e.g., 1 to 18, 4 to 16, 5 to 12, 6 to 10, 1 to 8, 2 to 8, 1 to 6, 2 to 6, 2 to 4, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR can also be calculated as the average DAR of the population of molecules in the product, i.e., the total ratio of Cn conjugated to Fc moieties as described herein to Fc moieties in the product, or the total ratio of Cn moieties conjugated to antibodies as described herein to antibodies in the product, or the total ratio of Cn moieties conjugated to fusion proteins as described herein to fusion proteins in the product, as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), and such DAR is referred to herein as the average DAR.In some embodiments, the average DAR value in the conjugates of the invention is 1 to 20, e.g., 2 to 18, 4 to 16, 5 to 12, 6 to 10, 2 to 8, 3 to 8, 2 to 6, 4 to 6, 6 to 10, e.g., 1.0 to 8.0, 2.0 to 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, 4.1, 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, and ranges consisting of any two of these values.

[0094] As used herein, the term "drug" encompasses any substance effective in the prevention or treatment of diseases associated with impaired glucose metabolism, cardiovascular disease, cerebrovascular disease, renal disease, and retinopathy. Diseases associated with impaired glucose metabolism include, for example, diabetes (e.g., type 1 diabetes, type 2 diabetes), obesity, hypertension, dyslipidemia, obesity, glucose intolerance, hyperglycemia, hyperinsulinemia, and cardiovascular disease.

[0095] As used herein, the term "active ingredient" refers to a substance, typically an active peptide substance, having any physiologically active function beneficial to a cell and / or organism (e.g., regulating gene expression and physiological functions, correcting abnormal conditions due to insufficient or excessive secretion of components involved in in vivo functional regulation, etc.). Examples of active ingredients include, but are not limited to, enzymes, enzyme inhibitors, antigens, antibodies, antibody fragments, hormones, glucagon-like peptide-1 (GLP-1), glucagon, interferons, cytokines, growth factors and / or differentiation factors, factors involved in cell motility or migration, factors involved in bone formation / resorption, chemokines, plasma or interstitial adhesion molecules or extracellular matrix, bactericidal or antifungal factors, etc.

[0096] The term "pharmaceutical composition" refers to a composition in which the biological activity of the active ingredient contained therein is present in a form that allows it to be effective, and which 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.

[0097] The term "pharmaceutical excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, buffer, surfactant, carrier, stabilizer, or the like, used in conjunction with an active ingredient for administration.

[0098] 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 the same or different time intervals, such that such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient. In some embodiments, the molecules of the present invention and other therapeutic agents used in the pharmaceutical combination are administered at levels equivalent to or lower than when each is administered 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 dosages and / or time intervals of the two or more active agents are preferably selected so that the combined use of the components results in a greater effect in treating a disease or disorder than would be achieved by the use of either component alone. The components may each be in separate formulations, which may be the same or different formulations.

[0099] The term "combination therapy" refers to the administration of two or more therapeutic agents or therapies (e.g., radiation therapy or surgery) for the treatment of a disease described herein. Such administration includes co-administration of the therapeutic agents substantially simultaneously, e.g., in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of the active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, liquids). Powders and / or liquids may be reconstituted or diluted to the desired dosage before administration. Furthermore, such administration also includes sequential use of each type of therapeutic agent, either at approximately the same time or at different times. In either case, the treatment regimen provides the beneficial effect of the drug combination in treating the disorder or condition described herein.

[0100] The terms "individual" and "subject" are used interchangeably herein to refer to mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, 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.

[0101] As used herein, "treating" refers to slowing, interrupting, halting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. As used herein, "preventing" includes inhibiting the occurrence or onset of a disease or disorder, or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of a disease associated with impaired glucose metabolism (e.g., diabetes) are candidates for a preventive regimen. Generally, in the context of a disease associated with impaired glucose metabolism (e.g., diabetes), the term "prevention" refers to the administration of an agent prior to the onset of signs or symptoms of a disease associated with impaired glucose metabolism (e.g., diabetes), particularly in subjects at risk of a disease associated with impaired glucose metabolism (e.g., diabetes).

[0102] As used herein, the term "effective amount" refers to an amount or dosage of the conjugate of the present invention, or a composition thereof, or a combination thereof, that exerts a desired effect in a patient in need of treatment or prevention when administered once or multiple times to the patient. As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic effect. A therapeutically effective amount is also an amount in which the toxic or detrimental effects of a conjugate of the present invention or a composition or combination thereof are less than the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits or reduces a measurable parameter (e.g., glucagon secretion, blood glucose concentration) by at least about 30%, more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%, compared to an untreated individual.

[0103] As used herein, a "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic effect. Generally, the prophylactically effective amount is less than the therapeutically effective amount, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease.

[0104] II. Pharmaceutical Compositions and Kits 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 compositions, preferably pharmaceutical compositions or pharmaceutical formulations, comprising any of the conjugate molecules described herein and their pharmaceutically acceptable salts.

[0105] In one embodiment, the pharmaceutical composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., the pharmaceutical composition, comprises a conjugate molecule of the invention in combination with one or more other therapeutic agents.

[0106] The compositions disclosed herein may also contain suitable pharmaceutical excipients known in the art, such as pharmaceutical carriers, excipients, such as buffers.

[0107] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, and the like.

[0108] For a discussion of the use of pharmaceutical excipients and their applications, see Handbook of Pharmaceutical Excipients, 8th Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0109] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), powders or suspensions, liposomal preparations, and suppositories. The preferred form depends on the intended method of administration and therapeutic use.

[0110] A medicament comprising the conjugate molecule of the present invention according to the present invention can be prepared by mixing the conjugate molecule of the present invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0111] Depending on the specific indication to be treated, the pharmaceutical composition or formulation of the present invention may contain more than one active ingredient, preferably ingredients with complementary activities that do not adversely affect each other. For example, it may be desirable to provide other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). The active ingredients are suitably present in a combination in amounts effective for the intended use.

[0112] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the conjugate molecule, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0113] 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.

[0114] It is yet another object of the present invention to provide a pharmaceutical kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, such that the dosage unit can be provided according to a dosing regimen or interval for administration of the drug.

[0115] In one embodiment, the pharmaceutical kit of the present invention comprises, in the same package: - a first container comprising a conjugate molecule of the invention; - a second container containing a pharmaceutical composition further comprising a therapeutic agent. Includes.

[0116] III. Uses and Methods of Treatment The long-acting platforms provided by the present invention, i.e., 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 extend the serum half-life of the active molecule. Specifically, by constructing the active molecule into a conjugate molecule having an "active molecule-Fc-Cn" structure, a conjugate molecule having an "active molecule-fusion protein-Cn" structure, or a conjugate molecule having an "antibody-Cn" structure using the methods disclosed in this application, the serum half-life of the active molecule can be effectively improved, reducing the administration frequency, reducing the dosage, and saving costs.

[0117] According to the present invention, the long-acting platform extends the serum half-life of the active molecule without affecting the biological function of the active molecule. The resulting conjugate molecules having an "active molecule-Fc-Cn" structure, an "active molecule-fusion protein-Cn" structure, and an "antibody-Cn" structure have low immunogenicity to living organisms, thereby effectively avoiding the negative effect that common conjugate molecules have, which is prone to inducing immune responses in living organisms.

[0118] The conjugate molecules of "active molecule-Fc-Cn," "active molecule-fusion protein-Cn," and "antibody-Cn" obtained by the present application can be used to prevent or treat various diseases in subjects. Those skilled in the art can easily identify diseases that can be treated or prevented with the conjugate molecule based on the biological function of the active molecule. For example, when the active molecule is GLP-1, the conjugate molecule can be used to effectively treat metabolic diseases and / or disorders such as diabetes, e.g., type I diabetes, type II diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity, metabolic syndrome, and cardiovascular disease.

[0119] When the active molecule is an anti-PD-1 antibody or an antigen-binding fragment thereof, the conjugated molecule can be used for the effective prevention or treatment of diseases associated with abnormal expression of PD-1, such as various tumors and cancers, including melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, Hodgkin's lymphoma, head and neck cancer, ovarian cancer, and brain cancer.

[0120] When the active molecule is an anti-VEGF antibody or an antigen-binding fragment thereof, the conjugated molecule can be effectively used in the prevention or treatment of diseases associated with the abnormal expression of VEGF, such as various angiogenesis-related diseases, e.g., ocular diseases such as wet or neovascular age-related macular degeneration (AMD) and diabetic macular edema (DME), most cancers, and cardiovascular diseases.

[0121] The present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a conjugated molecule of the present invention or a pharmaceutically acceptable salt thereof, pharmaceutical composition, pharmaceutical combination, or kit.

[0122] In the therapeutic methods of the invention, administration of a conjugated molecule of the invention can comprise 1) a therapeutic measure to cure, delay, alleviate the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder; or 2) a prophylactic measure to prevent and / or delay the onset of a pathological condition or disorder. In some embodiments, in the therapeutic methods of the invention, the subject benefits from the therapeutic or prophylactic measure and exhibits a reduction or amelioration of the occurrence, recurrence, or development of a disease, disorder, condition, and / or symptom compared to a subject not receiving treatment.

[0123] The pharmaceutical compositions of the present invention can be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusion includes intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, or subcutaneous administration. Administration may be by any suitable route, including, for example, injection, e.g., intravenous or subcutaneous, depending in part on whether administration is short-term or long-term. Various administration schedules are encompassed herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0124] The appropriate dosage of the pharmaceutical compositions of the invention (when used alone or in combination with one or more other therapeutic agents) for the prevention or treatment of disease will depend on the type of disease being treated, the particular active moiety of the conjugate molecule, the severity and course of the disease, the therapeutic objectives, previous treatment history, the patient's clinical history and response to the drugs, and the judgment of the treating physician.

[0125] In some embodiments, the present invention also provides the use of a pharmaceutical composition of the present invention in the manufacture of a medicament for use in the above-described treatment and prevention methods.

[0126] The above and other aspects and embodiments of the present invention are illustrated in the following examples. All or part of the features described above and throughout this application may be combined in various embodiments of the present invention. The following examples further describe the present invention, but these examples are illustrative and should not be construed as limiting in any way.

[0127] Abbreviations used in the specification and claims have the following meanings: Area under the AUC 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-propyleneoxy)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'-tetramethylurea hexafluorophosphate HATU 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HPLC High Performance Liquid Chromatography TBTU O-Benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate HOBT 1-Hydroxybenzotriazole HOAT 1-hydroxy-7-azobenzotriazole TFA trifluoroacetic acid TIS Triisopropylsilane TCEP Tris(2-carboxyethyl)phosphine hydrochloride TSTU O-(N-Succinimido)-1,1,3,3-tetramethyluronium tetrafluoroborate [Example]

[0128] Example 1. Preparation of long-chain fatty acid chains In this example, various examples of long-chain fatty acid chains that can react with different sites on the Fc region (or fusion protein), such as free thio and amino groups, were constructed.

[0129] 1.1 Preparation of long-chain fatty acid chain TM1 The fatty acid chain TM1 was prepared according to the following technical procedure: [ka]

[0130] (1) Compound 1 (5.0 g, 29.40 mmol) was dissolved in anhydrous DCM (50 mL) with vigorous stirring, and then EDCI (5.62 g, 29.4 mmol) and compound 1-2 (4.35 g, 29.40 mmol) were added and reacted at 25° C. for 2 h. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give 2 (5.5 g, 62.5%) as a colorless oil.

[0131] (2) Compound 2 (5.5 g, 18.30 mmol) was dissolved in anhydrous DCM (60 mL) with vigorous stirring, and then EDCI (3.50 g, 18.30 mmol) and compound 2-1 (2.98 g, 18.30 mmol) were added and reacted for 2 h at 25 °C. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give 3 (4.8 g, 59.3%) as a colorless oil.

[0132] (3) Compound 3 (4.8 g, 10.78 mmol) was dissolved in anhydrous DCM (40 mL) with vigorous stirring, and then EDCI (2.06 g, 10.78 mmol) and compound 3-1 (2.18 g, 10.78 mmol) were added and reacted for 12 h at 25 °C. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give 4 (4.0 g, 58.9%) as a colorless oil.

[0133] (4) Compound 4 (4.0 g, 6.34 mmol) was dissolved in anhydrous DCM (30 mL) with vigorous stirring, and then TFA (5 mL) was added and the reaction was allowed to proceed at 25° C. for 0.5 h. After completion of the reaction, the reaction mixture was directly concentrated to give 5 (3.2 g, 88%) as a brown oil.

[0134] (5) Compound 5 (3.2 g, 5.58 mmol) was dissolved in anhydrous DMF (30 mL) with vigorous stirring, and then DCC (1.14 g, 5.58 mmol), DMAP (0.14 g, 1.16 mmol), and compound 5-1 (1.75 g, 5.58 mmol) were added and reacted at 25 °C for 2 h. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give TM1 (2.1 g, 43.7%) as a colorless oil with a molecular weight of 870.05.

[0135] Structure determination of TM1: Mass spectrometry: MS-ESI(m / z):870.4[M+H]+

[0136] Nuclear magnetic hydrogen spectrum: 1 H NMR (400MHz, DMSO-d6) δ 8.02 (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)

[0137] Nuclear magnetic carbon spectrum: 13 C NMR (100MHz, DMSO-d6) δ 174.91, 173.91, 172.88, 171.95, 171.13, 169.77, 134.94, 70.63, 70.38, 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

[0138] 1.2 Preparation of long-chain fatty acid C18 esters Fatty acid chain C18 esters (i.e. C18-tert-butanol esters) were prepared according to the following technical procedure: [ka]

[0139] (1) Compound 1 (5.0 g, 29.40 mmol) was dissolved in anhydrous DCM (50 mL) with vigorous stirring, and then EDCI (5.62 g, 29.4 mmol) and compound 1-2 (4.35 g, 29.40 mmol) were added and reacted at 25° C. for 2 h. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give 2 (5.5 g, 62.5%) as a colorless oil.

[0140] (2) Compound 2 (5.5 g, 18.30 mmol) was dissolved in anhydrous DCM (60 mL) with vigorous stirring, and then EDCI (3.50 g, 18.30 mmol) and compound 2-1 (2.98 g, 18.30 mmol) were added and reacted for 2 h at 25 °C. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give 3 (4.8 g, 59.3%) as a colorless oil.

[0141] (3) Compound 3 (4.8 g, 10.78 mmol) was dissolved in anhydrous DCM (40 mL) with vigorous stirring, and then EDCI (2.06 g, 10.78 mmol) and compound 3-1 (2.18 g, 10.78 mmol) were added and reacted for 12 h at 25 °C. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give 4 (4.0 g, 58.9%) as a colorless oil.

[0142] (4) Compound 4 (4.0 g, 6.35 mmol) was dissolved in anhydrous DMF (40 mL) with vigorous stirring, and then DCC (1.31 g, 6.35 mmol), DMAP (0.155 g, 1.27 mmol), and compound 4-1 (2.35 g, 6.35 mmol) were added and reacted at 25 °C for 2 h. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give the C18 ester (1.8 g, 28.8%) as a colorless oil with a molecular weight of 982.27.

[0143] Structure determination of C18 ester: Mass spectrometry: MS-ESI(m / z):982.5[M+H]+

[0144] Nuclear magnetic hydrogen spectrum: 1 H NMR (400MHz, DMSO-d6) δ 8.11 (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) 1.92~1.85 (m, 2H, CH2) 1.40~1.35 (m, 22H, CH 2, CH3) 1.30~1.23 (m, 24H, CH 2, CH3)

[0145] Nuclear magnetic carbon spectrum: 13 C NMR (100MHz, DMSO-d6) δ 172.78, 171.78, 171.19, 169.95, 169.70, 135.02, 80.75, 79.76, 70.62, 70.42, 69.94, 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

[0146] 1.3 Preparation of long-chain fatty acid C16-NHS The fatty acid chain C16-NHS was prepared according to the following technical procedure: [ka]

[0147] (1) Compound 1 (3.0 g, 11.7 mmol) was dissolved in anhydrous DCM (30 mL) with vigorous stirring, and then 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 completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give 2 (2.1 g, 41.2%) as a colorless oil.

[0148] (2) Compound 2 (2.1 g, 4.76 mmol) was dissolved in anhydrous DCM (20 mL) with vigorous stirring, and then TSTU (1.43 g, 4.76 mmol), DIPEA (0.614 g, 4.76 mmol), and compound 2-1 (1.64 g, 14.28 mmol) were added and reacted at 25° C. for 1 h. After completion of the reaction, the reaction mixture was directly concentrated to give 3 (2.0 g, 78.1%) as a brown oil.

[0149] (3) Compound 3 (2.0 g, 3.71 mmol) was dissolved in anhydrous DCM (20 mL) with vigorous stirring, and then 2 mL of TFA was added and reacted at 25° C. for 1 hour. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give the C16 ester (also known as C16-NHS) as a colorless oil (1.2 g, 70.5%) with a molecular weight of 482.26.

[0150] Structure determination of C16-NHS ester: Mass spectrometry: MS-ESI(m / z):483.3[M+H]+

[0151] Nuclear magnetic hydrogen spectrum: 1 H NMR (400MHz, DMSO-d6) δ 12.71 (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)

[0152] Nuclear magnetic carbon spectrum: 13 C NMR (100MHz, DMSO-d6) δ 173.43, 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

[0153] 1.4 Preparation of long-chain fatty acid C20-NHS The fatty acid chain C20-NHS was prepared according to the following technical procedure: [ka]

[0154] (1) Compound 1 (5.0 g, 12.56 mmol) was dissolved in anhydrous DCM (50 mL) with vigorous stirring, and then DCC (2.58 g, 12.56 mmol), DMAP (0.306 g, 2.51 mmol), and compound 1-2 (2.54 g, 12.56 mmol) were added and reacted at 25° C. for 2 h. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give 2 (6.1 g, 83.56%) as a colorless oil.

[0155] (2) Compound 2 (6.1 g, 10.4 mmol) was dissolved in anhydrous DCM (60 mL) with vigorous stirring, and then DCC (2.14 g, 10.4 mmol), DMAP (0.53 g, 2.08 mmol), and compound 2-1 (3.2 g, 10.4 mmol) were added and reacted for 2 h at 25 °C. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give 3 (4.5 g, 51%) as a colorless oil.

[0156] (3) Compound 3 (4.5 g, 5.15 mmol) was dissolved in anhydrous DCM (40 mL) with vigorous stirring, and then 4 mL of TFA was added and reacted at 25° C. for 1 h. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give 4 (3.2 g, 82%) as a brown oil.

[0157] (4) Compound 4 (3.2 g, 4.20 mmol) was dissolved in anhydrous DMF (40 mL) with vigorous stirring, and then DCC (0.865 g, 4.20 mmol), DMAP (0.103 g, 0.84 mmol), and compound 4-1 (0.483 g, 4.2 mmol) were added and reacted at 25 °C for 2 h. After completion of the reaction, the reaction mixture was directly isolated by preparative HPLC and concentrated to give the C20 ester (also known as C20-NHS) as a white solid (1.5 g, 41.7%) with a molecular weight of 859.02.

[0158] Structure determination of C20-NHS ester: Mass spectrometry: MS-ESI(m / z):859.4[M+H] +

[0159] Nuclear magnetic hydrogen spectrum: 1 H NMR (400MHz, DMSO-d6) δ 12.41 (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)

[0160] Nuclear magnetic carbon spectrum: 13 C NMR (100MHz, DMSO-d6) δ 174.95, 174.00, 172.81, 171.87, 170.50, 169.72, 167.05, 70.84, 70.62, 70.41, 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

[0161] Example 2. Preparation of GLP-1-Fc-TM1 sample and detection of its DAR value In this example, a conjugate based on the Fc-long fatty acid chain platform of the present application was constructed using the commercially available GLP-1-Fc fusion protein dulaglutide as an example. Dulaglutide is composed of two identical long chains, one of which has the amino acid sequence shown below: [ka]

[0162] The Fc contained in dulaglutide has the following amino acid sequence: [ka]

[0163] 1.1 Conjugation of GLP-1-Fc fusion protein to TM1 7.5 mg of GLP-1-Fc fusion protein (dulaglutide, homemade) was exchanged into reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) through a 15 ml 30 kD ultra-filtered tube. This procedure was repeated four times; the protein concentration of the resulting sample, with a final volume of approximately 1 ml, was determined. A three-fold molar excess of TCEP was added to the sample and incubated in a 25°C water bath for 2 hours. The sample was then exchanged into conjugation buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) through a 15 ml 30 kD ultra-filtered tube. This procedure was repeated four times; the protein concentration and number of free thiol groups of the sample were then determined.

[0164] To the GLP-1-Fc fusion protein sample, TM1 was added in a 5-fold molar excess based on the protein amount, and the mixture was mixed uniformly and reacted for 2 hours at 25°C while shaking on a microplate shaker. The sample was purified by cation chromatography immediately after conjugation.

[0165] The purified sample was named GLP-1-Fc-TM1 (also called GLP1-Fc-TM1) and exchanged into a temporary buffer (25 mM phosphate, 150 mM NaCl, pH 7.0) for further detection.

[0166] 1.2 Determination of DAR value of GLP1-Fc-TM1 sample In this example, the conjugation ratio (DAR) of GLP1-Fc and TM1 was determined by HIC-HPLC. The analytical column used was a TSKgel Butyl-NPR (4.6 mm × 3.5 cm), and the analytical method was a known method described in "Drug-to-Antibody Ratio (DAR) and Drug Load Distribution by Hydrophobic Interaction Chromatography and Reversed Phase High-Performance Liquid Chromatography." The detection results are shown in Figure 1, and the average DAR value of TM1 conjugated to GLP1-Fc protein is 3.3.

[0167] Example 3. Preparation and detection of GLP-1-Fc-C18 In this example, a conjugate based on the Fc-long fatty acid chain platform of the present application was constructed using the commercially available GLP-1-Fc fusion protein dulaglutide as an example.

[0168] 8.34 mg of GLP1-Fc fusion protein (dulaglutide, homemade) was exchanged into reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) via 15 ml of a 30 kD ultra-filtered tube, and the exchange was repeated four times. The protein concentration was determined for the resulting sample, with a final volume of approximately 3 ml. TCEP was added to the antibody in a three-fold molar excess and the reaction was allowed to proceed in a 25°C water bath for 2 hours. The mixture was then exchanged into conjugation buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) via 15 ml of a 30 kD ultra-filtered tube, and the exchange was repeated four times. The protein concentration and number of free thiol groups in the sample were then determined.

[0169] To the GLP-1-Fc fusion protein sample, a three-fold molar excess of C18-tert-butanol ester was added based on the protein amount, and the mixture was mixed uniformly and reacted for 2 hours at 25°C while shaking on a miniature shaker. The sample was purified by cation chromatography immediately after conjugation.

[0170] The purified protein was named GLP-1-Fc-C18-tert-butanol ester (also called GLP1-Fc-C18 tert-butanol ester) and was exchanged into a temporary buffer for further detection.

[0171] In this example, the conjugation ratio (DAR) of GLP1-Fc and C18-tert-butanol ester was determined by HIC-HPLC according to the method in Example 2. The results are shown in Figure 8, and the average DAR value was 1.26.

[0172] Example 4. Preparation and detection of sample GLP1-Fc-C16-NHS In this example, a conjugate based on the Fc-long fatty acid chain platform of the present application was constructed using the commercially available GLP-1-Fc fusion protein dulaglutide as an example, in which the conjugate was linked via the amino group of a lysine residue on the Fc.

[0173] 4.56 mg of GLP-1-Fc fusion protein was exchanged into conjugation buffer (0.1 M MOPS, 20 mM Tris, pH 7.5) via a 30 KD ultrafiltration tube, and the resulting sample, with a final volume of 0.9 ml, was assayed for protein concentration. A 6-fold molar excess of C16-NHS was added to the fusion protein sample. The mixture was mixed homogeneously and incubated at 25°C for 2 hours with shaking on a miniature shaker. The sample was purified by cation chromatography immediately after conjugation, then exchanged into temporary buffer via a 30 KD ultrafiltration tube, and the concentration was assayed. The purified product was designated GLP1-Fc-C16-NHS or GLP1-Fc-C16.

[0174] In this example, detection was performed by HIC-HPLC according to the method in Example 2. The results are shown in Figure 9, and the conjugate ratio of C16-NHS and GLP1-Fc was 83.07%.

[0175] Example 5. Preparation and detection of sample GLP1-Fc-C20-NHS In this example, a conjugate based on the Fc-long fatty acid chain platform of the present application was constructed using the commercially available GLP-1-Fc fusion protein dulaglutide as an example, in which the conjugate was linked via the amino group of a lysine residue on the Fc.

[0176] 4.56 mg of GLP1-Fc fusion protein was exchanged into conjugation buffer (0.1 M MOPS, 20 mM Tris, pH 7.5) via a 30 KD ultrafiltration tube, and the resulting sample, with a final volume of 0.9 ml, was analyzed for protein concentration. A 6-fold molar excess of C20-NHS was added to the fusion protein sample. The mixture was mixed homogeneously and incubated at 25°C for 2 hours with shaking on a miniature shaker. The sample was purified by cation chromatography immediately after conjugation, then exchanged into temporary buffer via a 30 KD ultrafiltration tube, and the concentration was analyzed. The purified product was designated GLP1-Fc-C20-NHS or GLP1-Fc-C20.

[0177] In this example, detection was performed by HIC-HPLC according to the method in Example 2. The results are shown in Figure 10, and the conjugate ratio of C20-NHS and GLP1-Fc was 96.38%.

[0178] Example 6. Binding activity of GLP1-Fc-C16-NHS and GLP1-Fc-C20-NHS to HSA

[0179] In this example, the binding activity of GLP1-Fc-C16-NHS and GLP1-Fc-C20-NHS, which were obtained by conjugating lysine to the GLP1-Fc fusion protein, to HSA was detected by ELISA.

[0180] Eight ELISA plates were coated with HSA-His diluted to 0.5 μg / ml in coating solution (carbonate buffer) and incubated overnight at 4°C. The plates were washed three times with PBST (PBS containing 0.05% Tween 20), and then 200 μl / well of blocking solution (PBST containing 1% BSA) was added and incubated at 37°C for 1 hour. The plates were washed four times with PBST. GLP1-Fc-C16-NHS and GLP1-Fc-C20-NHS were each diluted to an initial concentration of 1000 nM in diluent (PBST solution containing 1% BSA), followed by seven 5-fold serial dilutions to create a total of eight concentration gradients. 100 μl / well of each dilution was added to the plate and incubated at 37°C for 1 hour. The plate was washed five times with PBST, and 100 μl / well of a working solution of HRP-goat anti-human 10000X was added and incubated for 1 hour at 37°C. The plate was washed six times with PBST, and 100 μl / well of freshly prepared color development solution (5 ml of substrate solution, 250 μl of TMB stock solution, 16 μl of 0.75% HO) was added and incubated for 10 minutes at 37°C. After adding 50 μl / well of stop solution (2 M HSO), the OD value was measured at 450 nm.

[0181] The results are shown in Figure 11. The results indicated that sample GLP1-Fc-C20-NHS after conjugation with C20-NHS exhibited strong binding to HSA-his. Sample GLP1-Fc-C16-NHS conjugated with C16-NHS showed weaker binding to HSA-his than GLP1-Fc-C20-NHS.

[0182] Example 7. Preparation of samples conjugated with TM1 and HX006 antibodies In this example, the anti-VEGF antibody HX006 disclosed in Chinese Patent Application Publication No. 104804088 is used as an example to construct a conjugate based on the Fc-long chain fatty acid chain platform of the present application, in which the HX006 antibody is an IgG1 type, and the heavy chain sequence and light chain sequence are as shown in the table below.

[0183] [Table 1]

[0184] 6.53 mg of HX006 antibody protein was exchanged into reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) via 15 ml of a 30 kD ultra-filtered tube, and the resulting sample (approximately 2 ml in final volume) was analyzed for protein concentration. A 2-fold molar excess of TCEP was added to the antibody and reacted in a 25°C water bath for 2 hours. The solution was then exchanged into conjugation buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) via 15 ml of a 30 kD ultra-filtered tube, and the analysis was repeated four times. The protein concentration and number of free thiol groups in the sample were then analyzed.

[0185] A two-fold molar excess of TM1 was added to the antibody sample based on the protein amount, and the mixture was mixed uniformly and reacted for 2 hours at 25°C while shaking on a microplate shaker. The sample was purified by cation chromatography immediately after conjugation.

[0186] The purified protein was designated HX006-TM1-1 and exchanged into a temporary buffer for further detection.

[0187] In this example, the conjugate ratio (DAR) of HX006 to TM1 in the HX006-TM1-1 molecule was determined by HIC-HPLC according to the method in Example 2.

[0188] The detection results are shown in Figure 2. The average DAR value of TM1 conjugated to the HX006 antibody in the HX006-TM1-1 molecule is 3.0.

[0189] Example 8. Preparation of HX006 antibody and TM1 conjugate sample In this example, a conjugate of HX006 antibody and TM1 was also prepared.

[0190] 6.56 mg of HX006 antibody protein was exchanged into reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) via 15 ml of a 30 kD ultra-filtered tube, and the resulting sample (approximately 2 ml in final volume) was analyzed for protein concentration. A three-fold molar excess of TCEP was added to the antibody and reacted for 2.5 hours in a 25°C water bath. The solution was then exchanged into conjugation buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) via 15 ml of a 30 kD ultra-filtered tube, and the exchange was repeated four times. The protein concentration and number of free thiol groups in the sample were then analyzed.

[0191] A four-fold molar excess of TM1 was added to the antibody sample based on the protein amount, and the mixture was mixed uniformly and incubated for 2 hours at 25°C while shaking on a microplate shaker. The sample was purified by cation chromatography immediately after conjugation.

[0192] The purified protein was designated HX006-TM1-2 and exchanged into a temporary buffer for further detection.

[0193] In this example, the conjugation ratio (DAR) of HX006 and TM1 within the HX006-TM1-2 molecule was determined by HIC-HPLC according to the method in Example 2.

[0194] The detection results are shown in Figure 3, and the average DAR value of TM1 conjugated to the HX006 antibody in the HX006-TM1-2 molecule was 4.67.

[0195] Example 9 Preparation of a conjugate sample of C18-tert-butanol ester and HX006 antibody In this example, a conjugate of HX006 antibody and C18 tert-butanol ester was prepared.

[0196] 4.3 mg of HX006 antibody protein was exchanged into reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) via 15 ml of a 30 kD ultra-filtered tube, and the resulting sample (approximately 2 ml in final volume) was analyzed for protein concentration. A three-fold molar excess of TCEP was added to the antibody and reacted in a 25°C water bath for 2 hours. The solution was then exchanged into conjugation buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) via 15 ml of a 30 kD ultra-filtered tube, and the analysis was repeated four times. The protein concentration and number of free thiol groups in the sample were then analyzed.

[0197] A three-fold molar excess of C18 tert-butanol ester was added to the antibody sample based on the protein amount, and the mixture was mixed uniformly and reacted for 2 hours at 25°C while shaking on a microplate shaker. The sample was purified by cation chromatography immediately after conjugation.

[0198] The purified protein was designated HX006-C18-tert-butanol ester (also known as HX006-C18) and exchanged into a temporary buffer for further detection.

[0199] In this example, the conjugation ratio (DAR) of HX006 and C18-tert-butanol ester was determined by HIC-HPLC according to the method in Example 2. The results are shown in Figure 15, and the average DAR value was 2.95.

[0200] Example 10 Preparation of a conjugate sample of C16-NHS and HX006 antibody Four milligrams of HX006 antibody protein was exchanged into conjugation buffer (0.1 M MOPS, 20 mM Tris, pH 7.5) via a 30 KD ultra-filtration tube, and the resulting sample, with a final volume of 0.9 ml, was assayed for protein concentration. A six-fold molar excess of C16-NHS was added to the fusion protein sample. The mixture was mixed homogeneously and incubated at 25°C for 2 hours with shaking on a miniature shaker. The sample was purified by cation chromatography immediately after conjugation, then exchanged into temporary buffer via a 30 KD ultra-filtration tube, and the protein concentration was assayed. This protein was designated HX006-C16-NHS.

[0201] Example 11. Preparation of C20-NHS and HX006 antibody conjugate sample Four milligrams of HX006 antibody protein was exchanged into conjugation buffer (0.1 M MOPS, 20 mM Tris, pH 7.5) via a 30 KD ultra-filtration tube, and the resulting sample, with a final volume of 0.9 ml, was assayed for protein concentration. A six-fold molar excess of C20-NHS was added to the fusion protein sample. The mixture was mixed homogeneously and incubated at 25°C for 2 hours with shaking on a miniature shaker. The sample was purified by cation chromatography immediately after conjugation, then exchanged into temporary buffer via a 30 KD ultra-filtration tube, and the protein concentration was assayed. This protein was designated HX006-C20-NHS.

[0202] Example 12. Binding activity of HX006-C16-NHS and HX006-C20-NHS to HSA In this example, the binding activity of samples HX006-C16-NHS and HX006-C20-NHS, obtained by conjugating lysine at the Fc of the HX006 antibody protein, to HSA was detected by ELISA.

[0203] Eight ELISA plates were coated with HSA-His diluted to 0.5 μg / ml in coating solution and incubated overnight at 4°C. The plates were washed three times with PBST (PBS containing 0.05% Tween 20), then 200 μl / well of blocking solution was added and incubated at 37°C for 1 hour. The plates were washed four times with PBST. HX006-C16-NHS and HX006-C20-NHS were each diluted to an initial concentration of 1000 nM in diluent, followed by seven 5-fold serial dilutions, for a total of eight serial dilutions. 100 μl / well of each dilution was added to the plate and incubated at 37°C for 1 hour. The plates were washed five times with PBST, and 100 μl / well of a working solution of HRP-goat anti-human 10000X was added and incubated at 37°C for 1 hour. The plate was washed six times with PBST, and 100 μl / well of freshly prepared color development solution was added and incubated for 10 minutes at 37° C. After adding 50 μl / well of stop solution (2M H2SO4), the OD value was measured at 450 nm.

[0204] The results are shown in Figure 12. The results showed that sample HX006-C20-NHS after conjugation with C20-NHS exhibited strong binding to HSA-his. Sample HX006-C16-NHS after conjugation with C16-NHS exhibited weaker binding to HSA-his than HX006-C20-NHS.

[0205] Example 13. Preparation of TM1 and HX008 antibody conjugate sample In this example, the anti-PD-1 antibody H8L2 (hereinafter referred to as HX008) disclosed in Chinese Patent Application Publication No. 108299560 is used as an example to construct a conjugate based on the Fc-long fatty acid chain platform of the present application. The HX008 antibody is an IgG4 type and has the following sequence:

[0206] The sequence of the heavy chain of the HX008 antibody is as follows: [ka]

[0207] The sequence of the heavy chain variable region of the HX008 antibody is as follows: [ka]

[0208] The sequence of the Fc region is as follows: [ka]

[0209] The sequence of the light chain variable region of the HX008 antibody is as follows: [ka]

[0210] The sequence of the light chain of the HX008 antibody is as follows: [ka]

[0211] Four milligrams of HX008 antibody protein was exchanged into a reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) via a 15 ml 30 kD ultra-filtered tube. This procedure was repeated four times; the protein concentration of the resulting sample, approximately 1 ml in final volume, was determined. An 8-fold molar excess of TCEP was added to the antibody, and the mixture was incubated in a 30°C water bath for 2.5 hours. The mixture was then exchanged four times into a conjugation buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) via a 15 ml 30 kD ultra-filtered tube. The protein concentration and number of free thiol groups in the sample were then determined.

[0212] TM1 was added to the fusion protein sample in a four-fold molar excess based on the amount of protein, and the mixture was mixed uniformly and reacted for 2 hours at 25°C while shaking on a microplate shaker. The sample was purified by cation chromatography immediately after conjugation.

[0213] The purified protein was designated HX008-TM1-2 and exchanged into a temporary buffer for further detection.

[0214] In this example, the HX008-TM1-2 intramolecular conjugate ratio (DAR) of HX008 and TM1 was determined by HIC-HPLC according to the method in Example 2.

[0215] The results are shown in Figure 4, where the average DAR value of TM1 conjugated to the HX008 antibody in the HX008-TM1-2 molecule was 1.45.

[0216] Example 14. Preparation of TM1 and HX008 antibody conjugate sample In this example, a conjugate of HX008 antibody and TM1 was also prepared.

[0217] Four milligrams of HX008 antibody protein was exchanged four times into a reducing buffer (25 mM sodium borate, 30 mM NaCl, 5 mM EDTA, pH 8.0) via a 15 ml 30 kD ultra-filtered tube; the protein concentration of the resulting sample, approximately 1 ml in final volume, was determined. A 10-fold molar excess of TCEP was added to the antibody and reacted for 2.5 hours in a 30°C water bath; the solution was then exchanged four times into a conjugation buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) via a 15 ml 30 kD ultra-filtered tube. The protein concentration and number of free thiol groups in the sample were then determined.

[0218] TM1 was added to the fusion protein sample in a 5-fold molar excess based on the amount of protein, and the mixture was mixed uniformly and reacted for 2 hours at 25°C while shaking on a microplate shaker. The sample was purified by cation chromatography immediately after conjugation.

[0219] The purified protein was designated HX008-TM1-3 and exchanged into a temporary buffer for further detection.

[0220] In this example, the HX008-TM1-3 intramolecular conjugate ratio (DAR) of HX008 and TM1 was determined by HIC-HPLC according to the method in Example 2.

[0221] The results are shown in Figure 4, where the average DAR value of TM1 conjugated to the HX008 antibody in the HX008-TM1-3 molecule was 2.0.

[0222] Example 15. Binding activity of GLP-1-Fc-TM1 to HSA The fatty acid chains contained in TM1 can bind to serum albumin (HSA). In this example, binding of the fusion GLP-1-Fc-TM1 to HSA was detected by ELISA. Three ELISA plates were coated with HSA-His diluted to 0.5 μg / ml in coating solution (carbonate buffer) and incubated overnight at 4°C. After washing the plates three times with PBST (PBS containing 0.05% Tween 20), 200 μl / well of blocking solution (PBST containing 1% BSA) was added and incubated at 37°C for 1 hour. The plates were washed four times with PBST. GLP-1-Fc-TM1 was initially diluted to 1000 nM in diluent (PBST containing 1% BSA), followed by seven 5-fold serial dilutions to create a total of eight concentration gradients. 100 μl / well of each dilution was added to the plate and incubated at 37°C for 1 hour. The plate was washed five times with PBST, and 100 μl / well of a working solution of HRP-goat anti-human 10000X was added and incubated for 1 hour at 37° C. The plate was washed six times with PBST, and 100 μl / well of freshly prepared color development solution (5 ml of substrate solution, 250 μl of TMB stock solution, 16 μl of 0.75% HO) was added and incubated for 10 minutes at 37° C. 50 μl / well of stop solution (2 M HSO) was added, and the OD value was measured at 450 nm.

[0223] The results are shown in Figure 5. The results showed that the sample GLP1-Fc-TM1 after conjugation with TM1 exhibited strong binding to HSA-his.

[0224] Example 16. Binding activity of GLP-1-Fc-C18-tert-butanol ester to HAS In this example, the binding of fused GLP-1-Fc-C18-tert-butanol ester to HSA was detected by ELISA. Three ELISA plates were coated with HSA-His diluted to 0.5 μg / ml in coating solution and incubated overnight at 4°C. After washing the plates three times with PBST (PBS solution containing 0.05% Tween 20), 200 μl / well of blocking solution was added and incubated at 37°C for 1 hour. The plates were washed four times with PBST. GLP-1-Fc-C18-tert-butanol ester was diluted to an initial concentration of 1000 nM in diluent, followed by seven 5-fold serial dilutions to create a total of eight gradient steps. Each dilution was added to the plate at 100 μl / well and incubated at 37°C for 1 hour. The plate was washed five times with PBST, and 100 μl / well of a working solution of HRP-goat anti-human 10000X was added and incubated for 1 hour at 37° C. The plate was washed six times with PBST, and 100 μl / well of freshly prepared color development solution was added and incubated for 10 minutes at 37° C. After adding 50 μl / well of stop solution (2M H2SO4), the OD value was measured at 450 nm.

[0225] The results are shown in Figure 13. The results show that the sample GLP-1-Fc-C18-tert-butanol ester exhibits strong binding to HSA-his after conjugation with C18-tert-butanol ester.

[0226] Example 17. Binding activity of HX006-TM1 to HSA In this example, binding of fusion HX006-TM1 to HAS was detected by ELISA. Eight ELISA plates were coated with HSA-His diluted to 0.5 μg / ml in coating solution and incubated overnight at 4°C. The plates were washed three times with PBST (PBS containing 0.05% Tween 20), and then 200 μl / well of blocking solution was added and incubated at 37°C for 1 hour. The plates were washed four times with PBST. HX006-TM1 was diluted to an initial concentration of 1000 nM in diluent, followed by seven 5-fold dilutions, for a total of eight gradient steps. 100 μl / well of each dilution was added to the plate and incubated at 37°C for 1 hour. The plates were washed five times with PBST, and 100 μl / well of a working solution of HRP-goat anti-human 10000X was added and incubated at 37°C for 1 hour. The plate was washed six times with PBST, and 100 μl / well of freshly prepared color development solution was added and incubated for 10 minutes at 37° C. After adding 50 μl / well of stop solution (2M H2SO4), the OD value was measured at 450 nm.

[0227] The results are shown in Figure 6. The results showed that sample HX006-TM1 after conjugation with TM1 exhibited strong binding to HSA-his, and the binding activity increased with increasing DAR value.

[0228] Example 18. Binding activity of HX006-C18-tert-butanol ester to HSA In this example, the binding of fused HX006-C18-tert-butanol ester to HSA was detected by ELISA. Eight ELISA plates were coated with HSA-His diluted to 0.5 μg / ml in coating solution and incubated overnight at 4°C. The plates were washed three times with PBST (PBS containing 0.05% Tween 20), then 200 μl / well of blocking solution was added and incubated at 37°C for 1 hour. The plates were washed four times with PBST. HX006-TM1 was diluted to an initial concentration of 1000 nM in diluent, followed by seven 5-fold dilutions, for a total of eight gradient steps. 100 μl / well of each dilution was added to the plate and incubated at 37°C for 1 hour. The plates were washed five times with PBST, and 100 μl / well of a working solution of HRP-goat anti-human 10000X was added and incubated at 37°C for 1 hour. The plate was washed six times with PBST, and 100 μl / well of freshly prepared color development solution was added and incubated for 10 minutes at 37° C. After adding 50 μl / well of stop solution (2M H2SO4), the OD value was measured at 450 nm.

[0229] The results are shown in Figure 14. The results show that sample HX006-C18-tert-butanol ester after conjugation with C18-tert-butanol ester exhibits stronger binding to HSA-his than the naked antibody HX006-DS.

[0230] Example 19. Binding activity of HX008-TM1 to HSA In this example, the binding of fused HX008-TM1 to HSA was detected by ELISA. For specific procedures, see Example 15, except that the test sample was changed to sample HX008-TM1-3. The results are shown in Figure 7, where sample HX008-TM1 conjugated with TM1 exhibited stronger binding to HSA-his than the naked antibody HX008-DS, and the binding activity increased with increasing DAR.

[0231] Example 20. Effect of GLP-1-Fc-TM1 on HEK293-CRE-Luc-GLP1R cell line The biological activity of test substance I (GLP-1-Fc-TM1) was detected in HEK-293 cells (HEK293-CRE-Luc-GLP1R, available from Nanjing GenScript Biotechnology Co., Ltd.) stably expressing the human GLP-1 receptor (GLP-1R) and an intracellular CRE-luciferase reporter gene (CRE4-luciferase). The biological activity of GLP-1-Fc-TM1 was detected by GLP-1-Fc specifically binding to GLP-1R, producing cAMP and activating the reporter gene. HEK293-CRE-Luc-GLP1R cells were harvested and cultured in DMEM (containing 10% FBS, 400 μg / ml G418, and 200 μg / ml hygromycin B) at 37°C in an incubator maintained at 5% CO2. Logarithmically growing cells were harvested, counted, resuspended in complete medium, adjusted to the appropriate concentration, and plated at 2 × 10 cells in a 384-well plate. 3 Cells were seeded at 20 μL of cell suspension per well. The cells were incubated overnight in a 100% relative humidity incubator maintained at 37°C and 5% CO2. Test compounds were diluted to their corresponding working concentrations with culture medium, and 30 μL of test substance solution was added to each well (the final working concentration and dilution gradient of the test compound depended on the specific requirements). A total of nine concentration gradients were set up, with two replicate wells for each concentration. Semaglutide (commercially available) and dulaglutide (homemade) were used as positive controls. The cells were incubated for 6 hours in a 100% relative humidity incubator maintained at 37°C and 5% CO2. The supernatant was discarded, and 40 μL of One-Glo test solution was added per well. The cells were incubated on a vortex shaker for 5 minutes, and luminescence (RLU) was measured using an ENVISION2104 plate reader.

[0232] The results of the activation effect of the CRE-luciferase reporter gene are shown in Figure 16. EC of test substance I (GLP-1-Fc-TM1) 50 is 8.4 x 10 -3nM, EC of dulaglutide 50 is 4.4 x 10 -3 nM, EC 50 is 4.7 x 10 -3 nM, and the EC 50 EC values ​​of dulaglutide and semaglutide under in vitro reporter gene assay conditions 50 This indicates that test substance I (GLP-1-Fc-TM1) has potent biological activity.

[0233] Example 21. Interaction of test substances with human serum albumin determined by surface plasmon resonance (SPR) The scanning reagent was 10 mM N-(2-hydroxyethyl)piperazine-N-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 in fixation reagent (10 mM sodium acetate, pH 4.5). First, the surface of a CM5 chip was activated by flowing 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 420 seconds. Next, 50 μg / mL mouse anti-His antibody was injected into the channel at a flow rate of 10 μL / min for approximately 420 seconds, immobilizing approximately 7,000-15,000 RU. Finally, the chip was blocked with 1 M ethanolamine at a flow rate of 10 μL / min for 420 seconds. Human serum albumin (HSA) was buffer-exchanged using a desalting column and the corresponding scanning reagents, and the concentration of the exchanged samples was measured using a SPECTROstar Nano. The ligands (test substances GLP-1-Fc, GLP-1-Fc-TM1, HX006-DS, HX006-TM1-1, HX006-TM1-2, HX008-DS, HX008-TM1-2, and HX008-TM1-3) were diluted to 5 μg / mL with the scanning reagents and injected into the His capture chip assay channel (Fc2) at a flow rate of 10 μL / min at approximately 400 RU. No ligand (test substance) capture was required in the reference channel (Fc1). Human serum albumin (HSA) was diluted 2-fold with the scanning reagent to create a total of seven dilutions. The diluted HSA was sequentially injected into the assay and reference channels at a flow rate of 30 μL / min, with corresponding binding (120 s) and dissociation (300 s) times. All binding and dissociation steps were performed in the scanning reagent. After each concentration analysis, the chip was regenerated with a pH 1.5 glycine-HCl solution at a flow rate of 20 μL / min for 30 s, followed by washing away the ligand and undissociated analyte. For the next concentration analysis, the same amount of ligand (test substance) had to be recaptured in the assay channel. 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.

[0234] The affinity of the test substances for human serum albumin detected by Biacore8K is as follows: the Kd value of unmodified GLP-1-Fc, HX006-DS, or HX008-DS is 0, i.e., no binding is observed; the Kd values ​​of GLP-1-Fc-TM1, HX006-TM1-1, HX006-TM1-2, HX008-TM1-2, and HX008-TM1-3 are each 1.01 × 10 -2 nM, 1.14 × 10 -3 nM, 6.92 × 10 -4 nM, 4.87 × 10 -3 nM, and 4.05 × 10 -3 nM, which indicates a strong affinity for human serum albumin.

[0235] Example 22. Efficacy study of GLP-1-Fc-TM1 after multiple administration in type II diabetic db / db mice Twelve male m / m mice (normal control) and 60 male db / db mice were purchased and bred accordingly. After the blood glucose levels of the db / db mice reached normal levels (approximately 8-10 weeks of age), 10 eligible m / m mice and 50 eligible db / db mice were selected and divided into a normal control group (m / m mice), a model group, a positive control group (dulaglutide), a low-dose test substance group, a medium-dose test substance group, and a high-dose test substance group, with 10 mice in each group. After grouping, mice were administered the following: the positive control group received TRULICITY (dulaglutide) subcutaneously at 10 nmol / kg / dose twice weekly for 4 weeks; the low, medium, and high dose groups received test substance I (GLP-1-Fc-TM1) subcutaneously at 3, 10, and 30 nmol / kg / dose twice weekly for 4 weeks; the db / db model group and normal control group received the corresponding vehicle (PBS) subcutaneously twice weekly for 4 weeks. All groups received 5 ml / kg / dose of the corresponding vehicle. Clinical observations were performed daily. Serum insulin and glycated hemoglobin were measured at the end of the treatment period, followed by an OGTT test. During the treatment period, body weight and food intake were monitored twice weekly. Random blood glucose levels were measured at 0 h (0–60 min before administration), 0.5 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h after the first and last administration, and pre-administration fasting blood glucose levels were measured once weekly (4 h fasting). At the end of the treatment period, random blood glucose levels, fasting blood glucose levels, and serum insulin levels were measured.

[0236] The results showed that compared with the positive control drug (dulaglutide), test substance I (GLP-1-Fc-TM1) showed an overall superior effect in reducing glycated hemoglobin, lowering random and fasting blood glucose levels, stimulating serum insulin secretion, and reducing body weight and food intake; low, medium, and high doses of test substance I (GLP-1-Fc-TM1) showed efficacy in a dose-dependent manner, indicating that test substance I (GLP-1-Fc-TM1) has a significant blood glucose lowering effect.

[0237] Specifically, the test substance GLP-1-Fc-TM1 dose-dependently increased the 4-hour fasting blood glucose level (Figure 19-1), the random blood glucose level after the first administration and the final administration (Figures 19-2 and 19-3), and the blood glucose AUC in the OGTT test after the final administration in db / db mice. 0-180分間 (Figures 19-4 and 19-5), it can reduce glycated hemoglobin content in db / db mice (Figure 19-6), increase insulin levels in db / db mice (Figure 19-7), and reduce the average daily intake in db / db mice (Figure 19-8). At the same dose, the test substance GLP-1-Fc-TM1 showed better effects on the above indicators than the positive control dulaglutide.

[0238] Example 23. Efficacy test of the effect of GLP-1-Fc-TM1 on body weight in DIO model mice Seventy-two male C57BL / 6J mice were purchased, of which 12 were used as normal controls (fed on a standard diet), and the remaining 60 were fed a high-fat diet to create DIO model mice. The mice had free access to food and water for approximately 10 weeks. Food intake and body weight were monitored twice weekly during the development period. When body weight exceeded 20% of normal mouse weight, DIO model criteria were met and mice were divided into groups. Ten mice were selected from the normal control group (fed on a standard diet) and 50 mice from the DIO model group. These mice were then divided into groups containing 10 mice each: normal control (fed on a standard diet), model group, positive control (dulaglutide), low-dose test substance group, medium-dose test substance group, and high-dose test substance group. After grouping, mice were treated with TRULICITY (dulaglutide) at 10 nmol / kg / dose by subcutaneous injection twice weekly for 4 weeks in the positive control group; test substance I (GLP-1-Fc-TM1) at 3, 10, and 30 nmol / kg / dose by subcutaneous injection twice weekly for 4 weeks in the low, medium, and high dose groups, respectively; and the obese DIO model and normal control groups received the corresponding vehicle (PBS) by subcutaneous injection twice weekly for 4 weeks. All groups were administered with a volume of 5 ml / kg / dose. All DIO mice continued to consume a high-fat diet throughout the treatment period, which was continued throughout the entire experimental period. Body weight and food intake were monitored twice weekly during the treatment period. Before treatment, fasting blood glucose levels (4-hour fasting) were measured, and blood samples were collected for four blood lipid parameters and liver serum biochemistry tests. Treatment began on day 1, and continued for 28 days. After administration (D28), 4-hour fasting blood glucose levels were measured, blood was collected and serum was isolated, and four blood lipid parameters, liver serum biochemistry tests, and serum insulin concentrations were measured. Finally, abdominal adipose tissue was collected and weighed, and the liver was removed and weighed. The organ coefficient was calculated using the following formula: The organ index was calculated as follows: organ weight (g) / mouse body weight (g). A portion of the liver was collected and fixed in formalin for histopathological examination (HE staining, oil red-O staining).

[0239] The results showed that compared with the positive control drug (dulaglutide), test substance I (GLP-1-Fc-TM1) showed overall superior effects in reducing fasting blood glucose levels, improving four blood lipids, liver serum chemistry indicators, and liver pathology, stimulating serum insulin secretion, and reducing body weight and food intake; dose-dependent effects were observed at low, medium, and high doses of test substance I (GLP-1-Fc-TM1), indicating that test substance I (GLP-1-Fc-TM1) has significant hypoglycemic and anti-obesity effects.

[0240] Specifically, GLP-1-Fc-TM1 dose-dependently reduced body weight in DIO mice (Figure 20-1), food intake (Figure 20-2), body fat content (Figure 20-3), and fasting blood glucose levels (Figure 20-4). Furthermore, the test substance GLP-1-Fc-TM1 dose-dependently reduced blood lipids (Figure 20-5) and serum liver function indicators ALT and AST levels (Figure 20-6), demonstrating an improvement in hepatocellular balloon lesions and hepatocellular lipid metabolism abnormalities in DIO mice (Figure 20-7). At the same dose, the test substance GLP-1-Fc-TM1 was more effective in improving hepatic steatosis than the positive control dulaglutide.

[0241] Example 24. Glucose tolerance test (IVGTT) study of GLP-1-Fc-TM1 in rats Fifty-eight male SD rats, 6-8 weeks old and weighing 180-220 g, were purchased and, after one week of acclimation, 50 eligible rats were selected and divided into a vehicle control group, a positive control group (dulaglutide), a low-dose test substance group, a medium-dose test substance group, and a high-dose test substance group (10 rats per group). The positive control group received a single subcutaneous injection of TRULICITY (dulaglutide) at 10 nmol / kg; the low-, medium-, and high-dose test substance groups received single subcutaneous injections of test substance I (GLP-1-Fc-TM1) at 3, 10, and 30 nmol / kg, respectively; and the vehicle control group received a single subcutaneous injection of the corresponding vehicle (PBS). All groups were dosed in a volume of 5 ml / kg / dose. The rats were fasted for 16 hours (but without fluid restriction) and then administered intravenously. After 24 and 72 hours, glucose (0.5 g / kg) was administered intravenously for stimulation; blood samples were taken at 2, 4, 6, 10, 20, and 30 minutes after glucose injection, respectively, to measure blood glucose and insulin levels, and AUC was calculated.

[0242] The results showed that compared with the positive control drug (dulaglutide), test substance I (GLP-1-Fc-TM1) showed equivalent or no significant difference in reducing fasting blood glucose levels and stimulating serum insulin secretion; low, medium, and high doses of test substance I (GLP-1-Fc-TM1) showed that the effect was dose-related.

[0243] Specifically, the test substance GLP-1-Fc-TM1 administered at a single dose of 3, 10, or 30 nmol / kg significantly reduced blood glucose AUC 24 and 72 hours after administration in SD rats. 0-30分間 Dose-dependent reductions in (Figures 21-1, 21-2, 21-3, and 21-4) and serum insulin AUC 0-30分間 (Figures 21-5, 21-6, 21-7 and 21-8).

[0244] Example 25. Pharmacokinetic study of a single subcutaneous injection of the test substance in SD rats Twelve SD rats (6-8 weeks old, weighing 180-220 g, half male and half female) were randomly divided into two groups (6 rats per group, half male and half female) after adaptive breeding: a test substance I (GLP-1-Fc-TM1) group and a positive control group (dulaglutide, homemade). Test substance I (GLP-1-Fc-TM1) and the positive control (dulaglutide) were administered by single subcutaneous injection at a dose of 0.1 mg / kg and a volume of 4 ml / kg. Approximately 0.5 mL of blood was collected from the jugular vein before administration and at 24 hours (±10 min, day 1), 48 hours (±10 min, day 2), 72 hours (±15 min, day 3), 96 hours (±15 min, day 4), 144 hours (±15 min, day 6), 192 hours (±30 min, day 8), 240 hours (±30 min, day 10), and 336 hours (±30 min, day 14) after administration. Anticoagulated with EDTA-K2, the concentrations of test substance I (GLP-1-Fc-TM1) and the positive control (dulaglutide) were measured in plasma at each time point by ELISA. The standard curve range for the three test substances was 3.13–200 ng / mL, and pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2.

[0245] The results are shown in Figure 17 and Table 1. After a single subcutaneous injection of test substance I (GLP-1-Fc-TM1) and dulaglutide at 0.1 mg / kg in SD rats, the mean plasma T max The mean C of test substance I (GLP-1-Fc-TM1) and dulaglutide over 24 hours max The mean T of test substance I (GLP-1-Fc-TM1) and dulaglutide was 225 ng / mL and 191 ng / mL, respectively. 1 / 2 The mean AUC of test substance I (GLP-1-Fc-TM1) and dulaglutide at 45 and 28 hours 0-t were 15700 and 7540 h*ng / mL, and the mean AUC of test substance I (GLP-1-Fc-TM1) and dulaglutide 0-∞ The mean Vz_ of test substance I (GLP-1-Fc-TM1) and dulaglutide was 16100 and 7840 h*ng / mL, respectively. F_obs The mean Cl values ​​for test substance I (GLP-1-Fc-TM1) and dulaglutide were 428 and 533 mL / kg, respectively. _F_obsThe MRTs of test substance I (GLP-1-Fc-TM1) and dulaglutide were 6.42 and 13.3 mL / h / kg, respectively. (0-t) The MRT of test substance I (GLP-1-Fc-TM1) and dulaglutide was measured at 70 and 39 hours. (0-∞) The half-life of test substance I (GLP-1-Fc-TM1) was 77 hours and 44 hours. 1 / 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 those of dulaglutide, respectively, indicating that test substance I (GLP-1-Fc-TM1) can extend the half-life by reducing the clearance rate.

[0246] [Table 2]

[0247] Example 26. Pharmacokinetic study of a single subcutaneous injection of test substance in cynomolgus monkeys Four cynomolgus monkeys (5-6 years old, weighing approximately 6.5 kg, half male and half female) were adaptively housed and then randomly assigned to two groups (half male and half female)—a reference formulation control group (dulaglutide, commercially available from Trulicity) and a test substance I (GLP-1-Fc-TM1) group, each consisting of two animals (half male and half female). The reference formulation control, dulaglutide, and test substance I (GLP-1-Fc-TM1) were administered via a single subcutaneous injection at a molar dose of 1.676 nmol / kg (i.e., a dulaglutide dose of 0.1 mg / kg and a GLP-1-Fc-TM1 dose of 0.101 mg / kg) in a volume of 4 mL / kg. Approximately 0.5 mL of blood was collected from the jugular vein before administration and at 4, 8, 24, 48, 96, 144, 240, and 336 hours after administration and anticoagulated with EDTA-K2. Plasma concentrations of the reference formulation control, dulaglutide, and test substance I (GLP-1-Fc-TM1), at each time point were measured by ELISA. The limit of detection (LLOQ) of the standard curve for the test substance was 15.625 ng / mL, and pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2.

[0248] The results are shown in Figure 18 and Table 2: Mean plasma T values ​​of Test Article I (GLP-1-Fc-TM1) and dulaglutide following a single subcutaneous injection of 676 nmol / kg of Test Article I (GLP-1-Fc-TM1) and the reference formulation control, dulaglutide, in cynomolgus monkeys. max The mean C values ​​for test substance I (GLP-1-Fc-TM1) and dulaglutide were 16 and 8 hours, respectively. max The mean T values ​​for test substance I (GLP-1-Fc-TM1) and dulaglutide were 760 and 602 ng / mL, respectively. 1 / 2 The mean AUC values ​​of test substance I (GLP-1-Fc-TM1) and dulaglutide were 97.9 and 48.2 hours, respectively. 0-t were 98546 and 49118 h*ng / mL, and the mean AUC of test substance I (GLP-1-Fc-TM1) and dulaglutide 0-∞The mean Vz_F_obs of test substance I (GLP-1-Fc-TM1) and dulaglutide were 140678 and 50947 h*ng / mL, the mean Cl_F_obs of test substance I (GLP-1-Fc-TM1) and dulaglutide were 0.74 and 1.97 mL / h / kg, and the MRT of test substance I (GLP-1-Fc-TM1) and dulaglutide were 0.74 and 1.97 mL / h / kg. (0-t) The MRT of test substance I (GLP-1-Fc-TM1) and dulaglutide was measured at 88 and 68 hours. (0-∞) The half-life of test substance I (GLP-1-Fc-TM1) was 163 hours and 77 hours. max , C max , T 1 / 2 , AUC 0-t , AUC 0-∞ , Vz-F, Cl, MRT (0-t) , and MRT (0-∞) were 2.00, 1.26, 2.03, 2.01, 2.76, 0.70, 0.38, 1.29, and 2.12 times those of dulaglutide, respectively, indicating that test substance I (GLP-1-Fc-TM1) can extend the half-life by reducing the clearance rate.

[0249] [Table 3]

Claims

1. A conjugate molecule having the structure "active molecule-Fc-Cn", wherein the active molecule is selected from any molecule beneficial to an organism, Fc is immunoglobulin IgG Fc, and Cn is C １４－２４ The conjugate molecule is a modified moiety that includes a fatty acid chain.

2. A conjugate molecule having the structure "antibody-Cn", where Cn is C １４－２４ The conjugate molecule is a modified moiety that includes a fatty acid chain.

3. A conjugate molecule having the structure "active molecule-fusion protein-Cn", wherein the active molecule is selected from any molecule beneficial to an organism, and Cn is C １４－２４ The conjugate molecule is a modified moiety that includes a fatty acid chain.

4. Cn is a compound of formula (I): -Z-Y (I) having the structure During the ceremony, Z is the following structure: -Z １ -Z ２ -Z ３ -Z ４ - where Z １ is a sulfur atom, a nitrogen atom, or an oxygen atom in Fc, Z ２ is —C(═O)— or a 5- to 10-membered heterocyclyl containing 1 or 2 heteroatoms preferably selected from N, S, and O; Z ３ is a bond, -C(=O)-, -C １ -C １０ Alkylene -C(=O)-, -C ３ -C １０ Alkynylene-C(═O)-, —C ３ -C １０ Alkenylene-C(═O)-, —C １ -C １０ Heteroalkylene -C(=O)-, -C ３ -C ８ Cycloalkylene -C(=O)-, -O-C １ -C ８ Alkylene-C(=O)-, -allylidene-C(=O)-, -C １ -C １０ Alkylene-arylidene-C(═O)-, -arylidene-C １ -C １０ Alkylene -C(=O)-, -C １ -C １０ Alkylene-C ３ -C ８ Cycloalkylene -C(=O)-, -C ３ -C ８ Cycloalkylene-C １ -C １０ Alkylene -C(=O)-, -C ３ -C ８ Heterocyclylene-C(═O)-, —C １ -C １０ Alkylene-C ３ -C ８ Heterocyclylene-C(═O)-, —C ３ -C ８ Heterocyclylene-C １ -C １０ alkylene-C(═O)—, wherein said alkylene, alkynylene, alkenylene, heteroalkylene, cycloalkylene, arylidene, and heterocyclylene are optionally substituted; Z ４ is a bond or the following expression 【Chemistry 1】 is a PEG unit represented by R １ is C １－４ Alkylene, —NH—, —NH—C １－４ Alkylene-, -NH-C １－４ alkylene-heteroaryl-, where heteroaryl is a 5- or 6-membered nitrogen-containing heteroaryl; R ２ is -C(=O)-, -C １－４ Alkylene, -C １－４ Alkylene -C(=O)-, -C １－４ Alkylene -NH-C(=O)-(CH ２ OCH ２ ) ｐ -C １－４ Alkylene-, -C １－４ Alkylene-C(=O)-NH-(CH ２ OCH ２ ) ｐ -C １－４ alkylene-, m is an integer from 2 to 6, and p is an integer from 1 to 3; Y, 【Chemistry 2】 and Y is connected to Z via X. ４ and k is an integer from 10 to 30; where R is independently hydrogen, C １－６ Alkyl, C １－６ Aminoalkyl, C １－６ Haloalkyl, C １－６ represents hydroxyalkyl, The conjugate molecule according to any one of claims 1 to 3.

5. Z ２ is a maleimide group 【Transformation 3】 and the wavy line on the left is Z １ indicates the bonding position, and the wavy line on the right is Z ３ The conjugate molecule according to any one of claims 1 to 4, wherein:

6. Z ３ Ga-C １ -C １０ alkylene-C(=O)-, wherein said alkylene is optionally substituted; Z ３ is connected to Z via -C-(=O)- ４ The conjugate molecule according to any one of claims 1 to 5, wherein the conjugate molecule is bound to

7. Z ２ is a maleimide group, and Z ３ Ga-C １－６ The conjugate molecule of any one of claims 1 to 6, which is alkylene-C(=O)-.

8. Z ４ is a bond, and Z ３ The conjugate molecule of any one of claims 1 to 7, wherein is directly attached to Y in formula (I).

9. Z ４ is the following formula: 【Chemistry 4】 is a PEG unit represented by In the formula, R １ is -NH- and -NH- Ｃ１－４ alkylene-, R ２ Ha-C １－４ Alkylene or -C １－４ Alkylene -NH-C(=O)-(CH ２ OCH ２ ) ｐ -C １－４ The conjugate molecule of any one of claims 1 to 8, which is alkylene-, where m is an integer from 2 to 6 and p is an integer from 1 to 3.

10. Z ４ The conjugate molecule of any one of claims 1 to 9, wherein is a unit comprising 2 to 6 PEG.

11. Z ４ but 【Transformation 5】 where m=1 to 4 and the asterisk on the left represents Z ３ The conjugate molecule of any one of claims 1 to 10, wherein the asterisk on the right indicates the position where Y is attached in formula II.

12. In formula (I), Z is the following structure: 【Transformation 6】 and In the formula, R Ｅ is hydrogen, C １－６ Alkyl, C １－６ Aminoalkyl, C １－６ Haloalkyl, C １－６ hydroxyalkyl, y=0-4, m=1-4, the left asterisk indicates the position where Ab is attached, and the right asterisk indicates the position where Y is attached.

13. Y through X to Z ４ and X is -NH-(C=O)- or -(C=O)-NH-.

14. Cn is C １６ fatty acid chain, C １８ Fatty acid chain, or C ２０ The conjugate molecule of any one of claims 1 to 13, comprising a fatty acid chain.

15. Cn is C １８ A conjugate molecule according to any one of claims 1 to 14, comprising a fatty acid chain and conjugated to a sulfur atom of a free thiol group of Fc.

16. Cn 【Transformation 7】 The conjugate molecule according to any one of claims 1 to 15, selected from:

17. The conjugated molecule according to any one of claims 1 to 16, wherein said Fc is an IgG1 Fc or an IgG4 Fc, preferably a human IgG1 Fc or a human IgG4 Fc.

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

19. 18. The conjugated molecule of any one of claims 1 to 17, wherein the Fc region has an alteration at an amino acid selected from positions 228, 233, 234, 235, 252, 254, 256, 297, 307, 308, 311, 380, 385, 386, 389, 428, 434, and 447 (according to EU numbering).

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

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

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

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

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

25. 25. The conjugated 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 differentiation factor, a factor involved in cell motility or migration, a factor involved in bone formation / resorption, a chemokine, a plasma or stromal adhesion molecule or extracellular matrix, a bactericidal factor or an antifungal factor.

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

27. The conjugate molecule of 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, an anti-VEGF antibody or an antigen-binding fragment thereof.

28. The peptide linker has the amino acid sequence (G ４ S) ｎ wherein n is an integer equal to or greater than 1, and preferably the peptide linker is (G ４ S) ３ , (G ４ S) ４ , (G ４ S) ６ , GS (G ４ S) ４ , DAAALEAAALDAAAREAAAARDAAAL, NVDHLPSNTLVDLA, (G ３ S) 2, (G ４ S) ２ , (G ３ S) ３ , (G ４ S) ３ , (G ３ S) ４ , (G ４ S) ４ , (G ３ S) ５ , (G ４ S) ５ , (G ３ S) ６ , (G ４ S) ６ , GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP, and GSTSGSGK PGSGEGSTKG.

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

30. The conjugate molecule of any one of claims 1 to 29, which is GLP-1-IgG4 Fc-TM1, GLP-1-IgG4 Fc-C18 tert-butanol ester, GLP-1-IgG4 Fc-C16-NHS, and GLP1-IgG4 Fc-C20-NHS.

31. 31. The conjugated 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 in the GLP-1-IgG4 Fc-TM1, GLP-1-IgG4 Fc-C18 tert-butanol ester, GLP-1-IgG4 Fc-C16-NHS, and GLP1-IgG4 Fc-C20-NHS conjugate molecules, the GLP-1-IgG4 Fc portion comprises the amino acid sequence set forth in SEQ ID NO:

1.

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

11.

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

12.

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

10.

36. The conjugate molecule of claim 35, which is a HX008-TM1 conjugate molecule, preferably a HX008-TM1-2 conjugate molecule or a HX008-TM1-3 conjugate molecule.

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

14.

39. 39. The conjugated molecule of claim 37 or 38, wherein the Fc comprises the sequence set forth in SEQ ID NO:

15.

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

41. A method for preparing a conjugate molecule having the structure "active molecule-Fc-Cn", comprising: (a) conjugating an active molecule polypeptide to an immunoglobulin Fc region to prepare an "active molecule-Fc" fusion; and (b) subjecting the "active molecule-Fc" fusion and a Cn comprising a fatty acid chain to a conjugation reaction under conditions that allow the Fc region to be conjugated to the Cn, to obtain an "active molecule-Fc-Cn" conjugate molecule.

42. 1. A method for preparing a conjugate molecule having an "active molecule-Fc-Cn" structure, comprising: (a) conjugating an antibody Fab fragment to an immunoglobulin Fc region to prepare an "Fab-Fc" fusion; (b) subjecting the Fab-Fc fusion to a conjugation reaction under conditions that allow the Fc region to be conjugated to a Cn containing a fatty acid chain, to obtain a Fab-Fc-Cn conjugate molecule.

43. A method for preparing a conjugate molecule having an "active molecule-Fc-Cn" structure or an "antibody-Cn" structure, the method comprising the step of subjecting a whole antibody to a conjugation reaction under conditions in which the Fc region can be conjugated to a Cn containing a fatty acid chain, to obtain an "active molecule-Fc-Cn" conjugate molecule.

44. A pharmaceutical composition comprising a conjugate molecule according to any one of claims 1 to 40.

45. A method for effectively extending the serum half-life of an active molecule, comprising the step of constructing the active molecule as a conjugate molecule having an "active molecule-Fc-Cn" structure or an "antibody-Cn" structure 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 a conjugated molecule according to any one of claims 1 to 40 or a pharmaceutical composition according to claim 44 in the manufacture of a medicament for the treatment of a human disease.

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

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