WO2024067401A1 - 包含Fc-高级脂肪酸链的超长效平台 - Google Patents

包含Fc-高级脂肪酸链的超长效平台 Download PDF

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WO2024067401A1
WO2024067401A1 PCT/CN2023/120731 CN2023120731W WO2024067401A1 WO 2024067401 A1 WO2024067401 A1 WO 2024067401A1 CN 2023120731 W CN2023120731 W CN 2023120731W WO 2024067401 A1 WO2024067401 A1 WO 2024067401A1
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molecule
antibody
conjugated
alkylene
conjugate
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PCT/CN2023/120731
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English (en)
French (fr)
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张发明
胡轶敏
周远
喻耀
彭飞宇
胡名龙
王小龙
赵阿龙
梁樱
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中美华世通生物医药科技(武汉)股份有限公司
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Publication of WO2024067401A1 publication Critical patent/WO2024067401A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to an ultra-long-acting platform for improving the half-life of active drug molecules, which comprises immunoglobulin Fc and higher fatty acid chains.
  • the present invention also relates to the preparation of the conjugate platform, a composition containing the conjugate and its therapeutic application.
  • peptide drugs are closer to endogenous substances in the body than chemical drugs, they have the advantages of less toxic side effects and stable efficacy. Therefore, they are widely used in a variety of clinical treatments such as cancer, cardiovascular disease, and autoimmune diseases. They have broad application prospects and are favored by pharmaceutical manufacturers and scientific researchers.
  • classic peptide drugs have low tolerance to proteases in the body and poor stability. They will be degraded quickly after entering the body, thus having a short plasma half-life; most bioactive peptide substances have poor bioavailability and cannot be taken orally. These problems have greatly hindered the clinical application of peptide drugs.
  • protein peptide drugs require more frequent dosing to maintain clinically effective drug concentrations.
  • peptide drugs are currently being modified to overcome them.
  • the modification of peptide drugs can be divided into two categories: one is to modify the peptide chain skeleton; the second is to optimize the structure and modify the performance by introducing other groups on the basis of keeping the peptide skeleton unchanged, including polyethylene glycol modification, glycosylation modification, protein fusion strategy, higher fatty acid modification, site-directed mutagenesis, cholesterol modification, etc.
  • Bioactive molecules are fused to the Fc region of immunoglobulins to form Fc fusion proteins, which combine the beneficial pharmacological properties of bioactive molecules with the additional properties of the Fc region, thereby increasing the serum half-life of physiologically active molecules and thereby reducing the frequency of drug administration.
  • Fc fusion proteins which combine the beneficial pharmacological properties of bioactive molecules with the additional properties of the Fc region, thereby increasing the serum half-life of physiologically active molecules and thereby reducing the frequency of drug administration.
  • the fusion of the Fc region with active peptides as ligands or receptors or with the extracellular domain (ECD) has greatly increased the clinical potential of active protein drugs. Specifically, for products with a molecular weight of less than 60 kDa, they can be easily cleared by the kidneys, thus having a short serum half-life. By coupling or fusing with the Fc region, their size is increased, exceeding the threshold of kidney filtration, thereby increasing their circulation time.
  • the Fc fusion protein When the Fc fusion protein is taken up by endothelial cells and enters the acidified endosome, the Fc fusion protein is protected from lysosomal degradation by binding to FcRn in the endosome. When the Fc is recycled and transported to the cell surface, under neutral and weakly alkaline conditions, Fc dissociates from FcRn and releases the Fc-fusion protein back into the blood circulation, thereby extending the half-life of the Fc-fusion protein and allowing the target tissue to contact the pharmacologically active portion of the Fc-fusion protein for a longer time, thereby enhancing the latter's therapeutic potential.
  • Fc fusion proteins represent a successful class of biopharmaceutical products, with 13 drugs approved in the EU and the US, and 3 biosimilars of etanercept. There is a great diversity of possible bioactive molecules, including the extracellular domains of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding structures that act as cytokine traps. Most Fc fusion proteins are produced by fusing the bioactive molecule to the N-terminus of the Fc domain. The strong interaction of the IgG-CH3 domain creates a stable Fc structure and allows more complex structures to be fused to locations such as the flexible hinge region, disulfide bonds, etc.
  • Eli Lilly and Company combined GLP-1 with IgG4 Dulaglutide, a glucose-lowering drug developed by fusion of GLP-1 with Fc, has a significantly increased molecular size, which reduces the renal clearance of GLP-1 and thus has a prolonged biological half-life.
  • Fatty acids are important components of human body fat, lipids and cell membrane phospholipids, and as endogenous components, they have low immunogenicity, so they are also used to modify bioactive molecules (such as peptide drugs).
  • bioactive molecules such as peptide drugs.
  • fatty acids are used to modify specific amino acid residues of peptide drugs, fatty acids increase the serum half-life of peptide drugs by reversibly binding to serum albumin.
  • fatty acid modification also has its own limitations: for example, it is easy to produce non-specific modification site products of fatty acids; because the binding of fatty acids to serum albumin is reversible, peptide drugs dissociated from albumin are easily eliminated through the kidneys, thus affecting or reducing the half-life of fatty acid-modified peptide drugs.
  • the ultra-long-acting platform provided by the present application has the following advantages:
  • Both the Fc component and the higher fatty acid chain component in the conjugated molecule can bind to FcRn directly or indirectly.
  • Fc binds directly to FcRn, while higher fatty acids bind to FcRn indirectly via serum albumin. Since Fc and serum albumin bind to different sites of FcRn respectively, they will not interfere with each other, thus providing a higher half-life for the active molecule conjugated thereto compared to Fc alone or higher fatty acids alone;
  • Fusion proteins, antibodies, Fc and higher fatty acids are all endogenous substances in the body and have low immunogenicity, which can reduce the heterologous nature of the conjugated molecules to the body and reduce the possibility of producing corresponding antibodies;
  • Conjugated molecules can increase the size of the contained active molecules and reduce their renal excretion rate, thereby prolonging the circulation time of the active molecules in the body;
  • the Fc in the conjugated molecule can be homologously paired with the cognate Fc fragment through its CH2-3 domain, thereby increasing stability and improving the concentration of the local conjugated molecule/active molecule.
  • the present invention provides a conjugate molecule having a structure of "active molecule-Fc-Cn", a conjugate molecule having a structure of "active molecule -fusion protein-Cn” conjugate molecule, a conjugate molecule with a structure of "antibody-Cn”, wherein the active molecule is selected from any molecule that is beneficial to the body, Fc is derived from the heavy chain constant region of immunoglobulin IgG, and Cn is a modified portion comprising a C 14 - 24 fatty acid chain.
  • the conjugate molecule with a structure of "active molecule-Fc-Cn”, the conjugate molecule with a structure of "active molecule-fusion protein-Cn”, and the conjugate molecule with a structure of "antibody-Cn” disclosed in the present invention has the structure of the following formula (I): -ZY (I),
  • Z1 is a sulfur atom, a nitrogen atom or an oxygen atom in Fc
  • Z4 is a bond or a PEG unit represented by the following formula
  • R1 is selected from C1-4 alkylene, -NH-, -NH- C1-4 alkylene-, -NH- C1-4 alkylene-heteroaryl-, wherein the heteroaryl is a 5-membered or 6-membered nitrogen-containing heteroaryl;
  • Y is connected to Z 4 through X
  • k is an integer from 10 to 30
  • R independently represents hydrogen, C 1-6 alkyl, C 1-6 aminoalkyl, C 1-6 haloalkyl, C 1-6 hydroxyalkyl.
  • Z 2 is maleimido
  • the wavy line on the left indicates the position connected to Z1 ; the wavy line on the right indicates the position connected to Z3 .
  • Z 3 is -C 1 -C 10 alkylene-C( ⁇ O)-, wherein the alkylene is optionally substituted and wherein Z 3 is linked to Z 4 through -C-( ⁇ O)-.
  • Z 4 is a bond and Z 3 is directly connected to Y in formula (I).
  • Z4 is a PEG unit represented by the formula
  • R 1 is selected from -NH- and -NH-C 1-4 alkylene-;
  • Z 4 is a unit comprising 2-6 PEGs. In some embodiments, Z 4 is
  • Z in formula (I) of the present invention has the following structure:
  • Y is connected to Z 4 via X
  • k is an integer between 10 and 30.
  • R independently represents hydrogen, C 1-6 alkyl, C 1-6 aminoalkyl, C 1-6 haloalkyl, C 1-6 hydroxyalkyl.
  • Cn is selected from
  • Fc is derived from the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4. In a specific embodiment, Fc is derived from the heavy chain constant region of IgG1 or IgG4. In another embodiment, the Fc region may further include a hinge region. In a specific embodiment, Fc comprises amino acid modifications. In a specific embodiment, the modification to the Fc region is modification at positions 254, 308 and 434 (according to EU numbering). In another specific embodiment, the modification to the Fc region is to replace the amino acids at positions 254, 308, and 434 with Thr, Pro, and Ala, respectively.
  • the modification of the Fc region is modification of position 228, 234, 235 and/or 447, for example modification S228P, F234A, L235A or modification S228P, F234A, L235A and deletion of 447.
  • the Fc region is selected from the sequence of SEQ ID NO: 10, 15 or 16.
  • the active molecule is a peptide active molecule.
  • the peptide active molecule is fused to an antibody, fusion protein or Fc region directly or through a peptide linker.
  • the C-terminus of the peptide active molecule and the N-terminus of the antibody, fusion protein or Fc region are fused together.
  • the N-terminus of the peptide active molecule and the C-terminus of the antibody, fusion protein or Fc region are fused together.
  • the peptide active molecule is connected to an antibody, fusion protein or Fc region in a monomeric form.
  • the peptide active molecule is connected to an antibody, fusion protein or Fc region in a multimeric form.
  • the active molecule is selected from an enzyme, an enzyme inhibitor, an antigen, an antibody or an antibody fragment, a hormone, glucagon-like peptide-1 (GLP-1), glucagon, an interferon, a cytokine, a growth factor and/or a differentiation factor, a factor involved in cell movement or migration, a factor involved in bone tissue development/resorption, a chemokine, a plasma or interstitial adhesion molecule or an extracellular matrix, a bactericidal or antifungal factor, and the like.
  • GLP-1 glucagon-like peptide-1
  • glucagon an interferon
  • a cytokine a growth factor and/or a differentiation factor
  • a factor involved in cell movement or migration a factor involved in bone tissue development/resorption
  • a chemokine a plasma or interstitial adhesion molecule or an extracellular matrix
  • bactericidal or antifungal factor and the like.
  • the active molecule is selected from GLP-1, antibody Fab fragments, antibody F(ab') 2 fragments. In another specific embodiment, the active molecule is selected from anti-PD-1 antibody Fab fragments, anti-PD-1 antibody F(ab') 2 fragments, anti-VEGF antibody Fab fragments, anti-VEGF antibody F(ab') 2 fragments.
  • the peptide linker comprises the amino acid sequence (G4S)n, wherein n is an integer equal to or greater than 1.
  • the peptide linker includes ( G4S ) 3 , ( G4S ) 4 , ( G4S ) 6 , GS( G4S ) 4 , DAAALEAAALDAAAREAAARDAAAL, NVDHLPSNTLVDLA, (G3S ) 2, ( G4S ) 2 , ( G3S ) 3 , ( G4S ) 3 , (G3S) 4 , ( G4S ) 4 , (G3S ) 5 , ( G4S ) 5 , ( G3S ) 6 , ( G4S ) 6 , GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP
  • the conjugate molecule with the structure of "active molecule-Fc-Cn" or “antibody-Cn” provided herein homodimerizes through its Fc region.
  • the present application provides a conjugated molecule having a structure of "active molecule-Fc-Cn", wherein Fc is selected from IgG1 or IgG4, and Cn comprises a fatty acid chain of 16, 18 or 20 carbons.
  • the Fc comprises a modification, such as the modification mentioned in the present application.
  • Cn comprises a fatty acid chain of 16 carbons
  • Cn comprises a fatty acid chain of 18 carbons
  • Cn comprises a fatty acid chain of 20 carbons.
  • the present application provides a conjugate molecule having a structure of "antibody-Cn", wherein the antibody is selected from IgG1 or IgG4, and Cn comprises a fatty acid chain of 16, 18 or 20 carbons.
  • the antibody The Fc comprises modifications, such as those mentioned herein.
  • Cn comprises a 16-carbon fatty acid chain
  • Cn comprises an 18-carbon fatty acid chain
  • Cn comprises a 20-carbon fatty acid chain.
  • the present application provides a conjugated molecule having a structure of "active molecule-Fc-Cn", wherein Fc is selected from IgG1 or IgG4, and Cn comprises a fatty acid chain of 16, 18 or 20 carbons and is coupled/conjugated to the free thiol of Fc.
  • the Fc comprises a modification, such as the modification mentioned in the present application.
  • Cn comprises a fatty acid chain of 16 carbons
  • Cn comprises a fatty acid chain of 18 carbons
  • Cn comprises a fatty acid chain of 20 carbons.
  • the present application provides a conjugate molecule having a structure of "antibody-Cn", wherein the antibody is selected from IgG1 or IgG4, and Cn comprises a fatty acid chain of 16, 18 or 20 carbons and is coupled/conjugated to the sulfur atom of the free thiol group of the antibody Fc.
  • the Fc of the antibody comprises a modification, such as the modification mentioned in the present application.
  • Cn comprises a fatty acid chain of 16 carbons
  • Cn comprises a fatty acid chain of 18 carbons
  • Cn comprises a fatty acid chain of 20 carbons.
  • the present application provides a conjugated molecule having a structure of "active molecule-fusion protein-Cn", wherein Cn comprises a 16, 18 or 20-carbon fatty acid chain and is coupled/conjugated to the sulfur atom of the free sulfhydryl group of the fusion protein.
  • Cn comprises a 16-carbon fatty acid chain
  • Cn comprises an 18-carbon fatty acid chain
  • Cn comprises a 20-carbon fatty acid chain.
  • the present application provides a conjugated molecule having a structure of "active molecule-IgG4 Fc-Cn", wherein Cn comprises a 16-, 18- or 20-carbon fatty acid chain and is coupled/conjugated to the free thiol group of IgG4 Fc.
  • the IgG4 Fc comprises a modification, such as the modification mentioned in the present application.
  • Cn comprises a 16-carbon fatty acid chain
  • Cn comprises an 18-carbon fatty acid chain
  • Cn comprises a 20-carbon fatty acid chain.
  • the present application provides a conjugate molecule having a structure of "antibody-Cn", wherein Cn comprises a fatty acid chain of 16, 18 or 20 carbons and is coupled/conjugated to the sulfur atom of the free thiol group of the IgG4 antibody.
  • the Fc of the IgG4 antibody comprises a modification, such as the modification mentioned in the present application.
  • Cn comprises a fatty acid chain of 16 carbons
  • Cn comprises a fatty acid chain of 18 carbons
  • Cn comprises a fatty acid chain of 20 carbons.
  • the present application provides a conjugated molecule having a structure of "active molecule-fusion protein-Cn", wherein Cn comprises a 16, 18 or 20-carbon fatty acid chain and is coupled/conjugated to the sulfur atom of the free sulfhydryl group of the fusion protein.
  • Cn comprises a 16-carbon fatty acid chain
  • Cn comprises an 18-carbon fatty acid chain
  • Cn comprises a 20-carbon fatty acid chain.
  • the present application provides a conjugated molecule having a structure of "active molecule-IgG1 Fc-Cn", wherein Cn comprises a 16-, 18- or 20-carbon fatty acid chain and is coupled/conjugated to the sulfur atom of the free thiol group of IgG1 Fc.
  • the IgG1 Fc comprises a modification, such as the modification mentioned in the present application.
  • Cn comprises a 16-carbon fatty acid chain
  • Cn comprises an 18-carbon fatty acid chain
  • Cn comprises a 20-carbon fatty acid chain.
  • the present application provides a conjugate molecule having a structure of "antibody-Cn", wherein Cn comprises a 16, 18 or 20 carbon fatty acid chain and is coupled/conjugated to the sulfur atom of the free sulfhydryl group of the IgG1 antibody.
  • the Fc of the IgG1 antibody comprises a modification, such as the modification mentioned in the present application.
  • Cn comprises a 16 carbon fatty acid chain
  • Cn comprises an 18 carbon fatty acid chain
  • Cn comprises a 20 carbon fatty acid chain.
  • the present application provides a conjugated molecule having a structure of "active molecule-fusion protein-Cn", wherein Cn comprises a 16, 18 or 20-carbon fatty acid chain and is coupled/conjugated to the sulfur atom of the free sulfhydryl group of the fusion protein.
  • Cn comprises a 16-carbon fatty acid chain
  • Cn comprises an 18-carbon fatty acid chain
  • Cn comprises a 20-carbon fatty acid chain.
  • the conjugated molecule with the structure of "active molecule-Fc-Cn" described in the present application is a GLP-1-Fc-Cn conjugated molecule, wherein the GLP-1 is any active GLP-1 known in the prior art.
  • Fc is selected from Fc of IgG1 or Fc of IgG4.
  • Cn in the conjugated molecule GLP-1-Fc-Cn comprises a fatty acid chain of 16, 18 or 20 carbons and is coupled/conjugated to the sulfur atom of the free thiol group of Fc or the nitrogen atom of Fc.
  • Cn in the conjugated molecule GLP-1-Fc-Cn is selected from TM1, C18-tert-butyl ester, C16-NHS or C20-NHS.
  • the conjugated molecule GLP-1-Fc-Cn is GLP-1-IgG4 Fc-TM1, GLP-1-IgG4 Fc-C18 tert-butyl alcohol lipid, GLP-1-IgG4 Fc-C16-NHS, GLP1-IgG4 Fc-C20-NHS.
  • Fc comprises the sequence shown in SEQ ID NO: 10, 15 or 16.
  • the GLP-1-IgG4 Fc in the GLP-1-IgG4 Fc-TM1, GLP-1-IgG4 Fc-C18, GLP-1-IgG4 Fc-C16-NHS, GLP1-IgG4 Fc-C20-NHS conjugated molecule is derived from dulaglutide, preferably dulaglutide, for example, having a structure as disclosed in CN1802167, preferably having a structure of Gly 8 -Glu 22 -Gly 36 -GLP-1(7-37)-1L-IgG4 (S228P, F234A, L235A).
  • the amino acid sequence of dulaglutide is as shown in SEQ ID NO: 1.
  • the GLP-1-Fc-Cn conjugated molecule is dulaglutide-Cn, such as dulaglutide-TM1, dulaglutide-C18 tert-butyl ester, dulaglutide-C16-NHS, dulaglutide-C20-NHS.
  • the active molecule in the conjugate molecule with the structure of "active molecule-Fc-Cn" of the present application is an anti-PD-1 antibody or an antigen-binding fragment thereof, and the anti-PD-1 antibody or its antigen-binding fragment may be any known anti-PD-1 antibody or its antigen-binding fragment.
  • the antibody in the sub-molecule is an anti-PD-1 antibody or an antigen-binding fragment thereof, and the anti-PD-1 antibody or its antigen-binding fragment may be any known anti-PD-1 antibody or its antigen-binding fragment.
  • Fc is selected from IgG1 Fc or IgG4 Fc.
  • the antibody is selected from IgG1 or IgG4.
  • the conjugated molecule is anti-PD-1 antibody antigen-binding fragment-IgG4 Fc-TM1, anti-PD-1 antibody antigen-binding fragment-IgG4 Fc-C18, anti-PD-1 antibody antigen-binding fragment-IgG4 Fc-C16-NHS, anti-PD-1 antibody antigen-binding fragment-IgG4 Fc-C20-NHS.
  • the conjugated molecule is anti-PD-1 antibody antigen-binding fragment-IgG1 Fc-TM1, anti-PD-1 antibody antigen-binding fragment-IgG1Fc-C18, anti-PD-1 antibody antigen-binding fragment-IgG1 Fc-C16-NHS, anti-PD-1 antibody antigen-binding fragment-IgG1 Fc-C20-NHS.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 9 and a light chain variable region as shown in SEQ ID NO: 11.
  • the Fc comprises a sequence as shown in SEQ ID NO: 10.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 8 and a light chain as shown in SEQ ID NO: 12.
  • the Cn is selected from TM1, C18-tert-butyl ester, C16-NHS, C20-NHS. In a more preferred embodiment, the Cn is selected from TM1.
  • the active molecule in the conjugated molecule with the structure of "active molecule-Fc-Cn" of the present application is an anti-VEGF antibody or an antigen-binding fragment thereof, and the anti-VEGF antibody or its antigen-binding fragment may be any known anti-VEGF antibody or its antigen-binding fragment.
  • the antibody in the conjugated molecule with the structure of "antibody-Cn" of the present application is an anti-VEGF antibody or its antigen-binding fragment, and the anti-VEGF antibody or its antigen-binding fragment may be any known anti-VEGF antibody or its antigen-binding fragment.
  • Fc is selected from IgG1 Fc or IgG4 Fc.
  • the antibody is selected from IgG1 or IgG4.
  • the conjugated molecule is anti-VEGF antibody antigen-binding fragment-IgG4 Fc-TM1, anti-VEGF antibody antigen-binding fragment-IgG4 Fc-C18, anti-VEGF antibody antigen-binding fragment-IgG4 Fc-C16-NHS, anti-VEGF antibody antigen-binding fragment-IgG4 Fc-C20-NHS.
  • the conjugated molecule is anti-VEGF antibody antigen-binding fragment-IgG1 Fc-TM1, anti-VEGF antibody antigen-binding fragment-IgG1 Fc-C18-tert-butyl alcohol ester, anti-VEGF antibody antigen-binding fragment-IgG1 Fc-C16-NHS, anti-VEGF antibody antigen-binding fragment-IgG1 Fc-C20-NHS.
  • the anti-VEGF antibody or its antigen-binding fragment comprises three heavy chain CDRs shown in SEQ ID NO: 2, 3 and 4 and three light chain CDRs shown in SEQ ID NO: 5, 6 and 7.
  • the Fc comprises the sequence shown in SEQ ID NO: 15.
  • the anti-VEGF antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 13 and the light chain shown in SEQ ID NO: 14.
  • the Cn is selected from TM1, C18-tert-butyl alcohol ester, C16-NHS, C20-NHS. In a more preferred embodiment, the Cn is selected from TM1.
  • the present application provides a method for preparing a conjugate molecule having a structure of "active molecule-Fc-Cn", comprising: (a) (a) coupling the "active molecule-Fc” fusion with a Cn containing a fatty acid chain under conditions that allow the Fc region to conjugate with the Cn to produce an "active molecule-Fc-Cn" conjugate molecule.
  • the present application also provides a method for preparing a conjugate molecule having a structure of "antibody-Cn", comprising (a) coupling an antibody with Cn containing a fatty acid chain under conditions that allow conjugation of the antibody and Cn to produce an "antibody-Cn" conjugate molecule.
  • the present application also provides a method for preparing a conjugated molecule with a structure of "active molecule-fusion protein-Cn", comprising (a) connecting an active molecule polypeptide to a fusion protein to prepare an "active molecule-fusion protein” fusion; and (b) subjecting the "active molecule-fusion protein” fusion to a coupling reaction with Cn containing a fatty acid chain under conditions that allow the fusion protein to conjugate with Cn, to produce an "active molecule-fusion protein-Cn" conjugated molecule.
  • the present application provides a method for preparing a conjugated molecule having a structure of "active molecule-Fc-Cn", which comprises the steps of (a) connecting an antibody Fab fragment to an immunoglobulin Fc region to prepare a "Fab-Fc” fusion, and (b) subjecting the "Fab-Fc" fusion to a coupling reaction under conditions that allow the Fc region to be conjugated to a Cn containing a fatty acid chain to produce a "Fab-Fc-Cn" conjugated molecule.
  • the Fab and Fc are derived from the same or different antibody molecules.
  • the present application provides a method for preparing a conjugated molecule having a structure of "active molecule-Fc-Cn", which includes the step of subjecting a whole antibody to a coupling reaction under conditions that allow conjugation of the Fc region with a Cn containing a fatty acid chain to produce the "active molecule-Fc-Cn" conjugated molecule.
  • the present application provides a method for preparing a conjugated molecule having a structure of "antibody-Cn”, which includes the step of subjecting a whole antibody to a coupling reaction under conditions that allow conjugation of the Fc region with a Cn containing a fatty acid chain to produce an "active molecule-Fc-Cn" conjugated molecule.
  • the present application provides a composition, such as a pharmaceutical composition, comprising the conjugated molecule of the first aspect.
  • a pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the present application provides a method for effectively prolonging the serum half-life of an active molecule, comprising the step of constructing the active molecule into a conjugated molecule having a structure of "active molecule-Fc-Cn", a conjugated molecule having a structure of "antibody-Cn”, or a conjugated molecule having a structure of "active molecule-fusion protein-Cn” according to the method of the second aspect, thereby effectively improving the serum half-life of the active molecule.
  • the present application provides use of the conjugated molecule described in the first aspect, or the composition described in the third aspect in the preparation of a drug for treating human diseases.
  • the present invention provides the conjugated molecule of the first aspect, or the composition of the third aspect for use in treat.
  • the present application provides a method for treating human diseases, comprising administering an effective amount of the conjugated molecule described in the first aspect of the present application, or the composition described in the third aspect to a subject.
  • Figure 1 shows the HIC-HPLC results of the GLP1-Fc-TM1 coupling products.
  • the upper figure is the HIC-HPLC result of GLP1-Fc without TM1 coupling
  • the lower figure is the HIC-HPLC result of GLP1-Fc-TM1 after TM1 coupling.
  • "0" in the lower figure represents GLP1-Fc without TM1 coupling
  • "2" represents GLP1-Fc coupled with 2 TM1s
  • "4" represents GLP1-Fc coupled with 4 TM1s.
  • Figure 2 shows the HIC-HPLC results of the HX006-TM1-1 coupling product.
  • the upper figure is the HIC-HPLC result of HX006 without coupling with TM1
  • the lower figure is the HIC-HPLC result of HX006-TM1-1 after coupling with TM1.
  • "0" in the lower figure represents HX006 without coupling with TM1
  • "2" represents HX006 coupled with 2 TM1s
  • "4" represents HX006 coupled with 4 TM1s
  • “6” represents HX006 coupled with 6 TM1s.
  • FIG. 3 shows the HIC-HPLC results of the HX006-TM1-2 coupling products, where "0” represents HX006 without TM1 coupling, "2” represents HX006 coupled with 2 TM1s, "4" represents HX006 coupled with 4 TM1s, “6” represents HX006 coupled with 6 TM1s, and “8” represents HX006 coupled with 8 TM1s.
  • Figure 4 shows the HIC-HPLC results of the HX008-TM1 coupling products
  • Figure 4A is the HIC-HPLC result of HX008 without TM1 coupling
  • Figure 4B is the HIC-HPLC result of HX008-TM1-2 after TM1 coupling
  • "0" in the figure represents HX008 without TM1 coupling
  • "2” represents HX008 coupled with 2 TM1s
  • "4" represents HX008 coupled with 4 TM1s
  • “6” represents HX008 coupled with 6 TM1s
  • Figure 4C is the HIC-HPLC result of HX008-TM1-3 after TM1 coupling
  • "0” in the figure represents HX008 without TM1 coupling
  • “2” represents HX008 coupled with 2 TM1s
  • "4" represents HX008 coupled with 4 TM1s
  • "6” represents HX008 coupled with 6 TM1s.
  • FIG5 shows the ELISA detection results of GLP1-Fc-TM1 binding to HSA, wherein HX042 represents GLP1-Fc.
  • FIG6 shows the ELISA test results of HX006-TM1 binding to HSA.
  • Figure 7 shows the ELISA test results of HX008-TM1 binding to HSA.
  • FIG8 shows the HIC-HPLC results after GLP1-Fc was coupled to C18-tert-butyl ester, where “0” indicates no coupling to C18-tert-butyl ester and “2” indicates coupling to two C18-tert-butyl esters.
  • FIG. 9 shows the HIC-HPLC results of the GLP1-Fc sample (upper panel) and the GLP1-Fc-C16-NHS sample (lower panel).
  • FIG. 10 shows the HIC-HPLC results of the GLP1-Fc-C20-NHS sample.
  • FIG11 shows the ELISA test results of GLP1-Fc-C16-NHS and GLP1-Fc-C20-NHS binding to HSA, Among them, HX042 represents GLP1-Fc.
  • FIG. 12 shows the ELISA test results of HX006-C16-NHS and HX006-C20-NHS binding to HSA.
  • FIG. 13 shows the binding activity of GLP1-Fc-C18-tert-butyl ester to HSA.
  • FIG. 14 shows the binding activity of HX006-C18-tert-butyl ester to HSA.
  • FIG. 15 shows the HIC-HPLC results after coupling HX006 with C18-tert-butyl alcohol ester.
  • FIG. 16 shows that GLP-1-Fc-TM1 can effectively activate the biological activity of the reporter gene.
  • FIG. 17 shows the plasma concentration-time curve of GLP1-Fc-TM1 after a single administration in rats, with dulaglutide as a positive control.
  • FIG. 18 shows the plasma concentration-time curve of GLP1-Fc-TM1 after a single administration in cynomolgus monkeys, with dulaglutide as a positive control.
  • Figure 19 shows the pharmacodynamic results of GLP-1-Fc-TM1 on type II diabetic db/db mice, wherein Figure 19-1 shows the results of lowering 4h fasting blood glucose in db/db mice; Figures 19-2 and 19-3 show the results of random blood glucose after the first and last administrations; Figures 19-4 and 19-5 show the results of blood glucose AUC 0-180min of the OGTT test after the last administration; Figure 19-6 shows the results of lowering the glycated hemoglobin content in db/db mice; Figure 19-7 shows the results of increasing the insulin level in db/db mice; and Figure 19-8 shows the results of reducing the average daily food intake in db/db mice.
  • Figure 20 shows the efficacy results of GLP-1-Fc-TM1 on DIO model mice, wherein Figure 20-1 shows the results of GLP-1-Fc-TM1 reducing the body weight of DIO mice; Figure 20-2 shows the results of GLP-1-Fc-TM1 reducing the food intake of DIO mice; Figure 20-3 shows the results of GLP-1-Fc-TM1 reducing the body fat content of mice; Figure 20-4 shows the results of GLP-1-Fc-TM1 reducing the fasting blood glucose of mice; Figure 20-5 shows the results of GLP-1-Fc-TM1 reducing the blood lipid content of mice; Figure 20-6 shows the results of ALT and AST levels of serum liver function indicators in mice; Figure 20-7 shows the results of GLP-1-Fc-TM1 improving the ballooning and fatty degeneration of liver tissue in DIO mice.
  • Figure 21 shows the pharmacological effects of GLP-1-Fc-TM1 on rats, wherein Figures 21-1 to 21-4 show the results of GLP-1-Fc-TM1 lowering blood glucose in SD rats, and Figures 21-5 to 21-8 show the results of GLP-1-Fc-TM1 increasing serum insulin.
  • the trade name includes product formulations of the trade name product, the generic drug, and the active pharmaceutical ingredient, unless the context indicates otherwise.
  • antibody is used in the broadest sense herein and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, complete antibodies or antibody fragments thereof that exhibit the desired antigen-binding activity.
  • a complete antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but may comprise fewer chains in certain cases, e.g., antibodies naturally occurring in camels may comprise only heavy chains.
  • the term "whole antibody” refers to an immunoglobulin molecule comprising at least two heavy chains (H) and two light chains (L).
  • Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the heavy chain of an antibody can be divided into mainly 5 different types based on the amino acid sequence of its constant region: IgA, IgD, IgE, IgG and IgM, and several of these types can be further divided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2.
  • antibody fragment and "antigen-binding fragment” of an antibody are used interchangeably and refer to a molecule that is not a complete antibody, which comprises a portion of a complete antibody that is used to bind to the antigen to which the complete antibody binds.
  • an antibody fragment generally comprises amino acid residues from a "complementarity determining region" or "CDR”.
  • Antibody fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of complete antibodies.
  • antibody fragments include, but are not limited to, Fab, scFab, disulfide-linked scFab, Fab', F(ab')2, Fab'-SH, Fv, scFv, disulfide-linked scFv.
  • the antibody fragment comprises a cysteine residue introduced into the Fc region to provide an amino acid residue site that can be used for sulfhydryl coupling chemistry.
  • an antibody when it is mentioned that an antibody is an IgG antibody, it refers to a heterotetrameric protein with an IgG class immunoglobulin structure.
  • IgG antibodies usually the VH-CH1 of the heavy chain is paired with the VL-CL of the light chain to form a Fab fragment that specifically binds to an antigen. Therefore, an IgG antibody is basically composed of two Fab molecules connected by the hinge region of the immunoglobulin and two dimerized Fc regions.
  • IgG immunoglobulins can be divided into subclasses based on the sequence of the heavy chain constant region, such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3), and ⁇ 4 (IgG4).
  • the antibody according to the present invention is an IgG antibody, such as an IgG1, IgG2, IgG3 or IgG4 antibody.
  • CDR region or “CDR” or “hypervariable region” refers to the regions of the antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen contact residues ("antigen contact points"). CDR is mainly responsible for binding to the antigen epitope.
  • variable region or “variable domain” is a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to its antigen.
  • the heavy chain variable region (VH) and light chain variable region (VL) can be further subdivided into hypervariable regions (HVRs, also known as complementarity determining regions (CDRs)), with more conserved regions (i.e., framework regions (FRs)) interspersed therebetween.
  • HVRs hypervariable regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • affinity refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. Affinity can be measured by common methods known in the art. One method for measuring affinity is the ELISA assay, and another method is the surface plasmon resonance (SPR) assay described in the Examples herein.
  • SPR surface plasmon resonance
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions, such as the Fc region sequences of various Ig subclasses 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.).
  • the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain.
  • the C-terminal terminal lysine (Lys447) of the Fc region may or may not be present.
  • the human IgG heavy chain Fc region carries a hinge sequence or a partial hinge sequence of a natural immunoglobulin at the N-terminus, such as a sequence from E216 to T225 or a sequence from D221 to T225 according to EU numbering.
  • the Fc region of an immunoglobulin comprises two constant domains, namely, CH2 and CH3, and in other embodiments, the Fc region of an immunoglobulin comprises three constant domains, namely, CH2, CH3, and CH4.
  • the EU numbering scheme is a widely adopted standard for numbering residues in antibodies in a consistent manner, developed based on sequence alignments.
  • the EU numbering scheme of Kabat is used to number and refer to amino acid residues in antibody or fusion protein regions (as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • Fc region mutant can be used interchangeably and refer to an Fc region that contains at least one amino acid modification and is different from the native sequence Fc region/wild-type Fc region. For example, by modifying the amino acids at multiple positions in the Fc region of the wild-type immunoglobulin IgG to reduce half-antibody formation, reduce or eliminate effector function, and increase serum half-life.
  • Various methods that have been disclosed in the prior art can be used to further modify the Fc region known in the prior art, the Fc region provided in the present application, and the fusion protein (such as an antibody) containing the Fc region, for example, to reduce immunogenicity, improve stability, solubility, function and other modifications of clinical benefits.
  • Such modifications include, but are not limited to, the following modifications, for example, of IgG Fc, for example, at amino acid residues at positions 214-238, 250-260, 297-299, 307-318, 322 or 327 to 331, 380-390, etc., specifically, for example, modifications to positions 228, 233, 234, 235, 252, 254, 256, 297, 307, 308, 311, 380, 385, 386, 389, 428, 434 and 447.
  • the hinge of native human IgG can be modified.
  • the chain sequence can be modified so that the Fc region is expressed as a homogeneous product.
  • the asparagine that is prone to deamidation can be modified, for example, replaced with glutamine, aspartic acid or glutamic acid, such as replacements including N297E, N315Q and N384Q.
  • the leucine at position 235 of the Fc region can be modified, such as L235K replacement.
  • the immunoglobulin Fc region can also be phosphorylated, sulfated, glycosylated, methylated, acetylated, amidated, etc. to meet specific needs.
  • the modification of the Fc region is a modification of positions 254, 308 and 434, such as the modifications disclosed in CN108299560A.
  • the modification of the Fc region is a modification as described in CN1802167.
  • the modification of the Fc region is a modification of positions 228, 234, 235 and/or 447, such as modification S228P, F234A, L235A or modification S228P, F234A, L235A and 447 deletion.
  • the modification of the Fc region is shown in brackets, such as IgG4 Fc (S228P, F234A, L235A) means that the amino acids at positions 228, 234 and 235 of the wild-type IgG4 Fc are substituted accordingly.
  • the wild-type immunoglobulin Fc region can be obtained from humans and animals (e.g., cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, etc.), or a recombinant form or derivative of the Fc region can be obtained from transformed animal cells or microorganisms.
  • the gene encoding the immunoglobulin can be isolated from the corresponding cDNA library using the PCR method, or the Fc region can be obtained by protease treatment of the intact immunoglobulin.
  • the technology for preparing Fc region derivatives can be found in WO 97/34631 and WO 96/32478.
  • the Fc region used in the present application is derived from an IgG immunoglobulin, such as an Fc region derived from IgG1, IgG2, IgG3, and IgG4 subclasses, preferably an Fc region derived from IgG1 and IgG4 subclasses.
  • the Fc region used in the present application is an Fc region derived from human IgG1 and IgG4 to reduce the immunogenicity of the conjugate of the present application.
  • the variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by one or more amino acid substitutions, deletions or additions. In some embodiments, the variant Fc region has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to a wild-type Fc region and/or a parent Fc region.
  • FcRn neonatal receptor
  • FcRn is responsible for transferring maternal IgG to the fetus and regulating the homeostasis of immunoglobulins in the body.
  • FcRn can bind to the Fc part of IgG, preventing IgG molecules from being lysosomal cleavage, and can increase the half-life of IgG in the body, and participate in the transport, maintenance and distribution metabolism of IgG in the body.
  • receptor-mediated endocytosis refers to a process in which a ligand/receptor complex is internalized and delivered into the cytosol or translocated to an appropriate intracellular compartment, triggered by the binding of a ligand to a corresponding receptor on the cell surface.
  • sequence identity refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison window.
  • the "percentage of sequence identity” can be calculated by comparing two optimally aligned sequences in a comparison window and determining the presence of identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, The number of the position of Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) is to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (that is, window size), and the result is multiplied by 100, to produce the sequence identity percentage.
  • sequence identity percentage In order to determine the best comparison carried out in the sequence identity percentage, it can be realized in many ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the suitable parameters for the alignment sequence, including any algorithm required for the maximum alignment in the full-length sequence range being compared or in the target sequence region for realizing.
  • the percentage of amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the given antibody sequence, in a preferred embodiment, according to the Kabat numbering convention. In this article, without specifying a comparison window (i.e., the target antibody region to be compared), it will be applicable to align the full length of the given antibody sequence.
  • the sequence identity may be distributed over the entire heavy chain variable region and/or the entire light chain variable region, or the sequence percentage identity may be limited to the framework region only, while the sequence in the corresponding CDR region remains 100% identical.
  • amino acid substitution refers to replacing at least one amino acid residue present in a predetermined amino acid sequence with another different “substitution” amino acid residue.
  • conservative substitution refers to substitution of one amino acid with another within the same class, such as substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid.
  • peptide linker refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and/or serine (S) and/or threonine residues (T), used alone or in combination, or a hinge region from an immunoglobulin, which connects the active ingredient and the Fc region.
  • the peptide linker that can be used in the present invention can be easily determined by those skilled in the art.
  • it comprises the amino acid sequence (G 4 S) n , wherein n is an integer equal to or greater than 1.
  • the peptide linker includes (G 4 S) 3 , (G 4 S) 4 , (G 4 S) 6 , GS(G 4 S) 4 , DAAALEAAALDAAAREAAARDAAAL, NVDHLPSNTLVDLA.
  • the peptide linker that can be used in the antibody molecules of the present invention can also be, for example but not limited to, the following amino acid sequence: ( G3S )2, ( G4S ) 2 , ( G3S ) 3 , ( G4S ) 3 , ( G3S ) 4 , ( G4S ) 4 , ( G3S ) 5 , ( G4S ) 5 , ( G3S ) 6 , ( G4S ) 6 , GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP and GSTSGSGK PGSGEGSTKG.
  • alkyl refers to a straight or branched saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms. Specifically, the alkyl group has 1-10 carbon atoms, such as 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 carbon atoms.
  • C 1 -C 6 alkyl refers to a straight or branched saturated hydrocarbon group having 1 to 6 carbon atoms.
  • Examples include methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl or tert-butyl), pentyl (including n-pentyl, isopentyl, neopentyl), hexyl (including n-hexyl, 2-methylpentyl, 3-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl), and the like.
  • alkylene refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight or branched saturated alkane. Specifically, the alkylene group has 1-10 carbon atoms, such as 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 carbon atoms.
  • C 1 -C 6 alkylene refers to a straight or branched alkylene group having 1 to 6 carbon atoms, including, but not limited to, methylene, ethylene, propylene, butylene, and the like.
  • cycloalkyl refers to a monocyclic, fused polycyclic, bridged polycyclic or spirocyclic non-aromatic monovalent hydrocarbon ring structure with a specified number of ring atoms, which may be saturated or unsaturated, for example, containing one or more double bonds.
  • the cycloalkyl group may contain 3 or more, for example, 3-18, 3-10, or 3-8 carbon atoms in the ring, for example, C 3-10 cycloalkyl, C 3-8 cycloalkyl, C 3-6 cycloalkyl, C 5-6 cycloalkyl.
  • Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl.
  • alkenyl refers to a straight or branched unsaturated hydrocarbon group consisting of carbon atoms and hydrogen atoms and containing at least one double bond. Specifically, the alkenyl group has 2-8, such as 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms.
  • C2 - C6 alkenyl refers to a straight or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.
  • alkenylene refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight or branched unsaturated olefin containing at least one double bond. Specifically, the alkenylene has 2-8, such as 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms.
  • C2 - C6 alkenylene refers to a straight or branched alkenylene having 2 to 6 carbon atoms, such as vinylene, propenylene, allylene, butenylene, pentenylene, and hexenylene.
  • alkynyl refers to a straight or branched unsaturated hydrocarbon group consisting of carbon atoms and hydrogen atoms and containing at least one triple bond. Specifically, the alkynyl group has 2-8, such as 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms.
  • C2 - C6alkynyl refers to a straight or branched 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, etc.
  • alkynylene refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight or branched unsaturated alkyne containing at least one triple bond. Specifically, the alkynylene group has 2-8, such as 2 to 6, 2 to 5, 2 to 4 or 2 to 3 carbon atoms.
  • C2 - C6 alkynylene refers to a straight or branched alkynylene group having 2 to 6 carbon atoms, such as ethynylene, propynylene, propargylene, butynylene, pentynylene and ethynylene. Hexynyl.
  • heterocycle refers to an aromatic or non-aromatic monocyclic, bicyclic or polycyclic ring system having 5-20 members (e.g., 5-14 members, 5-8 members, 5-6 members) of 1-4 heteroatom ring members independently selected from N, O or S. One or more N, C or S atoms in the heterocycle may be oxidized.
  • the heterocycle is a 5-10 ring system, which is a monocyclic or fused bicyclic ring.
  • Representative examples include, but are not limited to, pyrrolidine, azetidine, piperidine, morpholine, tetrahydrofuran, tetrahydropyran, benzofuran, benzothiophene, indole, benzopyrazole, pyrrole, thiophene (thiophene), furan, thiazole, imidazole, pyrazole, pyrimidine, pyridine, pyrazine, pyridazine, isothiazole and isoxazole. It should be understood that the term includes heteroaryl as defined herein.
  • aryl refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6-20, for example 6-12 carbon atoms in the ring portion.
  • the aryl group is a ( C6 - C10 ) aryl group.
  • Non-limiting examples include phenyl, biphenyl, naphthyl or tetrahydronaphthyl, each of which may be optionally substituted with 1-4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, sulfhydryl, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamido, heterocyclic radicals, etc.
  • substituents such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O
  • heteroaryl refers to a 5-20 membered (e.g., 5-14, 5-8, 5-6) aromatic monocyclic or polycyclic ring system containing 1-4 heteroatoms selected from N, O or S, which may be substituted or unsubstituted.
  • the heteroaryl is a 5-10 membered ring system, which is a monocyclic or fused bicyclic ring.
  • heteroaryl groups include 2- or 3-thienyl, 2- or 3-furanyl, 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-pyridinyl, 3- or 4-pyridazinyl, 3-, 4- or 5-pyrazinyl, 2-pyrazinyl, 2-, 4- or 5-pyrimidinyl.
  • heteroalkyl refers to a stable straight or branched chain hydrocarbon which is fully saturated or contains from 1 to 3 degrees of unsaturation, consisting of the indicated number of carbon atoms and from one to ten, preferably from one to three heteroatoms selected from O, N, Si and S, wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized.
  • the heteroatoms O, N, Si and S may be placed at any interior position of the heteroalkyl group or at the position at which the heteroalkyl group is attached to the rest of the molecule.
  • Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.
  • the C1 to C4 heteroalkyl or heteroalkylene group has 1 to 4 carbon atoms and 1 or 2 heteroatoms
  • the C1 to C3 heteroalkyl or heteroalkylene group has 1 to 3 carbon atoms and 1 or 2 heteroatoms.
  • the heteroalkyl and heteroalkylene groups are saturated.
  • substituted as used in the definition of each group herein means that the corresponding group may be substituted by, for example but not limited to, the following groups: alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, halogen, cyano, nitro, azido, carboxyl, hydroxyl, mercapto, amino, mono- or di-alkylamino, mono- or di-cyclo ...
  • cyclo- or di-cycloalkylamino cyclo- or di-cycloalkylamino, cyclo- or di-cycloalkylamino, cyclo- or di-cycloalkylamino, cyclo- or di-cycloalkylamino, cyclo- or di-cycloalkylamino, cyclo- or di-cycloalkylamino, cyclo- or di-cycloalkylamino, cyclo- or di-cycloalkylamino, cyclo- or di-cycloalkylamino, cyclo- or di-cycloalkylamino Arylamino, mono- or di-heterocyclylamino, mono- or di-heteroarylamino, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-oxy, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl-
  • substituents include, but are not limited to, one or more groups independently selected from the group consisting of halogen, OH, SH, CN, NH2 , NHCH3 , N( CH3 ) 2 , NO2 , N3 , C(O) CH3 , COOH, C(O)-amino, OCOCH3 , methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methylsulfonylamino, SO, SO2 , phenyl, piperidinyl, piperazinyl, and pyrimidinyl.
  • PEG unit refers to an organic moiety comprising repeated ethyleneoxy subunits (PEG or PEG subunits), which can be polydisperse, monodisperse or discrete (i.e., having a discrete number of ethyleneoxy subunits).
  • Polydisperse PEGs are heterogeneous mixtures of size and molecular weight, while monodisperse PEGs are typically purified from heterogeneous mixtures and thus have a single chain length and molecular weight.
  • Preferred PEG units include discrete PEGs, which are compounds synthesized in a stepwise manner rather than via a polymerization process. Discrete PEGs provide single molecules with defined and specified chain lengths.
  • DAR refers to the ratio of the Cn moiety coupled to the Fc described herein to the Fc, or the ratio of the Cn moiety coupled to the antibody described herein to the antibody, or the ratio of the Cn moiety coupled to the fusion protein described herein to the fusion protein.
  • DAR can be 1 to 20, such as 1-18, 4-16, 5-12, 6-10, 1-8, 2-8, 1-6, 2-6, 2-4, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • DAR can also be calculated as the average DAR of a population of molecules in the product, i.e., the total ratio of Cn coupled to the Fc portion described herein to the Fc portion, the total ratio of Cn coupled to the antibody described herein to the antibody, or the total ratio of Cn coupled to the fusion protein described herein to the fusion protein in the product measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and/or HPLC), which DAR is referred to herein as the average DAR.
  • a detection method e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and/or HPLC
  • the average DAR value of the conjugate of the invention is 1 to 20, e.g., 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.
  • drug encompasses any substance that is effective in preventing or treating diseases related to glucose metabolism disorders, cardiovascular and cerebrovascular diseases, kidney diseases, and retinopathy.
  • Diseases related to glucose metabolism disorders are, for example, diabetes (e.g., type I diabetes, type II diabetes), obesity, hypertension, dyslipidemia, obesity, glucose intolerance, hyperglycemia, hyperinsulinemia, cardiovascular diseases, etc.
  • active ingredient refers to a substance that has any physiologically active function (e.g., regulating gene expression and physiological function, correcting abnormal conditions caused by lack or excessive secretion of components involved in regulating body functions) that is beneficial to cells and/or organisms, usually an active peptide substance.
  • physiologically active function e.g., regulating gene expression and physiological function, correcting abnormal conditions caused by lack or excessive secretion of components involved in regulating body functions
  • it includes but is not limited to enzymes, enzyme inhibitors, antigens, antibodies, antibody fragments, hormones, glucagon-like peptide-1 (GLP-1), glucagon, interferon, cytokines, growth factors and/or differentiation factors, factors involved in cell movement or migration, factors involved in bone tissue generation/resorption, chemokines, plasma or interstitial adhesion molecules or extracellular matrix, bactericidal or antifungal factors, etc.
  • GLP-1 glucagon-like peptide-1
  • cytokines growth factors and/or differentiation factors
  • factors involved in cell movement or migration factors involved in bone tissue generation/resorption
  • chemokines chemokines
  • plasma or interstitial adhesion molecules or extracellular matrix bactericidal or antifungal factors, etc.
  • composition refers to a composition that is in a form that permits the biological activity of the active ingredients contained therein to be effective, and does not contain additional ingredients that are unacceptably toxic to a subject to which the composition is administered.
  • the pharmaceutical composition of the present invention comprises a conjugated molecule of the present invention and a pharmaceutical excipient.
  • pharmaceutical excipient refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, buffer, surfactant, carrier, stabilizer, etc., which is administered together with the active substance.
  • adjuvant eg, Freund's adjuvant (complete and incomplete)
  • excipient buffer, surfactant, carrier, stabilizer, etc.
  • drug combination refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition.
  • non-fixed combination means that the active ingredients are administered to the patient in sequence as separate entities simultaneously, without specific time limits or at the same or different time intervals, wherein such administration provides two or more active agents at effective levels of prevention or treatment in the patient.
  • the molecules of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those when they are used alone.
  • fixed combination means that two or more active agents are administered to the patient simultaneously in the form of a single entity.
  • the dosage and/or time interval of the two or more active agents be selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition.
  • Each component can be in the form of a separate formulation, which can be the same or different.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein.
  • This administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as a single capsule with a fixed ratio of active ingredients.
  • this administration includes the co-administration of each active ingredient in a variety of or in separate containers (e.g., tablets, capsules, powders, and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dose before administration.
  • this administration also includes the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating a disorder or condition described herein.
  • mammals include but are not limited to It is limited to domesticated animals (eg, cows, sheep, cats, dogs and horses), primates (eg, humans and non-human primates such as monkeys), rabbits and rodents (eg, mice and rats).
  • domesticated animals eg, cows, sheep, cats, dogs and horses
  • primates eg, humans and non-human primates such as monkeys
  • rabbits and rodents eg, mice and rats.
  • the subject is a human.
  • prevention includes the inhibition of the occurrence or development of a disease or disorder or the symptoms of a specific disease or disorder.
  • a subject with a family history of a disordered glucose metabolism-related disease e.g., diabetes
  • a preventive regimen e.g., a preventive regimen.
  • prevention refers to the administration of a drug before the signs or symptoms of a disordered glucose metabolism-related disease (e.g., diabetes) occur, particularly in a subject with a risk of a disordered glucose metabolism-related disease (e.g., diabetes).
  • the term "effective amount” refers to an amount or dosage of the conjugate/combination of the present invention or a composition or combination thereof, which produces the desired effect in a patient in need of treatment or prevention after administration to the patient in single or multiple doses.
  • the term "therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the conjugates/conjugates of the invention or compositions or combinations thereof are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably achieves at least about 30%, even more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100% inhibition or reduction of a measurable parameter (e.g., glucagon secretion, blood glucose concentration) relative to an untreated individual.
  • a measurable parameter e.g., glucagon secretion, blood glucose concentration
  • prophylactically effective amount refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • the present invention provides a composition comprising any of the conjugate molecules described herein and pharmaceutically acceptable salts thereof, preferably the composition is a pharmaceutical composition or pharmaceutical preparation.
  • the composition further comprises a pharmaceutical excipient.
  • a composition e.g., a pharmaceutical composition, comprises a conjugated molecule of the invention in combination with one or more other therapeutic agents.
  • compositions disclosed in the present invention may further comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories.
  • liquid solutions e.g., injectable solutions and infusible solutions
  • powders or suspensions e.g., liposomes and suppositories.
  • liposomes e.g., liposomes and suppositories.
  • suppositories e.g., suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use.
  • a medicament comprising the conjugated molecules described herein can be prepared by mixing the conjugated molecules 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.
  • the pharmaceutical composition or preparation of the present invention may also include more than one active ingredient, which is required for the specific indication to be treated, preferably with those active ingredients of complementary activities that do not adversely affect each other.
  • active ingredient may also include other therapeutic agents, including chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists), etc.
  • the active ingredient is suitably combined in an amount effective for the intended use.
  • sustained release preparations may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the conjugated molecules, which matrices are in the form of shaped articles, eg films, or microcapsules.
  • 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.
  • Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, so that the dosage unit can be provided according to the dosage regimen or the interval of drug administration.
  • kit of parts of the present invention comprises in the same package:
  • a second container of the pharmaceutical composition containing an additional therapeutic agent A second container of the pharmaceutical composition containing an additional therapeutic agent.
  • the long-acting platform provided in the present application namely the active molecule-fusion protein-Cn conjugate platform, the active molecule-Fc-Cn conjugate platform and the antibody-Cn conjugate platform can be used to effectively prolong the serum half-life of the active molecule.
  • the active molecule is constructed into a conjugated molecule with a structure of "active molecule-Fc-Cn", a conjugated molecule with a structure of "active molecule-fusion protein-Cn”, and a conjugated molecule with a structure of "antibody-Cn” through the method disclosed in the present application, thereby effectively improving the serum half-life of the active molecule, thereby reducing the frequency of administration, reducing the dosage, and saving costs.
  • the present application uses the above-mentioned long-acting platform to prolong the serum half-life of the active molecule without affecting the biological function of the active molecule, and the resulting conjugated molecules with the structure of "active molecule-Fc-Cn", the structure of "active molecule-fusion protein-Cn”, and the structure of "antibody-Cn” have low immunogenicity to the body, effectively avoiding the negative impact of common conjugated molecules that easily cause the body's immune response.
  • the conjugate molecules of "active molecule-Fc-Cn” and “active molecule-fusion protein-Cn” obtained in the present application can be used to prevent or treat a variety of diseases in a subject.
  • a person skilled in the art can easily determine the disease that the conjugate molecule can treat or prevent based on the biological function of the active molecule.
  • the conjugate molecule can be used to effectively treat metabolic diseases and/or disorders, such as diabetes, such as type I diabetes, type II diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity, metabolic syndrome, cardiovascular disease, etc.
  • the conjugated molecule can be used to effectively prevent or treat diseases associated with abnormal PD-1 expression, such as various tumors or cancers, such as melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, Hodgkin's lymphoma, head and neck cancer, ovarian cancer, and brain cancer.
  • diseases associated with abnormal PD-1 expression such as various tumors or cancers, such as melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, Hodgkin's lymphoma, head and neck cancer, ovarian cancer, and brain cancer.
  • the conjugated molecule can be used to effectively prevent or treat diseases associated with abnormal VEGF expression, such as various diseases associated with angiogenesis, for example, ophthalmic diseases, such as wet or neovascular age-related macular degeneration (AMD) and diabetic macular edema (DME); most cancers; and cardiovascular diseases.
  • diseases associated with abnormal VEGF expression such as various diseases associated with angiogenesis, for example, ophthalmic diseases, such as wet or neovascular age-related macular degeneration (AMD) and diabetic macular edema (DME); most cancers; and cardiovascular diseases.
  • AMD neovascular age-related macular degeneration
  • DME diabetic macular edema
  • the present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the conjugate molecule of the present invention or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, a pharmaceutical combination or a pharmaceutical kit.
  • the administration of the conjugated molecules of the present invention may include 1) therapeutic measures that cure, slow down, alleviate the symptoms of the diagnosed pathological condition or disease and/or stop the progression of the diagnosed pathological condition or disease; or 2) preventive or preventive measures that prevent and/or slow the development of the pathological condition or disease.
  • the individual will benefit from the therapeutic measures or preventive measures and show a reduction or improvement in the occurrence, recurrence or development of the disease, disorder, condition, and/or symptom compared to individuals who have not received the treatment.
  • compositions of the present invention can be administered by any suitable method, including parenteral administration, intratumoral administration and intranasal administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • the medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection.
  • Various medication schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration and pulse infusion.
  • the appropriate dosage of the pharmaceutical compositions of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the specific type of active molecule in the conjugated molecule, the severity and course of the disease, the purpose of the treatment, previous therapy, the patient's clinical history and response to the drugs, and the judgment of the attending physician.
  • the present invention also provides the use of the pharmaceutical composition of the present invention in the preparation of a medicament for the aforementioned treatment and prevention methods.
  • AUC Area under the curve CV column volume HSA Human serum albumin PBS Phosphate buffered saline tBu tert-butyl Pbf 2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl Trt Trityl Mmt 4-methoxytriphenyl Mtt methyl trityl 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"-tetramethyluronium hexafluorophosphate HATU 2-(7-Azobenzotriazole)-N,N,N,
  • the fatty acid chain TM1 was prepared according to the following technical route:
  • the fatty acid chain C18 ester (i.e., C18-tert-butyl alcohol ester) was prepared according to the following technical route:
  • the fatty acid chain C16-NHS was prepared according to the following technical route:
  • the fatty acid chain C20-NHS was prepared according to the following technical route:
  • Dulaglutide consists of two identical long chains, one of which has an amino acid sequence as shown below:
  • the amino acid sequence of Fc contained in dulaglutide is as follows:
  • the purified sample was named GLP-1-Fc-TM1 (also called GLP1-Fc-TM1) and replaced into a temporary buffer (25 mM phosphate, 150 mM sodium chloride, pH 7.0) for the next step of detection.
  • GLP-1-Fc-TM1 also called GLP1-Fc-TM1
  • a temporary buffer 25 mM phosphate, 150 mM sodium chloride, pH 7.0
  • the coupling ratio (DAR) of GLP1-Fc and TM1 was detected by HIC-HPLC analysis.
  • the analytical column used was TSKgel Butyl-NPR (4.6 mm*3.5 cm), and the analysis method was a known method, see literature: Dru-to-Antibody Ratio (DAR) and Drug Load Distribution by Hydrophobic Interaction Chromatography and Reversed Phase High-Performance Liquid Chromatography.
  • DAR Dru-to-Antibody Ratio
  • Drug Load Distribution by Hydrophobic Interaction Chromatography and Reversed Phase High-Performance Liquid Chromatography The test results are shown in Figure 1, and the average DAR value of TM1 coupled to the GLP1-Fc protein is: 3.3.
  • GLP1-Fc fusion protein (dulaglutide, homemade), use 15ml 30KD ultrafiltration tube to replace it in reducing buffer (25mM sodium borate, 30mM NaCl, 5mM EDTA pH8.0), replace it four times in total, the final volume is about 3ml, and the protein concentration is detected.
  • reducing buffer 25mM sodium borate, 30mM NaCl, 5mM EDTA pH8.0
  • the final volume is about 3ml
  • the protein concentration is detected.
  • the purified protein was named: GLP-1-Fc-C18-tert-butyl ester (also known as GLP1-Fc-C18 tert-butyl ester), and replaced into a temporary buffer for the next step of detection.
  • GLP-1-Fc-C18-tert-butyl ester also known as GLP1-Fc-C18 tert-butyl ester
  • the coupling ratio (DAR) of GLP1-Fc and C18-tert-butyl ester was detected by HIC-HPLC analysis according to the method of Example 2. The results are shown in FIG8 .
  • the average DAR value is 1.26.
  • GLP-1-Fc fusion protein 4.56 mg was taken and replaced into coupling buffer (0.1 M MOPS, 20 mM Tris, pH 7.5) using a 30KD ultrafiltration tube, with a final volume of 0.9 ml, and the protein concentration was tested. A 6-fold molar amount of C16-NHS was added to the fusion protein sample. After mixing, react at 25°C for 2h with micro shaker. After coupling, purify by cation chromatography immediately, and then replace the sample into temporary buffer with 30KD ultrafiltration tube to detect the sample concentration. The purified product is named: GLP1-Fc-C16-NHS or GLP1-Fc-C16.
  • HIC-HPLC analysis was performed according to the method of Example 2. The results are shown in FIG9 , and the coupling rate of C16-NHS to GLP1-Fc is 83.07%.
  • GLP1-Fc fusion protein Take 4.56mg GLP1-Fc fusion protein, replace it with coupling buffer (0.1M MOPS, 20mM Tris, pH7.5) using a 30KD ultrafiltration tube, the final volume is 0.9ml, and the protein concentration is detected. Add 6 times the molar amount of C20-NHS to the fusion protein sample. After mixing, react at 25°C for 2h and shake on a microshaker. Immediately after coupling, purify it by cationic chromatography, then replace the sample with a 30KD ultrafiltration tube into the temporary buffer and detect the sample concentration. The purified product is named: GLP1-Fc-C20-NHS or GLP1-Fc-C20.
  • HIC-HPLC analysis was performed according to the method of Example 2. The results are shown in FIG10 , and the coupling rate of C20-NHS to GLP1-Fc is 96.38%.
  • the ELISA method was used to detect the activity of samples GLP1-Fc-C16-NHS and GLP1-Fc-C20-NHS obtained by lysine coupling of GLP1-Fc fusion protein with HSA protein.
  • HSA-his Dilute HSA-his to 0.5ug/ml with coating solution (carbonate buffer), coat 8 ELISA plates, and incubate at 4°C overnight. Wash the plates 3 times with PBST (PBS containing 0.05% Tween20), add 200ul/well of blocking solution (1% BSA in PBST solution), and incubate at 37°C for 1h.
  • coating solution carbonate buffer
  • PBST PBS containing 0.05% Tween20
  • blocking solution 1% BSA in PBST solution
  • the plate was washed 6 times with PBST, 100ul/well of newly prepared colorimetric solution (5ml substrate solution, 250ul TMB stock solution, 16ul 0.75 % H2O2 ) was added, and incubated at 37°C for 10min. 50ul/well of stop solution ( 2M H2SO4 ) was added, and OD value was detected at 450nm.
  • HX006 disclosed in CN 104804088 A
  • a conjugate based on the Fc-higher fatty acid chain platform of the present application was constructed, wherein the HX006 antibody is of IgG1 type, and its heavy chain sequence and light chain sequence are shown in the following table.
  • the purified protein was named: HX006-TM1-1 and replaced into the temporary storage buffer for the next step of detection.
  • the coupling ratio (DAR) of HX006 and TM1 in the HX006-TM1-1 molecule was detected by HIC-HPLC analysis according to the method of Example 2.
  • test results are shown in Figure 2.
  • the average DAR value of TM1 coupled to the HX006 antibody is: 3.0.
  • TM1 4 times the molar amount of TM1 was added to the antibody sample, mixed and reacted at 25°C for 2 hours with shaking on a microshaker. After coupling, purification was performed immediately by cationic chromatography.
  • the purified protein was named: HX006-TM1-2 and replaced into the temporary storage buffer for the next step of detection.
  • the coupling ratio (DAR) of HX006 and TM1 in the HX006-TM1-2 molecule was determined by HIC-HPLC analysis according to the method of Example 2.
  • HX006 antibody protein Take 4.3mg HX006 antibody protein and replace it into reducing buffer (25mM sodium borate, 30mM NaCl, 5mM EDTA pH8.0) using a 15ml 30KD ultrafiltration tube. Replace it four times in total, and the final volume is about 2ml. Then test the protein concentration. Add 3 times the molar number of TCEP to the antibody and bathe it in 25°C water for 2h; then use a 15ml 30KD ultrafiltration tube to replace it into coupling buffer (50mM Tris, 150mM NaCl, 5mM EDTA, pH7.5) for a total of 4 times. Then test the sample protein concentration and the number of free thiol groups.
  • reducing buffer 25mM sodium borate, 30mM NaCl, 5mM EDTA pH8.0
  • the purified protein was named: HX006-C18-tert-butyl ester, also known as HX006-C18, and was replaced into the temporary buffer. In the flushing liquid, in preparation for the next step of testing.
  • the coupling ratio (DAR) of HX006 and C18-tert-butyl ester was detected by HIC-HPLC analysis according to the method of Example 2. The results are shown in FIG15 , and the average DAR value is: 2.95.
  • HX006 antibody protein Take 4mg HX006 antibody protein, replace it with coupling buffer (0.1M MOPS, 20mM Tris, pH7.5) using a 30KD ultrafiltration tube, the final volume is 0.9ml, and the protein concentration is detected.
  • the protein is named: HX006-C16-NHS.
  • HX006 antibody protein Take 4mg HX006 antibody protein, replace it with coupling buffer (0.1M MOPS, 20mM Tris, pH7.5) using a 30KD ultrafiltration tube, the final volume is 0.9ml, and the protein concentration is detected.
  • the protein is named: HX006-C20-NHS.
  • the ELISA method was used to detect the activity of samples HX006-C16-NHS and HX006-C20-NHS obtained by lysine coupling of HX006 antibody protein Fc with HSA protein.
  • HSA-his Dilute HSA-his to 0.5ug/ml with coating solution, coat 8 ELISA plates, and incubate at 4°C overnight. Wash the plate 3 times with PBST (PBS containing 0.05% Tween20), add 200ul/well of blocking solution, and incubate at 37°C for 1h. Wash the plate 4 times with PBST, dilute HX006-C16-NHS and HX006-C20-NHS to 1000nM as the starting concentration with diluent, and then dilute 7 dilutions by 5 times, a total of 8 gradients, and add 100ul/well to the wells of the ELISA plate, and incubate at 37°C for 1h.
  • PBST PBS containing 0.05% Tween20
  • the anti-PD-1 antibody H8L2 (referred to as HX008 in this application) disclosed in the CN108299560A patent was used as an example to construct a conjugate based on the Fc-higher fatty acid chain platform of this application, wherein the HX008 antibody is of IgG4 type and its sequence is The columns are as follows:
  • the heavy chain sequence of the HX008 antibody is:
  • the heavy chain variable region sequence of the HX008 antibody is:
  • the Fc region sequence is:
  • the light chain variable region sequence of the HX008 antibody is:
  • the light chain sequence of the HX008 antibody is:
  • the purified protein was named: HX008-TM1-2 and replaced into the temporary storage buffer for the next step of detection.
  • the coupling ratio (DAR) of HX008 and TM1 in the HX008-TM1-2 molecule was determined by HIC-HPLC analysis according to the method of Example 2.
  • Example 14 Sample preparation of HX008 antibody coupled to TM1
  • TM1 5 times the molar amount of TM1 was added to the fusion protein sample, mixed and reacted at 25°C for 2 hours with shaking on a microshaker. After coupling, it was immediately purified by cationic chromatography.
  • the purified protein was named: HX008-TM1-3 and replaced into the temporary storage buffer for the next step of detection.
  • the coupling ratio (DAR) of HX008 and TM1 in the HX008-TM1-3 molecule was detected by HIC-HPLC analysis according to the method of Example 2.
  • the fatty acid chain contained in TM1 can bind to serum albumin (HSA).
  • HSA serum albumin
  • the ELISA method was used to detect the binding of the fusion GLP-1-Fc-TM1 to HSA.
  • HSA-his was diluted to 0.5ug/ml with a coating solution (carbonate buffer), coated on 3 ELISA plates, and incubated at 4°C overnight.
  • the plate was washed 3 times with PBST (PBS solution containing 0.05% Tween20), and 200ul/well was added to the blocking solution (1% BSA in PBST solution), and incubated at 37°C for 1h.
  • PBST PBS solution containing 0.05% Tween20
  • the plate was washed 4 times with PBST, and the GLP-1-Fc-TM1 sample was diluted to 1000nM as the starting concentration with a diluent (1 ⁇ BSA in PBST solution), and then 5-fold gradient dilution was performed to 7 dilutions, a total of 8 gradients, and 100ul/well was added to the wells of the ELISA plate, respectively, and incubated at 37°C for 1h. Wash the plate 5 times with PBST, add 100ul/well of HRP-Goat anti-Huamn 10000X working solution, and incubate at 37°C for 1h.
  • This example uses the ELISA method to detect the binding of the fusion GLP-1-Fc-C18-tert-butyl ester to HSA.
  • HSA-his was diluted to 0.5ug/ml with the coating solution, and three ELISA plates were coated at 4°C overnight. The plate was washed 3 times with PBST (PBS solution containing 0.05% Tween20), and 200ul/well of the blocking solution was added, and incubated at 37°C for 1h.
  • PBST PBS solution containing 0.05% Tween20
  • the plate was washed 4 times with PBST, and the GLP-1-Fc-C18-tert-butyl ester sample was diluted to 1000nM as the starting concentration with the diluent, and then 5-fold gradient dilution was performed to 7 dilutions, a total of 8 gradients, and 100ul/well was added to the ELISA plate wells, and incubated at 37°C for 1h.
  • the plate was washed 5 times with PBST, and 100ul/well of HRP-Goat anti-Huamn 10000X working solution was added, and incubated at 37°C for 1h.
  • the plate was washed 6 times with PBST, 100ul/well of the newly prepared colorimetric solution was added, and the plate was incubated at 37°C for 10min. 50ul/well of the stop solution (2M H 2 SO 4 ) was added, and the OD value was detected at 450nm.
  • This example uses the ELISA method to detect the binding of the fusion HX006-TM1 to HSA.
  • HSA-his was diluted to 0.5ug/ml with the coating solution, and 8 ELISA plates were coated at 4°C overnight. The plate was washed 3 times with PBST (0.05% Tween20inPBS), 200ul/well was added to the blocking solution, and incubated at 37°C for 1h.
  • PBST 0.05% Tween20inPBS
  • the plate was washed 4 times with PBST, and the HX006-TM1 sample was diluted to 1000nM as the starting concentration with the diluent, and then 7 dilutions were diluted 5 times, a total of 8 gradients, and 100ul/well were added to the ELISA plate wells, and incubated at 37°C for 1h.
  • the plate was washed 5 times with PBST, and HRP-Goat anti-Huamn 10000X working solution was added to 100ul/well, and incubated at 37°C for 1h.
  • the plate was washed 6 times with PBST, and 100ul/well was added to the newly prepared color development solution, and incubated at 37°C for 10min. 50ul/well of stop solution (2M H 2 SO 4 ) was added, and the OD value was detected at 450nm.
  • the ELISA method was used to detect the binding of the fusion HX006-C18-tert-butyl ester to HSA.
  • HSA-his was diluted to 0.5ug/ml with coating solution, and 8 ELISA plates were coated at 4°C overnight. The plates were washed 3 times with PBST (0.05% Tween20inPBS), 200ul/well of blocking solution was added, and incubated at 37°C for 1h.
  • the plates were washed 4 times with PBST, and the HX006-TM1 sample was diluted to 1000nM as the starting concentration with diluent, and then 7 dilutions were made by 5-fold gradient dilution, for a total of 8 gradients, and 100ul/well were added to the wells of the ELISA plate, and incubated at 37°C for 1h.
  • the plates were washed 5 times with PBST, and 100ul/well of HRP-Goat anti-Huamn 10000X was added.
  • the plate was washed 6 times with PBST, and 100ul/well of the newly prepared color developing solution was added, and the plate was incubated at 37°C for 10min. 50ul/well of the stop solution (2M H 2 SO 4 ) was added, and the OD value was detected at 450nm.
  • This example uses ELISA to detect the binding of the fusion HX008-TM1 to HSA.
  • the specific steps are shown in Example 15, except that the test sample is replaced with the HX008-TM1-3 sample.
  • the results are shown in Figure 7.
  • the sample HX008-TM1 coupled with TM1 can bind to HSA-his more strongly, and the binding activity increases with the increase of DAR.
  • Example 20 Effect of GLP-1-Fc-TM1 on the activity of HEK293-CRE-Luc-GLP1R cell line
  • HEK-293 cells HEK293-CRE-Luc-GLP1R, from Nanjing GenScript Biotechnology Co., Ltd.
  • GLP-1R human GLP-1 receptor
  • CRE4-luciferase CRE4-luciferase
  • the biological activity was detected by using the method that GLP-1-Fc can specifically bind to GLP-1R to produce cAMP, thereby activating the reporter gene, thereby evaluating the ability of test substance I (GLP-1-Fc-TM1) to activate human GLP-1 receptor.
  • HEK293-CRE-Luc-GLP1R was revived, it was cultured in DMEM (containing 10% FBS, 400 ⁇ g/ml G418, 200 ⁇ g/ml HygromycinB) at 37°C, 5% CO 2 in an incubator. Collect cells in logarithmic growth phase, count, resuspend cells with complete medium, adjust cell concentration to appropriate concentration, inoculate 384-well plates at 2 ⁇ 10 3 cells/well, and add 20 ⁇ l cell suspension to each well. Incubate cells in a 37°C, 100% relative humidity, 5% CO 2 incubator overnight.
  • DMEM containing 10% FBS, 400 ⁇ g/ml G418, 200 ⁇ g/ml HygromycinB
  • test compound Dilute the test compound to the corresponding action concentration set with culture medium, 30 ⁇ l test solution per well (the final action concentration and dilution gradient of the test compound depend on specific requirements), set 9 concentration gradients in total, and 2 replicates for each concentration.
  • Semaglutide commercially available
  • Dulaglutide homemade were used as positive controls.
  • Cells were placed in a 37°C, 100% relative humidity, 5% CO 2 incubator for 6 hours. Discard the supernatant, add 40 ⁇ L/well of One-Glo detection solution, shake for 5 minutes, and measure luminescience (RLU) on an ENVISION 2104 microplate reader.
  • test substance I (GLP-1-Fc-TM1) was 8.4 ⁇ 10 -3 nM
  • the EC 50 of Dulaglutide was 4.4 ⁇ 10 -3 nM
  • the EC 50 of Semaglutide was 4.7 ⁇ 10 -3 nM.
  • the EC 50 of test substance I (GLP-1-Fc-TM1) was at the same order of magnitude as that of Dulaglutide and Semaglutide, indicating that test substance I (GLP-1-Fc-TM1) had stronger biological activity.
  • Example 21 Determination of the interaction between the test substance and human serum albumin by surface plasmon resonance (SPR) technology
  • HEPES N-(2-hydroxyethyl)piperazine-N-2-sulfonic acid
  • NaCl sodium chloride
  • EDTA 3mM ethylenediaminetetraacetic acid
  • Tween-20 0.005% Tween-20
  • pH 7.4 pH 7.4
  • Mouse anti-His antibody was diluted to 50 ⁇ g/mL with a fixation reagent (10mM sodium acetate, pH 4.5).
  • the surface of the CM5 chip was activated with 400mM EDC and 100mM NHS at a flow rate of 10 ⁇ L/min for 420s.
  • HSA Human serum albumin
  • the ligands (test substances GLP-1-Fc, GLP-1-Fc-TM1, HX006-DS, HX006-TM1-1, HX006-TM1-2, HX008-DS, HX008-TM1-2, HX008-TM1-3) were diluted to 5 ⁇ g/mL with the running reagent and injected into the His capture chip experimental channel (Fc2) at a flow rate of 10 ⁇ L/min, about 400 RU.
  • the reference channel (Fc1) does not need to capture the ligand (test substance).
  • Human serum albumin (HSA) was diluted 2-fold with the running reagent for a total of 7 concentrations.
  • the diluted human serum albumin (HSA) was injected into the experimental channel and the reference channel at a flow rate of 30 ⁇ L/min, and the binding (120s) and dissociation (300s) times were corresponding. The binding and dissociation steps were all performed in the running reagent. After each concentration analysis, the chip needs to be regenerated with glycine hydrochloride with a pH of 1.5 at a flow rate of 20 ⁇ L/min for 30s to wash off the ligand and undissociated analyte. When performing the next concentration analysis, the experimental channel needs to recapture the same amount of ligand (test substance). The KD value of each sample was calculated using the Biacore 8K analysis software Biacore Insight Evaluation Software. The reference channel (Fc1) was used for background subtraction.
  • the affinity of the test substances to human serum albumin detected by Biacore 8K was: the Kd values of unmodified GLP-1-Fc, HX006-DS, and HX008-DS were 0, i.e., no binding; the Kd values of GLP-1-Fc-TM1, HX006-TM1-1, HX006-TM1-2, HX008-TM1-2, and HX008-TM1-3 were 1.01 ⁇ 10 -2 nM, 1.14 ⁇ 10 -3 nM, 6.92 ⁇ 10 -4 nM, 4.87 ⁇ 10 -3 nM, and 4.05 ⁇ 10 -3 nM, respectively, showing strong affinity to human serum albumin.
  • Example 22 Study on the efficacy of GLP-1-Fc-TM1 in type II diabetic db/db mice after multiple administration
  • mice 12 m/m mice (normal control) and 60 male db/db mice were purchased and adaptively raised. After the blood sugar of db/db mice reached the standard (about 8-10 weeks of age), 10 m/m mice and 50 db/db mice were selected and grouped into normal control group (m/m mice), model group, positive control group (Dulaglutide), low-dose group of the test substance, medium-dose group of the test substance, and high-dose group of the test substance, with 10 mice in each group. After grouping, drug administration began.
  • the positive control group was given 10nmol/kg/dose of TRULICITY (Dulaglutide), subcutaneously injected twice a week for 4 weeks; the low-, medium-, and high-dose groups of the test substance were given 3, 10, and 30nmol/kg/dose of the test substance I (GLP-1-Fc-TM1), subcutaneously injected twice a week for 4 weeks; the db/db model group and normal control group were given the corresponding solvent (PBS), subcutaneously injected twice a week for 4 weeks.
  • the administration volume for all groups was 5 ml/kg/dose. Clinical observation was conducted once a day. Serum insulin and glycosylated hemoglobin were tested at the end of administration, followed by OGTT test.
  • test substance I (GLP-1-Fc-TM1) was better in reducing glycated hemoglobin, random blood glucose and fasting blood glucose, stimulating serum insulin secretion, and reducing body weight and food intake.
  • the effects of low, medium and high doses of the test substance I (GLP-1-Fc-TM1) showed a therapeutic effect relationship, indicating that the test substance I (GLP-1-Fc-TM1) had a significant hypoglycemic effect.
  • the test substance GLP-1-Fc-TM1 can dose-dependently reduce the 4h fasting blood glucose of db/db mice ( Figure 19-1), random blood glucose after the first and last doses ( Figures 19-2 and 19-3), and blood glucose AUC 0-180min of the OGTT test after the last dose ( Figures 19-4 and 19-5), reduce the content of glycated hemoglobin in db/db mice ( Figure 19-6), increase the insulin level of db/db mice ( Figure 19-7), and reduce the average daily food intake of db/db mice ( Figure 19-8); at the same dose, the effect of the test substance GLP-1-Fc-TM1 on the above indicators is better than that of the positive control Dulaglutide.
  • mice After 72 male C57BL/6J mice were purchased, except for 12 normal control mice (conventional feed), the remaining 60 mice were fed with high-fat feed to prepare DIO model mice, free to eat, and fed continuously for about 10 weeks. During the modeling period, the food intake was monitored twice a week, and the body weight was monitored twice a week. When the body weight exceeded 20% of the normal mice, the DIO model standard was reached, and then grouping began. Select 10 qualified normal control mice (conventional feed) and 50 DIO model mice for grouping, namely normal control group (conventional feed mice), model group, positive control group (dulaglutide), test low-dose group, test medium-dose group, and test high-dose group, 10 in each group. After grouping, the drug administration began.
  • the positive control group was given a dose of 10nmol/kg/dose of TRULICITY (dulaglutide), subcutaneously injected twice a week for 4 weeks; the low, medium and high dose groups of the test substance were given 3, 10, and 30nmol/kg/dose of the test substance I (GLP-1-Fc-TM1), subcutaneously injected twice a week for 4 weeks; the obese DIO model group and the normal control group were given the corresponding solvent (PBS), subcutaneously injected twice a week for 4 weeks.
  • the administration volume of all groups was 5ml/kg/dose. All DIO mice continued to be fed with high-fat diet during the administration period for the entire experimental period.
  • test substance I (GLP-1-Fc-TM1) was better in reducing fasting blood sugar, improving four blood lipids and liver biochemical indicators and liver pathology, stimulating serum insulin secretion, and reducing body weight and food intake.
  • the effects of low, medium and high doses of the test substance I (GLP-1-Fc-TM1) showed a therapeutic effect relationship.
  • Test substance I (GLP-1-Fc-TM1) has obvious hypoglycemic and weight-loss effects.
  • GLP-1-Fc-TM1 can reduce the body weight of DIO mice in a dose-dependent manner ( Figure 20-1), reduce the food intake of DIO mice (Figure 20-2), reduce their body fat content (Figure 20-3), and reduce fasting blood sugar (Figure 20-4); in addition, the test substance GLP-1-Fc-TM1 can also reduce blood lipid content ( Figure 20-5), serum liver function index ALT and AST levels ( Figure 20-6) in a dose-dependent manner, and improve the ballooning and fatty degeneration of liver tissue in DIO mice ( Figure 20-7). At the same dose, the test substance GLP-1-Fc-TM1 has a better effect on improving liver fatty lesions than the positive control Dulaglutide.
  • 58 male SD rats (6-8 weeks, weighing 180-220g) were purchased and adaptively raised for one week, and then 50 animals were selected for grouping, including vehicle control group, positive control group (Dulaglutide), low-dose test group, medium-dose test group, and high-dose test group, with 10 rats in each group. After grouping, drug administration began.
  • the positive control group was given a 10nmol/kg dose of TRULICITY (Dulaglutide), subcutaneously injected, and administered once; the low-, medium-, and high-dose test groups were given 3, 10, and 30nmol/kg doses of test substance I (GLP-1-Fc-TM1), subcutaneously injected, and administered once; the vehicle control group was given the corresponding vehicle (PBS), subcutaneously injected, and administered once.
  • the administration volume for all groups was 5ml/kg/dose. Rats were fasted for 16 hours (but not water deprivation) and then intravenously injected with glucose (0.5 g/kg). Blood samples were collected at 2, 4, 6, 10, 20 and 30 minutes after glucose injection to detect blood glucose concentration and insulin levels, and AUC was calculated.
  • test substance I (GLP-1-Fc-TM1) had equivalent or no significant difference in reducing fasting blood glucose and stimulating serum insulin secretion; and the effects of low, medium and high doses of the test substance I (GLP-1-Fc-TM1) showed a certain dose-effect relationship.
  • a single administration of the test substance GLP-1-Fc-TM1 at a dose of 3, 10, and 30 nmol/kg could dose-dependently reduce blood glucose AUC 0-30min in SD rats at 24h and 72h after administration ( Figure 21-1, Figure 21-2, Figure 21-3, and Figure 21-4) and increase serum insulin AUC 0-30min ( Figure 21-5, Figure 21-6, Figure 21-7, and Figure 21-8).
  • Example 25 Pharmacokinetic study of the test substance after a single subcutaneous injection in SD rats
  • test substance I GLP-1-Fc-TM1
  • positive control Dulaglutide, homemade
  • test substance I GLP-1-Fc-TM1
  • C max 225 and 191 ng/mL
  • T 1/2 45 and 28 h
  • AUC 0-t 15700 and 7540 h*ng/mL
  • AUC 0- ⁇ 16100 and 7840 h*ng/mL
  • Vz_ F_obs were 428 and 533 ml/kg
  • Cl _F_obs were 6.42 and 13.3 mL/h/kg, respectively
  • the MRT (0-t) were 70 and 39 h, respectively
  • MRT (0- ⁇ ) were 77 and 44 h, respectively.
  • test substance I (GLP-1-Fc-TM1) were 1.61, 1.18, 2.08, 2.05, 0.80, 0.48, 1.79 and 1.75 times that of dulaglutide, respectively, indicating that test substance I (GLP-1-Fc-TM1) can prolong the half-life by reducing the clearance rate.
  • Example 26 Pharmacokinetic study of a single subcutaneous injection of the test substance in cynomolgus monkeys
  • test substance I (GLP-1-Fc-TM1) were 2.00, 1.26, 2.03, 2.01, 2.76, 0.70, 0.38, 1.29 and 2.12 times that of dulaglutide, respectively, indicating that test substance I (GLP-1-Fc-TM1) can prolong the half-life by reducing the clearance rate.

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Abstract

本发明涉及用于改善药物活性分子半衰期的超长效平台，其包含免疫球蛋白Fc和高级脂肪酸链。本发明还涉及该缀合物平台的制备、含有所述缀合物的组合物及其治疗应用。本发明具体地涉及一种结构为"活性分子-Fc-Cn"的缀合分子，其中活性分子选自对机体有益的任何分子，Fc是免疫球蛋白IgG Fc，Cn为包含C14-24脂肪酸链的修饰部分，以及涉及该缀合分子的制备、含有所述缀合物的组合物及其治疗应用。

Description

包含Fc-高级脂肪酸链的超长效平台 技术领域
本发明涉及用于改善药物活性分子半衰期的超长效平台，其包含免疫球蛋白Fc和高级脂肪酸链。本发明还涉及该缀合物平台的制备、含有所述缀合物的组合物及其治疗应用。
背景技术
多肽类药物由于比化学药物更接近于机体内源性物质，具有毒副作用小、疗效稳定等优点，因而被广泛应用于诸如癌症、心血管疾病、自身免疫疾病等多种临床治疗中，具有广泛的应用前景，也备受制药厂家、科研工作者的青睐。然而，经典的多肽类药物对体内蛋白酶耐受性低，稳定性较差,进入体内将很快被降解，由此具有较短的血浆半衰期；大多生物活性肽类物质的生物利用度较差,无法口服，这些问题极大地阻碍了多肽类药物的临床应用。此外，由于靶组织暴露有限，许多蛋白质多肽类药物需要更频繁的给药次数来维持临床有效的药物浓度。
为了解决这些问题，目前通过对多肽类药物进行修饰加以克服。对多肽类药物的修饰可以分为两类，一类是对肽链骨架进行改造；第二类是在保持多肽骨架不变的基础上，通过引入其他基团进行结构优化和性能改造,包括聚乙二醇修饰、糖基化修饰、蛋白融合策略、高级脂肪酸修饰、定点突变、胆固醇修饰等。
生物活性分子与免疫球蛋白的Fc区融合形成Fc融合蛋白，其结合了生物活性分子的有益药理特性和Fc区的额外特性，从而增加生理活性分子的血清半衰期并由此减少药物施用的频率。目前，Fc区与作为配体或受体的活性肽或与细胞外结构域(ECD)的融合极大地提高了活性蛋白质药物的临床潜力。具体而言，对于分子量小于60kDa的产品，其可以容易地被肾脏清除，由此血清半衰期短，通过与Fc区偶联或融合，其大小得到增加，超过肾脏过滤的阈值，从而其循环时间得到增加。当Fc融合蛋白被内皮细胞摄取后而进入酸化内体，Fc融合蛋白通过Fc与内体中的FcRn结合而免遭溶酶体降解，当再循环内体转运到细胞表面时，在中性及弱碱性条件下，Fc与FcRn解离，将Fc-融合蛋白释放回血液循环中，从而延长Fc-融合蛋白的半衰期，使得靶组织接触Fc-融合蛋白的药理活性部分的时间更长，从而提高了后者的治疗潜力。
Fc融合蛋白代表了一类成功的生物制药产品，已经有13种药物在欧盟和美国获得批准，还有3种依那西普的生物仿制药。可能的生物活性分子有很大的多样性，包括自然受体的胞外结构域、功能活性肽、重组酶和作为细胞因子陷阱的基因工程结合结构。大多数Fc融合蛋白是由生物活性分子与Fc结构域的N端融合产生的。IgG-CH3结构域的强相互作用创造了稳定的Fc结构，并允许更复杂的结构融合到柔性铰链区、二硫键等位置。礼来公司将GLP-1与IgG4 (Fc)融合而开发的降糖药物杜拉鲁肽(dulaglutide)，由于分子尺寸显著增大,降低了肾对GLP-1的清除率,从而具有延长的生物半衰期。
脂肪酸是构成人体脂肪、类脂和细胞膜磷脂的重要成分，且其作为内源性成分而具有低的免疫原性，因此也被用于对生物活性分子(例如多肽药物)进行修饰。当利用脂肪酸对多肽药物的特定氨基酸残基进行修饰时，脂肪酸通过可逆地结合血清白蛋白而增加多肽药物的血清半衰期。但是脂肪酸修饰也有自身的局限性：例如容易产生脂肪酸的非特异性修饰位点产物；由于脂肪酸与血清白蛋白的结合是可逆的，因此与白蛋白解离的多肽药物容易通过肾脏被消除，因此影响或降低脂肪酸修饰的多肽药物的半衰期。
在保留多肽药物疗效的前提下，提高多肽药物的血清半衰期并同时降低药物的免疫原性一直是科研和制药领域的需求，本申请通过提供创新性的改善药物活性分子的超长效平台，满足了这一需求。
发明内容
本发明提供了具有如下结构的可以改善药物活性分子半衰期的超长效平台：活性分子-融合蛋白-Cn缀合物平台、活性分子-Fc-Cn缀合物平台和抗体-Cn缀合物平台，其中Cn表示包含n＝14-24的脂肪酸链的修饰部分，Fc为免疫球蛋白分子的Fc区。本申请提供的超长效平台具有如下优点：
1.缀合分子中的Fc组分和高级脂肪酸链组分都可以与FcRn直接或者间接结合，Fc与FcRn直接结合，而高级脂肪酸与FcRn借助血清白蛋白间接结合，其中由于Fc和血清白蛋白分别结合FcRn的不同位点，因而相互不会干扰，从而相较于Fc单独或者高级脂肪酸单独为与其缀合的活性分子提供更高的半衰期；
2.融合蛋白、抗体、Fc和高级脂肪酸都是体内的内源性物质，都具有低免疫原性，可以降低缀合分子对机体的异源性，降低产生相应抗体的可能；
3.缀合分子可以增加所包含的活性分子的大小，降低其肾排泄率,从而可以延长活性分子在体内的循环时间；
4.由于脂肪酸链的疏水性，有助于提高缀合分子的膜通透性，从而使更多的缀合分子进入循环系统；
5.缀合分子中的Fc通过其CH2-3结构域可以与同源的Fc片段同源配对，因此增加稳定性，提高局部缀合分子/活性分子的浓度。
第一方面，本发明提供了一种结构为“活性分子-Fc-Cn”的缀合分子、结构为“活性分子 -融合蛋白-Cn”的缀合分子、结构为“抗体-Cn”的缀合分子，其中活性分子选自对机体有益的任何分子，Fc衍生自免疫球蛋白IgG的重链恒定区，Cn为包含C14-24脂肪酸链的修饰部分。
在一些实施方案中，本发明公开的结构为“活性分子-Fc-Cn”的缀合分子、结构为“活性分子-融合蛋白-Cn”的缀合分子、结构为“抗体-Cn”的缀合分子中的Cn具有以下式(I)的结构：
-Z-Y                 (I)，
其中
Z具有以下结构：
-Z1-Z2-Z3-Z4-，
其中Z1为Fc中的硫原子或氮原子或氧原子，
Z2为-C(＝O)-或5-10元杂环基，优选地含1或2个选自N、S和O的杂原子；
Z3选自键、-C(＝O)-、-C1-C10亚烷基-C(＝O)-、-C3-C10亚炔基-C(＝O)-、-C3-C10亚烯基-C(＝O)-、-C1-C10亚杂烷基-C(＝O)-、-C3-C8亚环烷基-C(＝O)-、-O-C1-C8亚烷基-C(＝O)-、-亚芳基-C(＝O)-、-C1-C10亚烷基-亚芳基-C(＝O)-、-亚芳基-C1-C10亚烷基-C(＝O)-、-C1-C10亚烷基-C3-C8亚环烷基-C(＝O)-、-C3-C8亚环烷基-C1-C10亚烷基-C(＝O)-、-C3-C8亚杂环基-C(＝O)-、-C1-C10亚烷基-C3-C8亚杂环基-C(＝O)-、-C3-C8亚杂环基-C1-C10亚烷基-C(＝O)-，其中所述的亚烷基、亚炔基、亚烯基、亚杂烷基、亚环烷基、亚芳基和亚杂环基可任选地被取代；
Z4是键或是下式表示的PEG单元，
其中，R1选自C1-4亚烷基、-NH-、-NH-C1-4亚烷基-、-NH-C1-4亚烷基-杂芳基-，其中杂芳基为5元或6元的含氮杂芳基；R2为-C(＝O)-、-C1-4亚烷基、-C1-4亚烷基-C(＝O)-、-C1-4亚烷基-NH-C(＝O)-(CH2OCH2)p-C1-4亚烷基-、-C1-4亚烷基-C(＝O)-NH-(CH2OCH2)p-C1-4亚烷基-，其中m为2-6的整数，p为1-3的整数，
Y是
其中Y通过X与Z4连接，k是10-30的整数，
其中R独立地表示氢、C1-6烷基、C1-6氨基烷基、C1-6卤代烷基、C1-6羟基烷基。
在一些实施方案中，Z2为亚马来酰亚胺基，其中左侧波浪线表示与Z1连接的位置；右侧波浪线表示与Z3连接的位置。
在一些实施方案中，Z3为-C1-C10亚烷基-C(＝O)-，其中所述的亚烷基任选地被取代并且其中Z3通过-C-(＝O)-与Z4连接。
在一些优选实施方案中，Z2为亚马来酰亚胺基，Z3为-C1-6亚烷基-C(＝O)-。
在一些实施方案中，Z4为键，Z3与式(I)中的Y直接连接。
在一些实施方案中，Z4是下式表示的PEG单元，
其中，R1选自-NH-和-NH-C1-4亚烷基-；R2为-C1-4亚烷基或-C1-4亚烷基-NH-C(＝O)-(CH2OCH2)p-C1-4亚烷基-，其中m为2-6的整数，p为1-3的整数。
在一些实施方案中，Z4为包含2-6个PEG的单元。在一些实施方案中，Z4为
其中m＝1-4，左侧的星号表示与Z3连接的位置；右侧的星号表示与式II中的Y连接的位置。
在一些实施方案中，本发明式(I)中的Z具有以下结构：
其中RE是氢、C1-6烷基、C1-6氨基烷基、C1-6卤代烷基、C1-6羟基烷基，其中y＝0-4,m＝1-4，其中左侧的星号表示与Ab连接的位置，右侧的星号表示与Y连接的位置。
Y是
其中Y通过X与Z4连接，并且X是-NH-(C＝O)-或-(C＝O)-NH-，
k是10-30的整数，
其中R独立地表示氢、C1-6烷基、C1-6氨基烷基、C1-6卤代烷基、C1-6羟基烷基。
在一个具体的实施方案中，Cn选自
在一个实施方案中，Fc衍生自IgG1、IgG2、IgG3或IgG4的重链恒定区。在一个具体实施方案中，Fc衍生自IgG1或IgG4的重链恒定区。在又一个实施方案中，Fc区可进一步包括铰链区。在一个具体实施方案中，Fc包含氨基酸修饰。在一个具体实施方案中，对Fc区的修饰是对位置254，308和434处的修饰(根据EU编号)。在另一个具体实施方案中，对Fc区的修饰是将第254、308、434位氨基酸分别替换为Thr、Pro和Ala。在一个具体的实施方案中， 对Fc区的修饰是对位置228，234，235和/或447处的修饰，例如修饰S228P，F234A，L235A或修饰S228P，F234A，L235A和447缺失。在一个具体实施方案中，Fc区选自SEQ ID NO:10，15或16的序列。
在一个实施方案中，活性分子是肽类活性分子。在一个实施方案中，肽类活性分子直接或者通过肽接头与抗体、融合蛋白或Fc区融合在一起。在一个具体实施方案中将肽类活性分子的C端和抗体、融合蛋白或Fc区的N端融合到一起。备选地，将肽类活性分子的N端和抗体、融合蛋白或Fc区的C端融合到一起。在又一个具体实施方案中，肽类活性分子以单体形式连接到抗体、融合蛋白或Fc区。在又一个具体实施方案中，肽类活性分子以多聚体形式连接到抗体、融合蛋白或Fc区。
在一个实施方案中，活性分子选自酶、酶抑制剂、抗原、抗体或抗体片段、激素、胰高血糖素样肽-1(GLP-1)、胰高血糖素、干扰素、细胞因子、生长因子和/或分化因子、参与细胞运动或迁移的因子、参与骨组织发生/再吸收的因子、趋化因子、血浆或间质粘连分子或细胞外基质、杀细菌或抗真菌因子等。
在一个具体的实施方案中，活性分子选自GLP-1、抗体Fab片段、抗体F(ab’)2片段。在另一个具体的实施方案中，活性分子选自抗PD-1的抗体Fab片段、抗PD-1的抗体F(ab’)2片段、抗VEGF的抗体Fab片段、抗VEGF的抗体F(ab’)2片段。
在一个实施方案中，肽接头包含氨基酸序列(G4S)n，其中n是等于或大于1的整数。在一个具体实施方案中，肽接头包括(G4S)3、(G4S)4、(G4S)6、GS(G4S)4、DAAALEAAALDAAAREAAARDAAAL、NVDHLPSNTLVDLA，(G3S)2、(G4S)2、(G3S)3、(G4S)3、(G3S)4、(G4S)4、(G3S)5、(G4S)5、(G3S)6、(G4S)6、GGG、DGGGS、TGEKP、GGRR、EGKSSGSGSESKVD、KESGSVSSEQLAQFRSLD、GGRRGGGS、LRQRDGERP、LRQKDGGGSERP和GSTSGSGK PGSGEGSTKG。
在一个实施方案中，本申请提供的结构为“活性分子-Fc-Cn”或“抗体-Cn”的缀合分子通过其Fc区同二聚化。
在一个具体的实施方案中，本申请提供一种结构为“活性分子-Fc-Cn”的缀合分子，其中Fc选自IgG1或IgG4，Cn包含16、18或20碳的脂肪酸链。在一个优选的实施方案中，所述Fc包含修饰，例如本申请提及的修饰。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请提供一种结构为“抗体-Cn”的缀合分子，其中抗体选自IgG1或IgG4，Cn包含16、18或20碳的脂肪酸链。在一个优选的实施方案中，所述抗体 的Fc包含修饰，例如本申请提及的修饰。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请提供一种结构为“活性分子-Fc-Cn”的缀合分子，其中Fc选自IgG1或IgG4，Cn包含16、18或20碳的脂肪酸链且与Fc的游离巯基偶联/缀合。在一个优选的实施方案中，所述Fc包含修饰，例如本申请提及的修饰。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请提供一种结构为“抗体-Cn”的缀合分子，其中抗体选自IgG1或IgG4，Cn包含16、18或20碳的脂肪酸链且与抗体Fc的游离巯基的硫原子偶联/缀合。在一个优选的实施方案中，所述抗体的Fc包含修饰，例如本申请提及的修饰。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请提供一种结构为“活性分子-融合蛋白-Cn”的缀合分子，其中Cn包含16、18或20碳的脂肪酸链且与融合蛋白的游离巯基的硫原子偶联/缀合。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请提供一种结构为“活性分子-IgG4 Fc-Cn”的缀合分子，其中Cn包含16、18或20碳的脂肪酸链且与IgG4 Fc的游离巯基偶联/缀合。在一个优选的实施方案中，所述IgG4 Fc包含修饰，例如本申请提及的修饰。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请提供一种结构为“抗体-Cn”的缀合分子，其中Cn包含16、18或20碳的脂肪酸链且与IgG4抗体的游离巯基的硫原子偶联/缀合。在一个优选的实施方案中，所述IgG4抗体的Fc包含修饰，例如本申请提及的修饰。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请提供一种结构为“活性分子-融合蛋白-Cn”的缀合分子，其中Cn包含16、18或20碳的脂肪酸链且与融合蛋白的游离巯基的硫原子偶联/缀合。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请提供一种结构为“活性分子-IgG1 Fc-Cn”的缀合分子，其中Cn包含16、18或20碳的脂肪酸链且与IgG1 Fc的游离巯基的硫原子偶联/缀合。在一个优选的实施方案中，所述IgG1 Fc包含修饰，例如本申请提及的修饰。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请提供一种结构为“抗体-Cn”的缀合分子，其中Cn包含16、18或20碳的脂肪酸链且与IgG1抗体的游离巯基的硫原子偶联/缀合。在一个优选的实施方案中，所述IgG1抗体的Fc包含修饰，例如本申请提及的修饰。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请提供一种结构为“活性分子-融合蛋白-Cn”的缀合分子，其中Cn包含16、18或20碳的脂肪酸链且与融合蛋白的游离巯基的硫原子偶联/缀合。在一个具体的实施方案中，Cn包含16碳的脂肪酸链，在另一个具体的实施方案中，Cn包含18碳的脂肪酸链，在再一个具体的实施方案中，Cn包含20碳的脂肪酸链。
在一个具体的实施方案中，本申请所述结构为“活性分子-Fc-Cn”的缀合分子是GLP-1-Fc-Cn缀合分子，其中所述GLP-1为现有技术中已知的任何有活性的GLP-1。在一个实施方案中，Fc选自IgG1的Fc或IgG4的Fc。在一个具体实施方案中，缀合分子GLP-1-Fc-Cn中的Cn包含16、18或20碳的脂肪酸链且与Fc的游离巯基的硫原子或Fc的氮原子偶联/缀合。在一个具体实施方案中，缀合分子GLP-1-Fc-Cn中的Cn选自TM1,C18-叔丁醇酯，C16-NHS或C20-NHS。在一个优选的技术方案中，缀合分子GLP-1-Fc-Cn是GLP-1-IgG4 Fc-TM1、GLP-1-IgG4 Fc-C18叔丁醇脂、GLP-1-IgG4 Fc-C16-NHS、GLP1-IgG4 Fc-C20-NHS。在一个实施方案中，Fc包含SEQ ID NO:10,15或16所示的序列。在一个更优选的实施方案中，GLP-1-IgG4 Fc-TM1、GLP-1-IgG4 Fc-C18、GLP-1-IgG4 Fc-C16-NHS、GLP1-IgG4 Fc-C20-NHS缀合分子中的GLP-1-IgG4 Fc衍生自杜拉鲁肽，优选是杜拉鲁肽，例如具有如CN1802167中公开的结构，优选具有Gly8-Glu22-Gly36-GLP-1(7-37)-1L-IgG4(S228P，F234A，L235A)的结构。在一个实施方案中，杜拉鲁肽的氨基酸序列如SEQ ID NO:1所示。在一个具体的实施方案中，GLP-1-Fc-Cn缀合分子是杜拉鲁肽-Cn，例如杜拉鲁肽-TM1、杜拉鲁肽-C18叔丁醇脂、杜拉鲁肽-C16-NHS、杜拉鲁肽-C20-NHS。
在一个具体的实施方案中，本申请结构为“活性分子-Fc-Cn”的缀合分子中的活性分子为抗PD-1抗体或其抗原结合片段，所述抗PD-1抗体或其抗原结合片段可以为已知任何的抗PD-1抗体或其抗原结合片段。在一个具体的实施方案中，本申请结构为“抗体-Cn”的缀合分 子中的抗体为抗PD-1抗体或其抗原结合片段，所述抗PD-1抗体或其抗原结合片段可以为已知任何的抗PD-1抗体或其抗原结合片段。在一个优选的实施方案中，Fc选自IgG1 Fc或IgG4 Fc。在一个优选的实施方案中，抗体选自IgG1或IgG4。在一个优选的技术方案中，缀合分子是抗PD-1抗体抗原结合片段-IgG4 Fc-TM1、抗PD-1抗体抗原结合片段-IgG4 Fc-C18、抗PD-1抗体抗原结合片段-IgG4 Fc-C16-NHS、抗PD-1抗体抗原结合片段-IgG4 Fc-C20-NHS。在一个优选的技术方案中，缀合分子是抗PD-1抗体抗原结合片段-IgG1 Fc-TM1、抗PD-1抗体抗原结合片段-IgG1Fc-C18、抗PD-1抗体抗原结合片段-IgG1 Fc-C16-NHS、抗PD-1抗体抗原结合片段-IgG1 Fc-C20-NHS。在一个优选的实施方案中，所述抗PD-1抗体或其抗原结合片段包含SEQ ID NO:9所示的重链可变区和SEQ ID NO:11所示的轻链可变区。在一个优选实施方案中，所述Fc包含SEQ ID NO:10所示的序列。在一个更优选的实施方案中，所述抗PD-1抗体或其抗原结合片段包含SEQ ID NO:8所示的重链和SEQ ID NO:12所示的轻链。在一个更优选的实施方案中，所述Cn选自TM1、C18-叔丁醇脂、C16-NHS、C20-NHS。在一个更优选的实施方案中，所述Cn选自TM1。
在一个具体的实施方案中，本申请结构为“活性分子-Fc-Cn”的缀合分子中的活性分子为抗VEGF抗体或其抗原结合片段，所述抗VEGF抗体或其抗原结合片段可以为已知的任何抗VEGF抗体或其抗原结合片段。在一个具体的实施方案中，本申请结构为“抗体-Cn”的缀合分子中的抗体为抗VEGF抗体或其抗原结合片段，所述抗VEGF抗体或其抗原结合片段可以为已知任何的抗VEGF抗体或其抗原结合片段。在一个优选的实施方案中，Fc选自IgG1 Fc或IgG4 Fc。在一个优选的实施方案中，抗体选自IgG1或IgG4。在一个优选的技术方案中，缀合分子是抗VEGF抗体抗原结合片段-IgG4 Fc-TM1、抗VEGF抗体抗原结合片段-IgG4 Fc-C18、抗VEGF抗体抗原结合片段-IgG4 Fc-C16-NHS、抗VEGF抗体抗原结合片段-IgG4 Fc-C20-NHS。在一个优选的技术方案中，缀合分子是抗VEGF抗体抗原结合片段-IgG1 Fc-TM1、抗VEGF抗体抗原结合片段-IgG1Fc-C18-叔丁醇脂、抗VEGF抗体抗原结合片段-IgG1 Fc-C16-NHS、抗VEGF抗体抗原结合片段-IgG1 Fc-C20-NHS。在一个优选的实施方案中，所述抗VEGF抗体或其抗原结合片段包含SEQ ID NO:2、3和4所示的3个重链CDR和SEQ ID NO:5、6和7所示的3个轻链CDR。在一个优选实施方案中，所述Fc包含SEQ ID NO:15所示的序列。在一个更优选的实施方案中，所述抗VEGF抗体或其抗原结合片段包含SEQ ID NO:13所示的重链和SEQ ID NO:14所示的轻链。在一个更优选的实施方案中，所述Cn选自TM1、C18-叔丁醇脂、C16-NHS、C20-NHS。在一个更优选的实施方案中，所述Cn选自TM1。
第二方面，本申请提供了制备结构为“活性分子-Fc-Cn”的缀合分子的方法，包括(a)将活 性分子多肽连接至免疫球蛋白Fc区以制备“活性分子-Fc”融合物；和(b)将“活性分子-Fc”融合物与含有脂肪酸链的Cn在允许Fc区与Cn缀合的条件下发生偶联反应，产生“活性分子-Fc-Cn”缀合分子。
本申请还提供了制备结构为“抗体-Cn”的缀合分子的方法，包括(a)将抗体与含有脂肪酸链的Cn在允许抗体与Cn缀合的条件下发生偶联反应，产生“抗体-Cn”缀合分子。
本申请还提供了制备结构为“活性分子-融合蛋白-Cn”的缀合分子的方法，包括(a)将活性分子多肽连接至融合蛋白以制备“活性分子-融合蛋白”融合物；和(b)将“活性分子-融合蛋白”融合物与含有脂肪酸链的Cn在允许融合蛋白与Cn缀合的条件下发生偶联反应，产生“活性分子-融合蛋白-Cn”缀合分子。
在一个备选实施方案中，本申请提供了制备结构为“活性分子-Fc-Cn”的缀合分子的方法，其中包括(a)将抗体Fab片段连接至免疫球蛋白Fc区以制备“Fab-Fc”融合物，(b)将“Fab-Fc”融合物在允许Fc区与含有脂肪酸链的Cn缀合的条件下发生偶联反应产生“Fab-Fc-Cn”缀合分子的步骤。在一个实施方案中，所述Fab和Fc衍生自相同或者不同的抗体分子。
在一个备选实施方案中，本申请提供了制备结构为“活性分子-Fc-Cn”的缀合分子的方法，其中包括将全抗体在允许Fc区与含有脂肪酸链的Cn缀合的条件下发生偶联反应产生“活性分子-Fc-Cn”缀合分子的步骤。
在一个备选实施方案中，本申请提供了制备结构为“抗体-Cn”的缀合分子的方法，其中包括将全抗体在允许Fc区与含有脂肪酸链的Cn缀合的条件下发生偶联反应产生“活性分子-Fc-Cn”缀合分子的步骤。
第三方面，本申请提供了包括第一方面所述缀合分子的组合物，例如药物组合物。在一个实施方案中，所述药物组合物还包含可药用载体。
第四方面，本申请提供了一种有效延长活性分子血清半衰期的方法，包括根据第二方面的方法将所述活性分子构建成结构为“活性分子-Fc-Cn”、结构为“抗体-Cn”的缀合分子、结构为“活性分子-融合蛋白-Cn”的缀合分子的缀合分子的步骤，从而有效提高所述活性分子的血清半衰期。
第五方面，本申请提供了第一方面所述的缀合分子，或第三方面所述的组合物在制备用于治疗人类疾病中的用途。
一个实施方案中，本发明提供了第一方面所述的缀合分子，或第三方面所述的组合物用于 治疗。
第六方面，本申请提供了一种治疗人类疾病的方法，包括将本申请有效量的第一方面所述的缀合分子，或第三方面所述的组合物施用给受试者。
附图说明
图1显示了GLP1-Fc-TM1偶联产物的HIC-HPLC结果，上图为未偶联TM1的GLP1-Fc的HIC-HPLC结果，下图为偶联TM1后GLP1-Fc-TM1的HIC-HPLC结果，下图中的“0”表示未偶联TM1的GLP1-Fc，“2”表示偶联2个TM1的GLP1-Fc，“4”表示偶联4个TM1的GLP1-Fc。
图2显示了HX006-TM1-1偶联产物的HIC-HPLC结果，上图为未偶联TM1的HX006的HIC-HPLC结果，下图为偶联TM1后HX006-TM1-1的HIC-HPLC结果，下图中的“0”表示未偶联TM1的HX006，“2”表示偶联2个TM1的HX006，“4”表示偶联4个TM1的HX006，“6”表示偶联6个TM1的HX006。
图3显示了HX006-TM1-2偶联产物的HIC-HPLC结果，图中的“0”表示未偶联TM1的HX006，“2”表示偶联2个TM1的HX006，“4”表示偶联4个TM1的HX006，“6”表示偶联6个TM1的HX006，“8”表示偶联8个TM1的HX006。
图4显示了HX008-TM1偶联产物的HIC-HPLC结果，图4A为未偶联TM1的HX008的HIC-HPLC结果，图4B为偶联TM1后HX008-TM1-2的HIC-HPLC结果，图中的“0”表示未偶联TM1的HX008，“2”表示偶联2个TM1的HX008，“4”表示偶联4个TM1的HX008，“6”表示偶联6个TM1的HX008，图4C为偶联TM1后HX008-TM1-3的HIC-HPLC结果，图中的“0”表示未偶联TM1的HX008，“2”表示偶联2个TM1的HX008，“4”表示偶联4个TM1的HX008，“6”表示偶联6个TM1的HX008。
图5显示了GLP1-Fc-TM1结合HSA的ELISA检测结果，其中HX042代表GLP1-Fc。
图6显示了HX006-TM1结合HSA的ELISA检测结果。
图7显示了HX008-TM1结合HSA的ELISA检测结果.
图8显示了GLP1-Fc偶联C18-叔丁醇酯后的HIC-HPLC结果，图中的“0”表示未偶联C18-叔丁醇酯，“2”表示偶联2个C18-叔丁醇酯。
图9显示了GLP1-Fc样品(上图)和GLP1-Fc-C16-NHS样品(下图)的HIC-HPLC结果。
图10显示了GLP1-Fc-C20-NHS样品HIC-HPLC结果。
图11显示了GLP1-Fc-C16-NHS和GLP1-Fc-C20-NHS分别结合HSA的ELISA检测结果， 其中HX042代表GLP1-Fc。
图12显示了HX006-C16-NHS和HX006-C20-NHS分别结合HSA的ELISA检测结果。
图13显示了GLP1-Fc-C18-叔丁醇酯与HSA的结合活性。
图14显示了HX006-C18-叔丁醇酯与HSA的结合活性。
图15显示了HX006偶联C18-叔丁醇酯后的HIC-HPLC结果。
图16显示GLP-1-Fc-TM1可以有效激活报告基因的生物学活性。
图17显示GLP1-Fc-TM1在大鼠中单次给药后的血浆浓度-时间曲线，以杜拉鲁肽作为阳性对照。
图18显示GLP1-Fc-TM1在食蟹猴中单次给药后的血浆浓度-时间曲线，以杜拉鲁肽作为阳性对照。
图19显示了GLP-1-Fc-TM1对II型糖尿病db/db小鼠的药效结果，其中图19-1显示了降低db/db小鼠4h空腹血糖的结果；图19-2和图19-3显示了首次和末次给药后的随机血糖的结果；图19-4和图19-5显示了末次给药后的OGTT测试的血糖AUC0-180min的结果；图19-6显示了降低db/db小鼠糖化血红蛋白含量的结果；图19-7显示了提升db/db小鼠胰岛素水平的结果；图19-8显示了减少db/db小鼠日均摄食的结果。
图20显示了GLP-1-Fc-TM1对DIO模型小鼠的药效结果，其中图20-1显示了GLP-1-Fc-TM1减轻DIO小鼠体重的结果；图20-2显示了GLP-1-Fc-TM1减少DIO小鼠摄食量的结果；图20-3显示了GLP-1-Fc-TM1降低小鼠体脂含量的结果；图20-4显示了GLP-1-Fc-TM1降低小鼠空腹血糖的结果；图20-5显示了GLP-1-Fc-TM1降低小鼠血脂含量的结果；图20-6显示了小鼠血清肝功指标ALT、AST水平的结果；图20-7显示GLP-1-Fc-TM1改善了DIO小鼠肝脏组织气球样变和脂肪变性的结果。
图21显示了GLP-1-Fc-TM1对大鼠的药效结果，其中图21-1至21-4显示了GLP-1-Fc-TM1降低SD大鼠血糖的结果，图21-5至21-8显示了GLP-1-Fc-TM1升高血清胰岛素的结果。
发明详述
I.定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
当在本文中使用商品名时，除非上下文另有指出，否则该商品名包括商品名产品的产品配方、通用名药物和活性药物成分。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数 字数值大5％的上限的范围内的数字数值。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或多项的组合。
术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“抗体”在本文中以最广意义使用并且涵盖多种抗体结构物，包括但不限于单克隆抗体、多克隆抗体、重组抗体、人源化抗体、嵌合抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体或其显示出所需的抗原结合活性的抗体片段。完整抗体通常将包含至少两条全长重链和两条全长轻链，但在某些情况下可包括较少的链，例如骆驼中天然存在的抗体可仅包含重链。
术语“全抗体”指包含至少两条重链(H)和两条轻链(L)的免疫球蛋白分子。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。抗体的重链可以基于其恒定区的氨基酸序列而划分为主要5种不同的类型：IgA、IgD、IgE、IgG和IgM，并且这些类型中的几种可以进一步划分成亚类，如，IgG1、IgG2、IgG3和IgG4、IgA1以及IgA2。
术语抗体的“抗体片段”和“抗原结合片段”可互换使用，是指并非完整抗体的分子，其包含完整抗体中用于结合该完整抗体所结合的抗原的部分。如本领域技术人员理解的，为实现抗原结合目的，抗体片段通常包含来自“互补决定区”或“CDR”的氨基酸残基。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗体片段。抗体片段的例子包括但不限于，Fab、scFab、二硫键连接的scFab、Fab’、F(ab’)2、Fab’-SH、Fv、scFv、二硫键连接的scFv。在一些实施方案中，抗体片段包含引入Fc区的半胱氨酸残基，以提供可用于巯基偶联化学的氨基酸残基位点。
在本文中，当提及一个抗体是IgG抗体时，指该抗体具有IgG类免疫球蛋白结构的异四聚体蛋白。在IgG抗体中，通常重链的VH-CH1与轻链的VL-CL配对形成特异性结合抗原的Fab片段。因此，一个IgG抗体基本上由借助免疫球蛋白铰链区连接的两个Fab分子和两个二聚化的Fc区组成。IgG免疫球蛋白可以基于重链恒定区的序列，划分成亚类，例如γ1(IgG1)、γ2(IgG2)、γ3(IgG3)、和γ4(IgG4)。在一些实施方案中，根据本发明的抗体是IgG抗体，例如IgG1,IgG2,IgG3或IgG4抗体。
术语“互补决定区”或“CDR区”或“CDR”或“高变区”，是抗体可变结构域中在序列上高度可变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的 区域。CDR主要负责与抗原表位结合。
术语“可变区”或“可变结构域”是抗体的重链或轻链中参与抗体与其抗原的结合的结构域。重链可变区(VH)和轻链可变区(VL)可以进一步再划分为高变区(HVR，又称作互补决定区(CDR))，其间插有较保守的区域(即，构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。
术语“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个方法是ELISA测定法，另一方法是本文实施例描述的表面等离子共振技术(SPR)测定法。
术语“Fc区”指免疫球蛋白重链的C端区域，包括天然序列Fc区和变异Fc区，例如各种Ig亚型以及其同种异型的Fc区序列(Gestur Vidarsson等,IgG subclasses and allotypes:from structure to effector functions,20 October 2014,doi:10.3389/fimmu.2014.00520.)。在一些实施方案中，人IgG重链Fc区具有自Cys226或自Pro230延伸至重链羧基端的氨基酸序列。然而，Fc区的C端末端赖氨酸(Lys447)可以存在或不存在。在再一些实施方案中，人IgG重链Fc区在N端带有天然免疫球蛋白的铰链序列或部分铰链序列，例如根据EU编号，E216到T225的序列或D221到T225的序列。在某些实施方案中，免疫球蛋白的Fc区包含两个恒定结构域域，即CH2和CH3，在另一些实施方案中，免疫球蛋白的Fc区包含三个恒定结构域，即CH2、CH3和CH4。
EU编号方案是基于序列比对开发的、以一致的方式对抗体中的残基进行编号的广泛采用的标准。在本申请的上下文中，除非特别说明，否则，采用Kabat的EU编号方案对抗体或融合蛋白区中的氨基酸残基进行编号和提及(如Kabat等人，Sequences of Proteins of Immunological Interest,第5版，Public Health Service，National Institutes of Health，Bethesda，Md.(1991)所述)。
“Fc区突变体”、“突变Fc区”和“突变的Fc区”等类似术语可以互换使用，指包含至少一处氨基酸修饰而区别于天然序列Fc区/野生型Fc区的Fc区。例如通过对野生型免疫球蛋白IgG的Fc区中多个位置上的氨基酸进行修饰以减少半抗体形成、减轻或消除效应子功能、提高血清半衰期。可以利用现有技术中已经公开的多种方法，对现有技术中已知的Fc区、本申请提供的Fc区、包含Fc区的融合蛋白(例如抗体)等进行进一步的修饰，例如以用于降低免疫原性、提高稳定性、溶解性、功能和临床益处的其他修饰。此类修饰包括但不限于在例如IgG Fc的下列修饰，例如在位置214-238、250-260、297-299、307-318、322或327到331、380-390等处的氨基酸残基，具体地例如对位置228，233，234，235，252，254，256，297，307，308，311，380，385，386，389，428，434和447处的修饰。可以对天然人IgG的铰 链序列进行修饰，从而使Fc区表达为均质产物。为了改善Fc区的化学稳定性，可以对易发生脱酰胺的天冬酰胺进行修饰，例如替换为谷氨酰胺、天冬氨酸或谷氨酸，例如包括N297E、N315Q和N384Q的置换。为了改善Fc区的物理稳定性，可以对Fc区第235位的亮氨酸进行修饰，例如L235K置换。在一些实施方案中，还可以对免疫球蛋白Fc区进行磷酸化、硫酸化、糖基化、甲基化、乙酰化、酰胺化等修饰，以满足具体需求。
在一个实施方案中，对Fc区的修饰是对位置254，308和434处的修饰，例如CN108299560A中公开的修饰。在另一个实施方案中，对Fc区的修饰是如CN1802167中所述的修饰。在一个具体的实施方案中，对Fc区的修饰是对位置228，234，235和/或447处的修饰，例如修饰S228P，F234A，L235A或修饰S228P，F234A，L235A和447缺失。在本申请中，Fc区的修饰显示在括号内，例如IgG4 Fc(S228P，F234A，L235A)表示对野生型的IgG4 Fc的第228，234和235位的氨基酸进行相应的取代。
可以从人和动物(例如牛、山羊、猪、小鼠、兔、仓鼠、大鼠和豚鼠等)获得野生型免疫球蛋白Fc区，或者可以从转化的动物细胞或微生物获得的Fc区重组形式或衍生物。例如，可以使用PCR方法从相应的cDNA文库分离编码免疫球蛋白的基因而获得，或者可以通过对完整的免疫球蛋白进行蛋白酶处理而获得Fc区。制备Fc区衍生物的技术例如参见WO 97/34631和WO 96/32478。
在一些实施方案中，本申请中使用的Fc区来源于IgG免疫球蛋白，例如来源于IgG1、IgG2、IgG3和IgG4亚类的Fc区，优选来源于IgG1和IgG4亚类的Fc区。在一些实施方案中，本申请中使用的Fc区是人IgG1和IgG4衍生的Fc区，以降低本申请缀合物的免疫原性。
在一些实施方案中，变异Fc区包含与天然序列Fc区的氨基酸序列相差一处或多处氨基酸取代、缺失或添加的氨基酸序列。在一些实施方案中，变异Fc区与野生型Fc区和/或亲本Fc区具有至少约80％、90％、95％、96％、97％、98％、99％或更高的同源性。
术语“新生儿受体(FcRn)”指一种位于细胞膜表面的IgG抗体受体。FcRn负责将母体IgG转移给胎儿，并调节免疫球蛋白在体内的稳态。FcRn可以和IgG的Fc部分结合，阻止IgG分子被溶酶体裂解，可以起到增长IgG体内半衰期的作用，参与到IgG的体内转运、维持和分布代谢过程中。
术语“受体介导的内吞”是指，由配体与细胞表面上的相应受体结合所触发的、配体/受体复合物被内化并递送到细胞溶质中或转移至合适的细胞内区室的过程。
术语“序列同一性”是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、 Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
在本发明中，就抗体序列而言，氨基酸序列同一性百分数通过将候选抗体序列与给定抗体序列最佳比对后，在一个优选方案中按照Kabat编号规则进行最佳比对后，予以确定。在本文中，在不指定比较窗(即待比较的目标抗体区域)的情况下，将适用于在给定抗体序列的全长上进行比对。在一些实施方案中，就抗体而言，序列同一性可以分布在整个重链可变区和/或整个轻链可变区上，或序列百分数同一性可以仅限定于构架区，而对应CDR区的序列保持100％相同。
“氨基酸取代”指将预定的氨基酸序列中存在的至少一个氨基酸残基替代为另外的不同“取代”氨基酸残基。
术语“保守取代”是指一个氨基酸经相同类别内的另一氨基酸取代，例如一个酸性氨基酸经另一酸性氨基酸取代，一个碱性氨基酸经另一碱性氨基酸取代，或一个中性氨基酸经另一中性氨基酸取代。
在本申请述及缀合物的上下文中，术语“肽接头”指连接活性成分和Fc区的，由氨基酸组成的短氨基酸序列，例如单独或组合使用的甘氨酸(G)和/或丝氨酸(S)和/或苏氨酸残基(T)，或来自免疫球蛋白的铰链区。
可以用于本发明中的肽接头可以容易地被本领域技术人员确定。例如，包含氨基酸序列(G4S)n，其中n是等于或大于1的整数。在一个优选实施方案中，肽接头包括(G4S)3、(G4S)4、(G4S)6、GS(G4S)4、DAAALEAAALDAAAREAAARDAAAL、NVDHLPSNTLVDLA。可以用于本发明抗体分子的肽接头还可以是，例如但不限于，如下氨基酸序列：(G3S)2、(G4S)2、(G3S)3、(G4S)3、(G3S)4、(G4S)4、(G3S)5、(G4S)5、(G3S)6、(G4S)6、GGG、DGGGS、TGEKP、GGRR、EGKSSGSGSESKVD、KESGSVSSEQLAQFRSLD、GGRRGGGS、LRQRDGERP、LRQKDGGGSERP和GSTSGSGK PGSGEGSTKG。
术语“烷基”指由碳原子和氢原子组成的直链或支链的饱和烃基团。具体地，烷基具有1-10个碳原子，例如1至8个、1至6个、1至5个、1至4个、1至3个或1至2个碳原子。例如，如本文中所使用，术语“C1-C6烷基”指具有1至6个碳原子的直链或支链的饱和烃基团，其实 例例如甲基、乙基、丙基(包括正丙基和异丙基)、丁基(包括正丁基、异丁基、仲丁基或叔丁基)、戊基(包括正戊基、异戊基、新戊基)、己基(包括正己基、2-甲基戊基、3-甲基戊基、3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基)，等。
术语“亚烷基”指从直链或支链的饱和烷烃的相同或两个不同碳原子上移去两个氢原子得到的二价基团。具体地，亚烷基具有1-10个碳原子，例如1至6个、1至5个、1至4个、1至3个或1至2个碳原子。例如，如本文中所使用，术语“C1-C6亚烷基”指具有1至6个碳原子的直链或支链的亚烷基，包括，但不限于，亚甲基、亚乙基、亚丙基、亚丁基，等。
术语“环烷基”是指具有指定环原子数的单环、稠合多环、桥接多环或螺环非芳族单价烃环结构，其可以是饱和或不饱和的，例如包含1个或多个双键。环烷基基团在环中可包含3个或更多个例如3-18、3-10、或3-8个碳原子，例如C3-10环烷基、C3-8环烷基、C3-6环烷基、C5-6环烷基。环烷基基团的实例包括但不限于环丙基、环丁基、环戊基、环己基、环庚基。
术语“烯基”指由碳原子和氢原子组成的包含至少一个双键的直链或支链的不饱和烃基团。具体地，烯基具有2-8个，例如2至6个、2至5个、2至4个或2至3个碳原子。例如，如本文中所使用，术语“C2-C6烯基”指具有2至6个碳原子的直链或支链的烯基，例如乙烯基、丙烯基、烯丙基、1-丁烯基、2-丁烯基、1,3-丁二烯基、1-戊烯基、2-戊烯基、3-戊烯基、1,3-戊二烯基、1,4-戊二烯基、1-己烯基、2-己烯基、3-己烯基、1,4-己二烯基等。
术语“亚烯基”是指，从包含至少一个双键的直链或支链的不饱和烯烃的相同或两个不同碳原子上移去两个氢原子得到的二价基团。具体地，亚烯基具有2-8个，例如2至6个、2至5个、2至4个或2至3个碳原子。例如，如本文中所使用，术语“C2-C6亚烯基”指具有2至6个碳原子的直链或支链的亚烯基，例如亚乙烯基、亚丙烯基、亚烯丙基、亚丁烯基、亚戊烯基、和亚己烯基。
术语“炔基”指由碳原子和氢原子组成的包含至少一个叁键的直链或支链的不饱和烃基团。具体地，炔基具有2-8个，例如2至6个、2至5个、2至4个或2至3个碳原子。例如，如本文中所使用，术语“C2-C6炔基”指具有2至6个碳原子的直链或支链的炔基，例如乙炔基、丙炔基、炔丙基、1-丁炔基、2-丁炔基、1-戊炔基、2-戊炔基、3-戊炔基、4-甲基-1-戊炔基、1-己炔基、2-己炔基、3-己炔基、5-甲基-2-己炔基、等。
术语“亚炔基”指从包含至少一个叁键的直链或支链的不饱和炔烃的相同或两个不同碳原子上移去两个氢原子得到的二价基团。具体地，亚炔基具有2-8个，例如2至6个、2至5个、2至4个或2至3个碳原子。例如，如本文中所使用，术语“C2-C6亚炔基”指具有2至6个碳原子的直链或支链的亚炔基，例如亚乙炔基、亚丙炔基、亚炔丙基、亚丁炔基、亚戊炔基和亚 己炔基。
术语“杂环”或“杂环基”指具有1-4个独立地选自N、O或S的杂原子环成员的5-20元(例如5-14元、5-8元、5-6元)的芳族或非芳族单环、二环、或多环环系。杂环中的一个或多个N、C或S原子可被氧化。优选地，杂环是5-10元环系，为单环或稠合双环。代表性实例包括但不限于吡咯烷、氮杂环丁烷、哌啶、吗啉、四氢呋喃、四氢吡喃、苯并呋喃、苯并噻吩、吲哚、苯并吡唑、吡咯、噻吩(噻吩)、呋喃、噻唑、咪唑、吡唑、嘧啶、吡啶、吡嗪、哒嗪、异噻唑和异噁唑。应该理解，该术语包含本文所定义的杂芳基。
术语“芳基”指在环部分具有6-20例如6-12个碳原子的单环或多环芳香烃基团。优选地，芳基是(C6-C10)芳基。非限制性示例包括苯基、联苯基、萘基或四氢萘基，它们各自可以任选被1-4个取代基取代，所述取代基例如烷基、三氟甲基、环烷基、卤素、羟基、烷氧基、酰基、烷基-C(O)-O-、芳基-O-、杂芳基-O-、氨基、巯基、烷基-S-、芳基-S-、硝基、氰基、羧基、烷基-O-C(O)-、氨基甲酰基、烷基-S(O)-、磺酰基、磺酰氨基、杂环基等。
术语“杂芳基”指含有选自N、O或S的1-4个杂原子的5-20元(例如5-14元、5-8元、5-6元)的芳族单环状或多环状环系，其可以是取代的或未取代的。优选地，杂芳基是5-10元环系，为单环或稠合双环。代表性的杂芳基基团包括2-或3-噻吩基、2-或3-呋喃基、2-或3-吡咯基、2-、4-或5-咪唑基、3-、4-或5-吡唑基、2-、4-或5-噻唑基、3-、4-或5-异噻唑基、2-、4-或5-噁唑基、3-、4-或5-异噁唑基、3-或5-1,2,4-三唑基、4-或5-1,2,3-三唑基、四唑基、2-、3-或4-吡啶基、3-或4-哒嗪基、3-、4-或5-吡嗪基、2-吡嗪基、2-、4-或5-嘧啶基。
术语“杂烷基”指完全饱和或含1至3个不饱和度、由所示数量的碳原子和一至十个、优选一至三个选自O、N、Si和S的杂原子组成的稳定的直链或支链烃，其中的氮和硫原子可任选被氧化并且氮杂原子可任选被季铵化。杂原子O、N、Si和S可位于杂烷基基团的任何内部位置处或位于杂烷基基团与分子的其余部分连接的位置处。杂烷基的代表性实例包括–CH2-CH2-O-CH3、-CH2-CH2-NH-CH3、-CH2-CH2-N(CH3)-CH3、-CH2-S-CH2-CH3、-CH2-CH2-S(O)-CH3、-NH-CH2-CH2-NH-C(O)-CH2-CH3、-CH2-CH2-S(O)2-CH3、-CH＝CH-O-CH3、-Si(CH3)3、-CH2-CH＝N-O-CH3和–CH＝CH-N(CH3)-CH3。至多两个杂原子可以是连续的，如例如-CH2-NH-OCH3和–CH2-O-Si(CH3)3。通常，C1至C4杂烷基或亚杂烷基具有1至4个碳原子和1或2个杂原子，C1至C3杂烷基或亚杂烷基具有1至3个碳原子和1或2个杂原子。在一些方面，杂烷基和亚杂烷基是饱和的。
除非另外指出，否则本文定义各个基团时所使用的术语“被取代”，是指相应基团可以被例如但不限于以下的基团取代：烷基、烯基、炔基、环烷基、芳基、杂芳基、杂环基、卤素、氰基、硝基、叠氮基、羧基、羟基、巯基、氨基、单或二烷基氨基、单或二环烷基氨基、单或二 芳基氨基、单或二杂环基氨基、单或二杂芳基氨基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-氧基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-硫基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-酰基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-酰氨基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-酰氧基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-磺酰基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-磺酰氧基、烷基-或环烷基-或杂环基-或杂芳基-或芳基-磺酰氨基、或上述任选取代的氨基-甲酰基，以及其各自被其余可选取代基进一步取代的基团，其中的各类基团如本文所定义。取代基的实例包括但不限于一个或多个独立地选自以下的基团：卤素、OH、SH、CN、NH2、NHCH3、N(CH3)2、NO2、N3、C(O)CH3、COOH、C(O)-氨基、OCOCH3、甲基、乙基、丙基、异-丙基、丁基、异丁基、仲丁基、叔丁基、环丙基、甲氧基、乙氧基、丙氧基、氧代基、三氟甲基、二氟甲基、磺酰基氨基、甲磺酰基氨基、SO、SO2、苯基、哌啶基、哌嗪基和嘧啶基。
术语“PEG单元”是指包含重复的乙烯氧基亚单元(PEG或PEG亚单元)的有机部分，其可以是多分散的、单分散的或离散的(即，具有离散数目的乙烯-氧基亚单元)。多分散PEG为大小和分子量的非均匀混合物，而单分散PEG通常是从非均匀混合物纯化的并因此具有单一的链长和分子量。优选的PEG单元包含离散的PEG，其是以逐步的方式而非经由聚合过程合成的化合物。离散的PEG提供具有限定和指定链长的单一分子。
在根据上下文无矛盾的情况下，本文中“药学上可接受的”和“药用”可互换使用。
术语“DAR”在本申请中指在缀合物中，偶联于本文所述的Fc上的Cn部分与Fc的比例、或偶联于本文所述的抗体上的Cn部分与抗体的比例、或偶联于本文所述的融合蛋白上的Cn部分与融合蛋白的比例。在本文所述的一些实施方案中，DAR可以为1至20，例如1-18、4-16、5-12、6-10、1-8、2-8、1-6、2-6、2-4，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。DAR也可被计算为产品中分子群体的平均DAR，即通过检测方法(例如通过常规方法如质谱法、ELISA测定、电泳和/或HPLC)测得的产品中偶联于本文所述的Fc部分的Cn与Fc部分的总体比例、偶联于本文所述的抗体上的Cn部分与抗体的总体比例、或偶联于本文所述的融合蛋白上的Cn部分与融合蛋白的总体比例，此DAR在文中称为平均DAR。在一些实施方案中，本发明偶联物的平均DAR值是1至20，例如2-18、4-16、5-12、6-10、2-8、3-8、2-6、4-6、6-10，例如1.0-8.0，2.0-6.0，例如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或10.0，以这些数值中的两个作为端点的范围。
本文所述的术语“药物”涵盖在预防或治疗葡萄糖代谢紊乱相关的疾病、心脑血管疾病、肾脏疾病、视网膜病中有效的任何物质。葡萄糖代谢紊乱相关的疾病例如是糖尿病(例如I型糖尿病、II型糖尿病)、肥胖、高血压、血脂异常，肥胖，葡萄糖耐受不良，高血糖症，高胰岛素血症，心血管疾病等。
术语“活性成分”用在本发明中指对细胞和/或机体具有有益的，任何生理活性功能(例如调节基因表达和生理功能、纠正由缺乏或过多分泌参与调节体内功能的成分而引起的异常状况)的物质，通常为活性肽类物质。例如，包括但不限于酶、酶抑制剂、抗原、抗体、抗体片段、激素、胰高血糖素样肽-1(GLP-1)、胰高血糖素、干扰素、细胞因子、生长因子和/或分化因子、参与细胞运动或迁移的因子、参与骨组织发生/再吸收的因子、趋化因子、血浆或间质粘连分子或细胞外基质、杀细菌或抗真菌因子等。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。在一些实施方案中，本发明的药物组合物包含本发明的缀合分子和药物辅料。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、缓冲剂、表面活性剂、载体或稳定剂等。
术语“药物组合”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于患者，其中这类施用在患者体内提供预防或治疗有效水平的两种或更多种活性剂。在一些实施方案中，药物组合中使用的本发明的分子和其他治疗剂以不超过它们单独使用时的水平施用。术语“固定组合”意指两种或更多种活性剂以单个实体的形式被同时施用于患者。优选对两种或更多种活性剂的剂量和/或时间间隔进行选择，从而使各部分的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种成分所能达到的效果。各成分可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。
术语“组合疗法”是指施用两种或更多种治疗剂或治疗方式(例如放射疗法或手术)以治疗本文所述疾病。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的方式使用每种类型的治疗剂。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。
在本文中，术语“个体”或“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不 限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，受试者是人。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有葡萄糖代谢紊乱相关疾病(例如糖尿病)家族病史的受试者是预防性方案的候选。通常，在葡萄糖代谢紊乱相关疾病(例如糖尿病)的背景中，术语“预防”是指在葡萄糖代谢紊乱相关疾病(例如糖尿病)的病征或症状发生前，特别是在具有葡萄糖代谢紊乱相关疾病(例如糖尿病)风险的受试者中发生前的药物施用。
用于本文时，术语“有效量”指本发明的偶联物/缀合物或其组合物或组合的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。
用于本文时，术语“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中本发明的偶联物/缀合物或其组合物或组合的任何有毒或有害作用不及治疗有益作用。相对于未治疗的个体，“治疗有效量”优选地对可度量参数(例如胰高血糖素分泌、血液中血糖浓度)实现至少约30％、甚至更优选地至少约40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％甚至100％的抑制或降低。
用于本文时，术语“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
II.药物组合物、试剂盒
本发明缀合分子的可药用盐形式在本发明的范围内。在一些实施方案中，本发明提供包含本文所述的任何缀合分子及其可药用盐的组合物，优选该组合物为药物组合物或药物制剂。
在一个实施方案中，所述组合物还包含药用辅料。在一个实施方案中，组合物，例如，药物组合物，包含本发明的缀合分子，以及一种或多种其它治疗剂的组合。
本发明公开的这些组合物还可以包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。
对于药用辅料的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”,第八版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，可注射用溶液剂和可输注溶液剂)、散剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。
可以通过将具有所需纯度的本发明的缀合分子与一种或多种任选的药用辅料混合来制备包含本文所述的缀合分子的药物，优选地以冻干制剂或水溶液的形式。
本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它治疗剂，包括化疗剂、血管生成抑制剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫检查点抑制剂或激动剂)等。所述活性成分以对于目的用途有效的量合适地组合存在。
可制备持续释放制剂。持续释放制剂的合适实例包括含有缀合分子的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
在一些实施方案中，本发明还提供了药物组合或药物组合产品，其包含本发明的缀合分子以及一种或多种其它治疗剂。
本发明的另一个目的是提供一种成套药盒，其包含本发明的药物组合，优选地所述药盒为药物剂量单元形式。由此可以依据给药方案或药物施用间隔提供剂量单元。
在一个实施方案中，本发明的成套药盒在同一包装内包含：
-含有本发明的缀合分子的第一容器；
-含有其它治疗剂的药物组合物的第二容器。
III.用途和治疗方法
本申请提供的长效平台，即活性分子-融合蛋白-Cn缀合物平台、活性分子-Fc-Cn缀合物平台和抗体-Cn缀合物平台可以用于有效延长活性分子血清半衰期，具体地，通过本申请公开的方法将所述活性分子构建成结构为“活性分子-Fc-Cn”的缀合分子、结构为“活性分子-融合蛋白-Cn”的缀合分子、结构为“抗体-Cn”的缀合分子，从而有效提高所述活性分子的血清半衰期，由此可以降低给药频率，减少用药量，节约成本。
本申请通过上述长效平台在延长活性分子血清半衰期的同时，并不影响活性分子的生物功能，且产生的结构为“活性分子-Fc-Cn”的缀合分子、结构为“活性分子-融合蛋白-Cn”的缀合分子、结构为“抗体-Cn”的缀合分子对机体具有低的免疫原性，有效避免了常见缀合分子容易引起机体免疫反应的负面影响。
本申请获得的“活性分子-Fc-Cn”的缀合分子、“活性分子-融合蛋白-Cn”的缀合分子、 结构为“抗体-Cn”的缀合分子可以用于在受试者中预防或治疗多种疾病。本领域技术人员根据活性分子的生物功能可以容易地确定该缀合分子可以治疗或预防的疾病。例如当活性分子是GLP-1时，所述缀合分子可以用于有效治疗代谢疾病和/或紊乱，例如糖尿病，如I型糖尿病、II型糖尿病、糖耐量降低、高血糖症、血脂异常、肥胖症、代谢综合征、心血管疾病等。
当活性分子是抗PD-1抗体或其抗原结合片段时，所述缀合分子可以用于有效预防或者治疗与PD-1表达异常的疾病，例如各种肿瘤或癌症，例如黑色素瘤、非小细胞肺癌、肾细胞癌、膀胱癌、霍奇金淋巴瘤、头颈癌、卵巢癌和脑癌。
当活性分子是抗VEGF抗体或其抗原结合片段时，所述缀合分子可以用于有效预防或者治疗与VEGF表达异常的疾病，例如各种与血管发生相关的疾病，例如，眼科疾病，如，湿性或新生血管性年龄相关性黄斑变性(AMD)和糖尿病黄斑水肿(DME)；大多数癌症；心血管疾病。
本发明提供了在受试者中预防或治疗疾病的方法，包括向受试者施用有效量的本发明的缀合分子或其可药用盐、药物组合物、药物组合或药盒。
在本发明的治疗方法中，本发明缀合分子的施用可以包括1)治疗性措施，该措施治愈、减缓、减轻经诊断的病理状况或疾患的症状及/或停止该经诊断的病理状况或疾患的进展；或2)预防性或防范性措施，该措施预防及/或减缓病理状况或疾患的发展。在一些实施方案中，在本发明的治疗方法中，个体将受益于所述的治疗性措施或预防性措施，并相比于未接受所述处理的个体，表现出在疾病、病症、病状、和/或症状的发生、复发或发展上的减轻或改善。
本发明的药物组合物可以通过任何合适的方法给药，包括肠胃外给药，肿瘤内给药和鼻内给药。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明的药物组合物的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、缀合分子中活性分子的具体类型、疾病的严重性和进程、治疗目的、以前的治疗、患者的临床病史和对所述药物的应答，和主治医师的判断力。
在一些实施方案中，本发明也提供本发明药物组合物在制备用于前述治疗和预防方法的药物中的用途。
本发明的这些以及其它方面和实施方案示例于以下实施例中。上文以及整个本申请中所描述的任何或所有特征可以在本发明的各种实施方案中组合。以下实施例进一步说明本发明，然 而，应理解实施例以举例说明为目的，不应理解为构成任何限制。
说明书和权利要求书中所使用的缩写含义如下：
AUC     曲线下面积
CV      柱体积
HSA     人血清白蛋白
PBS     磷酸盐缓冲盐水
tBu     叔丁基
Pbf     2,2,4,6,7-五甲基苯并呋喃-5-磺酰基
Trt     三苯甲基
Mmt     4-甲氧基三苯基
Mtt     甲基三苯甲基
Alloc   (2-丙烯氧基)羰基
DCM     二氯甲烷
DCC     二环己基碳二亚胺
DMF     N,N-二甲基甲酰胺
DMAP    4-二甲氨基吡啶
DIPEA   N,N-二异丙基乙胺
DIC     N,N-二异丙基碳二亚胺
HBTU    苯并三氮唑-N,N,N”,N”-四甲基脲六氟磷酸盐
HATU    2-(7-偶氮苯并三氮唑)-N,N,N”,N”-四甲基脲六氟磷酸酯
HPLC    高效液相色谱法
TBTU    O-苯并三氮唑-N,N,N”,N”-四甲基脲四氟硼酸
HOBT    1-羟基苯并三唑
HOAT    1-羟基-7-偶氮苯并三氮唑
TFA     三氟乙酸
TIS     三异丙基硅烷
TCEP    三(2-羧乙基)膦盐酸盐
TSTU    O-(N-琥珀酰亚胺)-1,1,3,3-四甲基脲四氟硼酸酯
实施例
实施例1.制备高级脂肪酸链
在本实施例中，构建了可以与Fc区(或融合蛋白)上的不同位点，例如游离巯基、氨基反应的多种不同的高级脂肪酸链的示例。
1.1制备高级脂肪酸链TM1
按照如下技术路线制备脂肪酸链TM1：
(1)将化合物1(5.0g,29.40mmol)溶于无水DCM(50mL),搅拌均匀,加入EDCI(5.62g,29.4mmol),再加入化合物1-2(4.35g,29.40mmol)，25℃下反应2h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物2(5.5g，62.5％)。
(2)将化合物2(5.5g,18.30mmol)溶于无水DCM(60mL),搅拌均匀,再加入EDCI(3.50g,18.30mmol),再加入化合物2-1(2.98g,18.30mmol)，25℃下反应2h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物3(4.8g，59.3％)。
(3)将化合物3(4.8g,10.78mmol)溶于无水DCM(40mL),搅拌均匀,再加入EDCI(2.06g,10.78mmol),再加入化合物3-1(2.18g,10.78mmol)，25℃下反应12h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物4(4.0g，58.9％)。
(4)将化合物4(4.0g,6.34mmol)溶于无水DCM(30mL)，搅拌均匀,加入TFA(5ml)，25℃下反应0.5h。反应结束后，直接浓缩得到棕黑色油状物5(3.2g，88％)。
(5)将化合物5(3.2g，5.58mmol)溶于无水DMF(30mL),搅拌均匀,再加入DCC(1.14g,5.58mmol)和DMAP(0.14g,1.16mmol),再加入化合物5-1(1.75g,5.58mmol)，25℃下反应2h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物TM1(2.1g，43.7％)，分子量870.05。
TM1结构解析：
质谱：
MS-ESI(m/z)：870.4[M+H]+
核磁氢谱：
1H NMR(400MHz,DMSO-d6)δ8.02(S,2H,NH)7.88(S,1H,COOH)7.65(S,1H,COOH)6.97(S,2H,CH)4.13(S,1H,CH)3.67～3.69(S,2H,CH2)3.65～3.15(m,22H,CH2)2.33～1.17(m,10H,CH2)1.16(S,4H,CH2)1.21(S,26H,CH2)
核磁碳谱：
13C NMR(100MHz,DMSO-d6)δ174.91,173.91,172.88,171.95,171.13,169.77,134.94,70.63,70.38,69.33,51.94,40.45,40.25,39.83,39.20,35.53,34.20,32.15,29.57,27.49,25.70,24.95
1.2制备高级脂肪酸链C18酯
按照如下技术路线制备脂肪酸链C18酯(即，C18-叔丁醇酯)：
(1)将化合物1(5.0g,29.40mmol)溶于无水DCM(50mL),搅拌均匀,加入EDCI(5.62g,29.4mmol),再加入化合物1-2(4.35g,29.40mmol)，25℃下反应2h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物2(5.5g，62.5％)。
(2)将化合物2(5.5g,18.30mmol)溶于无水DCM(60mL),搅拌均匀,再加入EDCI(3.50g,18.30mmol),再加入化合物2-1(2.98g,18.30mmol)，25℃下反应2h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物3(4.8g，59.3％)。
(3)将化合物3(4.8g,10.78mmol)溶于无水DCM(40mL),搅拌均匀,再加入EDCI(2.06g,10.78mmol),再加入化合物3-1(2.18g,10.78mmol)，25℃下反应12h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物4(4.0g，58.9％)。
(4)将化合物4(4.0g，6.35mmol)溶于无水DMF(40mL),搅拌均匀,再加入DCC(1.31g,6.35mmol)和DMAP(0.155g,1.27mmol),再加入化合物4-1(2.35g,6.35mmol)，25℃下反应2h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物C18酯(1.8g，28.8％)，分子量982.27。
C18酯结构解析：
质谱：
MS-ESI(m/z)：982.5[M+H]+
核磁氢谱：
1H NMR(400MHz,DMSO-d6)δ8.11(m,2H,NH)7.91～7.62(m,2H,NH)7.01(S,2H,CH)3.87～3.59(m,1H,CH)3.57(S,2H,CH2)3.56～3.55(m,10H,CH2)3.50～3.28(m,6H,CH2)3.21～3.16(m,6H,CH2)2.25(S,2H,CH2)2.16～2.10(m,6H,CH2)1.92～1.85(m,2H,CH2)1.40～1.35(m,22H,CH2、CH3)1.30～1.23(m,24H,CH2、CH3)
核磁碳谱：
13C NMR(100MHz,DMSO-d6)δ172.78，171.78，171.19，169.95，169.70，135.02，80.75，79.76，70.62，70.42，69.94，69.81，69.47，69.35，52.77，38.94，35.23，34.53，29.50，29.38，29.09，28.81，28.23，28.09，25.06
1.3制备高级脂肪酸链C16-NHS
按照如下技术路线制备脂肪酸链C16-NHS：
(1)将化合物1(3.0g,11.7mmol)溶于无水DCM(30mL),搅拌均匀,加入DCC(2.41g,11.7mmol)和DMAP(0.285g,2.34mmol)，25℃下反应12h。反应结束后，直接过制备HPLC 分离，浓缩得到无色油状物2(2.1g，41.2％)。
(2)将化合物2(2.1g,4.76mmol)溶于无水DCM(20mL),搅拌均匀,再加入TSTU(1.43g,4.76mmol)和DIPEA(0.614g,4.76mmol),再加入化合物2-1(1.64g,14.28mmol)，25℃下反应1h。反应结束后，直接浓缩得到棕黑色油状物3(2.0g，78.1％)。
(3)将化合物3(2.0g,3.71mmol)溶于无水DCM(20mL),搅拌均匀,再加入TFA 2ml，25℃下反应1h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物，称为C16酯或C16-NHS(1.2g，70.5％)，分子量482.26。
C16-NHS酯结构解析：
质谱：
MS-ESI(m/z)：483.3[M+H]+
核磁氢谱：
1H NMR(400MHz,DMSO-d6)δ12.71(S,1H,COOH)8.20(S,1H,NH)4.32～4.31(m,1H,CH)2.88～2.71(m,6H,CH2)2.18～2.17(m,4H,CH2)1.61～1.52(m,2H,CH2)1.30～1.28(m,24H,CH2)0.94～0.90(m,3H,CH3)
核磁碳谱：
13C NMR(100MHz,DMSO-d6)δ173.43，172.95，170.59，168.87，51.21，35.52，31.77，29.52，29.18，29.05，27.60，26.42，25.90，25.64，22.56，14.41
1.4制备高级脂肪酸链C20-NHS
按照如下技术路线制备脂肪酸链C20-NHS：
(1)将化合物1(5.0g,12.56mmol)溶于无水DCM(50mL),搅拌均匀,加入DCC(2.58g,12.56mmol)和DMAP(0.306g,2.51mmol),再加入化合物1-2(2.54g,12.56mmol)，25℃下反应2h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物2(6.1g，83.56％)。
(2)将化合物2(6.1g,10.4mmol)溶于无水DCM(60mL),搅拌均匀,再加入DCC(2.14g,10.4mmol)和DMAP(0.53g,2.08mmol),再加入化合物2-1(3.2g,10.4mmol)，25℃下反应2h。反应结束后，直接过制备HPLC分离，浓缩得到无色油状物3(4.5g，51％)。
(3)将化合物3(4.5g,5.15mmol)溶于无水DCM(40mL),搅拌均匀,再加入TFA 4ml，25℃下反应1h。反应结束后，直接过制备HPLC分离，浓缩得到棕色油状物4(3.2g，82％)。
(4)将化合物4(3.2g,4.20mmol)溶于无水DMF(40mL),搅拌均匀,再加入DCC(0.865g,4.20mmol)和DMAP(0.103g,0.84mmol),再加入化合物4-1(0.483g,4.2mmol)，25℃下反应2h。反应结束后，直接过制备HPLC分离，浓缩得到白色固体，称为C20酯或C20-NHS(1.5g，41.7％)，分子量859.02。
C20-NHS酯结构解析：
质谱：
MS-ESI(m/z)：859.4[M+H]+
核磁氢谱：
1H NMR(400MHz,DMSO-d6)δ12.41(S,2H,COOH)8.11～7.72(m,3H,NH)4.66(S,1H,CH)3.92～3.61(m,4H,CH2)3.49～3.40(m,18H,CH2)3.44～3.33(m,4H,CH2)2.88～2.21(m,6H,CH2)2.18～2.10(m,2H,CH2)1.52～1.28(m,30H,CH2)
核磁碳谱：
13C NMR(100MHz,DMSO-d6)δ174.95，174.00，172.81，171.87，170.50，169.72，167.05，70.84，70.62，70.41，69.80，69.56，69.33，66.23，39.88，34.12，29.54，29.37，29.30，29.01，25.94，25.69，24.96
实施例2.GLP-1-Fc-TM1样品制备及其DAR值检测
在本实施例中，以商用GLP-1-Fc融合蛋白杜拉鲁肽(Dulaglutide)为例，构建了基于本申请Fc-高级脂肪酸链平台的缀合物。杜拉鲁肽由2条同样的长链组成，其中一条链的氨基酸序列如下所示：
杜拉鲁肽所包含的Fc的氨基酸序列如下所示：
1.1 GLP-1-Fc融合蛋白与TM1的偶联
取7.5mg GLP-1-Fc融合蛋白(杜拉鲁肽，自制)，使用15ml的30KD超滤管置换到还原缓冲液中(25mM硼酸钠，30mM NaCl,5mM EDTA，pH8.0)，共置换四次；终体积约为1ml，并检测蛋白质浓度。向样品中加入3倍摩尔数TCEP，25℃水浴2h；然后使用15ml的30KD超滤管置换到偶联缓冲液中(50mM Tris，150mM NaCl，5mMEDTA，pH7.5)，共置换4次。然后检测样品蛋白质浓度和自由巯基数。
根据蛋白质的量，向GLP-1-Fc融合蛋白样品中加入5倍摩尔量的TM1，混匀后25℃反应 2h，微型摇床震摇。偶联完后立刻通过阳离子层析进行纯化。
将纯化后的样品命名为GLP-1-Fc-TM1(也称为GLP1-Fc-TM1)，并置换到暂存缓冲液(25mM磷酸盐，150m氯化钠，pH7.0)中，以备下一步检测。
1.2 GLP1-Fc-TM1样品DAR值检测
在本实施例中通过HIC-HPLC分析检测了GLP1-Fc和TM1的偶联比例(DAR)。所采用的分析柱为：TSKgel Butyl-NPR(4.6mm*3.5cm)，分析方法为已知方法，参见文献:Dru-to-Antibody Ratio(DAR)and Drug Load Distribution by Hydrophobic Interaction Chromatography and Reversed Phase High-Performance Liquid Chromatography。检测结果如图1所示，TM1偶联到GLP1-Fc蛋白的平均DAR值为：3.3。
实施例3.GLP-1-Fc-C18的制备及检测
在本实施例中，以商用GLP-1-Fc融合蛋白杜拉鲁肽(Dulaglutide)为例，构建了基于本申请Fc-高级脂肪酸链平台的缀合物。
取8.34mg GLP1-Fc融合蛋白(杜拉鲁肽，自制)，使用15ml的30KD超滤管置换到还原缓冲液液中(25mM硼酸钠，30mM NaCl,5mM EDTA pH8.0)，共置换四次，终体积约为3ml，并检测蛋白质浓度。向抗体中加入3倍摩尔数TCEP，25℃水浴2h；然后使用15ml的30KD超滤管置换到偶联缓冲液中(50mM Tris，150mM NaCl，5mMEDTA，pH7.5)，共置换4次。然后检测样品蛋白质浓度和自由巯基数。
根据蛋白质的量，向GLP-1-Fc融合蛋白样品中加入3倍摩尔量的C18-叔丁醇酯，混匀后25℃反应2h，微型摇床震摇。偶联完后立刻通过阳离子层析进行纯化。
将纯化后的蛋白质命名为：GLP-1-Fc-C18-叔丁醇酯(也称为GLP1-Fc-C18叔丁醇酯)，并置换到暂存缓冲液中，以备下一步检测。
在本实施例中，参照实施例2的方法通过HIC-HPLC分析检测了GLP1-Fc和C18-叔丁醇酯的偶联比例(DAR)，结果如图8所示，平均DAR值为：1.26。
实施例4.GLP1-Fc-C16-NHS的样品制备及检测
在本实施例中，以商用GLP-1-Fc融合蛋白杜拉鲁肽(Dulaglutide)为例，构建了基于本申请Fc-高级脂肪酸链平台、通过Fc上赖氨酸残基的氨基连接的缀合物。
取4.56mg GLP-1-Fc融合蛋白，用30KD超滤管置换到偶联缓冲液中(0.1M MOPS，20mM Tris,pH7.5)，终体积0.9ml，并检测蛋白质浓度。向融合蛋白样品中加入6倍摩尔量的C16-NHS。 混匀后，25℃反应2h，微型摇床震摇。偶联完后立刻通过阳离子层析进行纯化，然后用30KD超滤管将样品置换到暂存缓冲液中，检测样品浓度。将纯化后的产品命名为：GLP1-Fc-C16-NHS或GLP1-Fc-C16。
在本实施例中，参照实施例2的方法通过HIC-HPLC分析检测。结果如图9所示，C16-NHS与GLP1-Fc的偶联率为：83.07％。
实施例5 GLP1-Fc-C20-NHS的样品制备及检测
在本实施例中，以商用GLP-1-Fc融合蛋白杜拉鲁肽(Dulaglutide)为例，构建了基于本申请Fc-高级脂肪酸链平台、通过Fc上赖氨酸残基的氨基连接的缀合物。
取4.56mg GLP1-Fc融合蛋白，用30KD超滤管置换到偶联缓冲液中(0.1M MOPS，20mM Tris,pH7.5)，终体积0.9ml，并检测蛋白质浓度。向融合蛋白样品中加入6倍摩尔量的C20-NHS。混匀后，25℃反应2h，微型摇床震摇。偶联完后立刻通过阳离子层析进行纯化，然后用30KD超滤管将样品置换到暂存缓冲液中，检测样品浓度。将纯化后的产品命名为：GLP1-Fc-C20-NHS或GLP1-Fc-C20。
在本实施例中，参照实施例2的方法通过HIC-HPLC分析检测。结果如图10所示，C20-NHS与GLP1-Fc的偶联率为：96.38％。
实施列6 GLP1-Fc-C16-NHS和GLP1-Fc-C20-NHS与HSA蛋白的结合活性
本实施例采用ELISA方法检测了通过GLP1-Fc融合蛋白的赖氨酸偶联获得的样品GLP1-Fc-C16-NHS和GLP1-Fc-C20-NHS分别与HSA蛋白活性的情况。
用包被液(碳酸盐缓冲液)将HSA-his稀释至0.5ug/ml，包被8条酶标板，4℃过夜。PBST(含0.05％Tween20的PBS)洗版3次，200ul/孔加入封闭液(1％BSA的PBST溶液)，37℃孵育1h。PBST洗板4次，用稀释液(1‰BSA的PBST溶液)分别将GLP1-Fc-C16-NHS和GLP1-Fc-C20-NHS样品稀释至1000nM作为起始浓度，再5倍梯度稀释7个稀释度，共8个梯度，100ul/孔分别加入酶标板孔中，37℃孵育1h。PBST洗板5次，100ul/孔加入HRP-Goat anti-Huamn 10000X工作液，37℃孵育1h。PBST洗板6次，100ul/孔加入新配显色液(5ml底物液，250ulTMB母液，16ul 0.75％H2O2)，37℃孵育10min。50ul/孔加入终止液(2M H2SO4)，于450nm检测OD值。
结果见图11。结果显示偶联C20-NHS后的样品GLP1-Fc-C20-NHS能够较强的跟HSA-his结合。偶联C16-NHS后的样品GLP1-Fc-C16-NHS跟HSA-his结合较弱。
实施例7.HX006抗体偶联TM1的样品制备
在本实施例中，以CN 104804088 A专利中公开的抗VEGF抗体HX006为例，构建了基于本申请Fc-高级脂肪酸链平台的缀合物，其中HX006抗体为IgG1型，其重链序列和轻链序列如下表所示。
取6.53mg HX006抗体蛋白，使用15ml的30KD超滤管置换到还原缓冲液液中(25mM硼酸钠，30mM NaCl,5mM EDTA，pH8.0)，共置换四次，终体积约为2ml，并检测蛋白质浓度。向抗体中加入2倍摩尔数TCEP，25℃水浴2h；然后使用15ml的30KD超滤管置换到偶联缓冲液中(50mM Tris，150mM NaCl，5mM EDTA，pH7.5)，共置换4次。然后检测样品蛋白质浓度和自由巯基数。
根据蛋白质的量，向抗体样品中加入2倍摩尔量的TM1，混匀后25℃反应2h，微型摇床震摇。偶联完后立刻通过阳离子层析进行纯化。
将纯化后的蛋白质命名为：HX006-TM1-1，并置换到暂存缓冲液中，以备下一步检测。
在本实施例中，参照实施例2的方法通过HIC-HPLC分析检测了HX006和TM1在HX006-TM1-1分子中的偶联比例(DAR)。
检测结果如图2所示，在HX006-TM1-1分子中，TM1偶联到HX006抗体的平均DAR值为：3.0。
实施例8 HX006抗体偶联TM1的样品制备
在本实施例中，进一步制备了HX006抗体与TM1的偶联物。
取6.56mg HX006抗体蛋白，使用15ml的30KD超滤管置换到还原缓冲液液中(25mM硼酸钠，30mM NaCl,5mM EDTA pH8.0)，共置换四次，终体积约为2ml，并检测蛋白质浓度。向抗体中加入3倍摩尔数TCEP，25℃水浴2.5h；然后使用15ml的30KD超滤管置换到偶联缓冲液中(50mM Tris，150mM NaCl，5mMEDTA，pH7.5)，共置换4次。然后检测样品蛋白质浓度和自由巯基数。
根据蛋白质的量，向抗体样品中加入4倍摩尔量的TM1，混匀后25℃反应2h，微型摇床震摇。偶联完后立刻通过阳离子层析进行纯化。
将纯化后的蛋白质命名为：HX006-TM1-2，并置换到暂存缓冲液中，以备下一步检测。
在本实施例中，参照实施例2的方法通过HIC-HPLC分析检测了在HX006-TM1-2分子中HX006和TM1的偶联比例(DAR)。
检测结果如图3所示，在HX006-TM1-2分子中，TM1偶联到HX006抗体的平均DAR值为：4.67。
实施例9 HX006抗体偶联C18-叔丁醇酯的样品制备
在本实施例中，制备了HX006抗体与C18叔丁醇酯的偶联物。
取4.3mg HX006抗体蛋白，使用15ml的30KD超滤管置换到还原缓冲液液中(25mM硼酸钠，30mM NaCl,5mM EDTA pH8.0)，共置换四次，终体积约为2ml，并检测蛋白质浓度。向抗体中加入3倍摩尔数TCEP，25℃水浴2h；然后使用15ml的30KD超滤管置换到偶联缓冲液中(50mM Tris，150mM NaCl，5mMEDTA，pH7.5)，共置换4次。然后检测样品蛋白质浓度和自由巯基数。
根据蛋白质的量，向抗体样品中加入3倍摩尔量的C18叔丁醇酯，混匀后25℃反应2h，微型摇床震摇。偶联完后立刻通过阳离子层析进行纯化。
将纯化后的蛋白质命名为：HX006-C18-叔丁醇酯，也称为HX006-C18，并置换到暂存缓 冲液中，以备下一步检测。
在本实施例中，参照实施例2的方法通过HIC-HPLC分析检测了HX006和C18-叔丁醇酯的偶联比例(DAR)，结果如图15所示，平均DAR值为：2.95。
实施例10 HX006抗体偶联C16-NHS的样品制备
取4mg HX006抗体蛋白，用30KD超滤管置换到偶联缓冲液中(0.1M MOPS，20mM Tris,pH7.5)，终体积0.9ml，并检测蛋白质浓度。向融合蛋白样品中加入6倍摩尔量的C16-NHS。混匀后，25℃反应2h，微型摇床震摇。偶联完后立刻通过阳离子层析进行纯化，然后用30KD超滤管将样品置换到暂存缓冲液中，检测样品蛋白浓度。蛋白质命名为：HX006-C16-NHS。
实施例11.HX006抗体偶联C20-NHS的样品制备
取4mg HX006抗体蛋白，用30KD超滤管置换到偶联缓冲液中(0.1M MOPS，20mM Tris,pH7.5)，终体积0.9ml，并检测蛋白质浓度。向融合蛋白样品中加入6倍摩尔量的C20-NHS。混匀后，25℃反应2h，微型摇床震摇。偶联完后立刻通过阳离子层析进行纯化，然后用30KD超滤管将样品置换到暂存缓冲液中，检测样品蛋白浓度。蛋白质命名为：HX006-C20-NHS。
实施列12 HX006-C16-NHS和HX006-C20-NHS与HSA蛋白的结合活性
本实施例采用ELISA方法检测了通过HX006抗体蛋白Fc的赖氨酸偶联获得的样品HX006-C16-NHS和HX006-C20-NHS与HSA蛋白活性的情况。
用包被液将HSA-his稀释至0.5ug/ml，包被8条酶标板，4℃过夜。PBST(含0.05％Tween20的PBS)洗版3次，200ul/孔加入封闭液，37℃孵育1h。PBST洗板4次，用稀释液分别将HX006-C16-NHS和HX006-C20-NHS稀释至1000nM作为起始浓度，再5倍梯度稀释7个稀释度，共8个梯度，100ul/孔分别加入酶标板孔中，37℃孵育1h。PBST洗板5次，100ul/孔加入HRP-Goat anti-Huamn 10000X工作液，37℃孵育1h。PBST洗板6次，100ul/孔加入新配显色液，37℃孵育10min。50ul/孔加入终止液(2M H2SO4)，于450nm检测OD值。
结果见图12。结果显示偶联C20-NHS后的样品HX006-C20-NHS能够较强的跟HSA-his结合。偶联C16-NHS后的样品HX006-C16-NHS跟HSA-his结合较弱。
实施例13.HX008抗体偶联TM1的样品制备
在本实施例中，以CN108299560A专利中公开的抗PD-1抗体H8L2(本申请中称为HX008)为例，构建了基于本申请Fc-高级脂肪酸链平台的缀合物，其中HX008抗体为IgG4型，其序 列如下所示：
HX008抗体的重链序列为：
HX008抗体的重链可变区序列为：
Fc区序列为：
HX008抗体的轻链可变区序列为：
HX008抗体的轻链序列为：
取4mg HX008抗体蛋白，使用15ml的30KD超滤管置换到还原缓冲液液中(25mM硼酸钠，30mM NaCl,5mM EDTA，pH8.0)，共置换四次；终体积约为1ml，并检测蛋白质浓度。向抗体中加入8倍摩尔数TCEP，30℃水浴2.5h；然后使用15ml的30KD超滤管置换到偶联缓冲液中(50mM Tris，150mM NaCl，5mM EDTA，pH7.5)，共置换4次。然后检测样品蛋白浓度和自由巯基数。
根据蛋白质的量，向融合蛋白样品中加入4倍摩尔量的TM1，混匀后25℃反应2h，微型 摇床震摇。偶联完后立刻通过阳离子层析进行纯化。
将纯化后的蛋白质命名为：HX008-TM1-2，并置换到暂存缓冲液中，以备下一步检测。
在本实施例中，参照实施例2的方法通过HIC-HPLC分析检测了在HX008-TM1-2分子中HX008和TM1的偶联比例(DAR)。
结果如图4所示，在HX008-TM1-2分子中，TM1偶联到HX008抗体的平均DAR值为：1.45。
实施例14.HX008抗体偶联TM1的样品制备
在本实施例中，进一步制备了HX008抗体与TM1的偶联物。
取4mg HX008抗体蛋白，使用15ml的30KD超滤管置换到还原缓冲液液中(25mM硼酸钠，30mM NaCl,5mM EDTA pH8.0)，共置换四次；终体积约为1ml，并检测蛋白质浓度。向抗体中加入10倍摩尔数TCEP，30℃水浴2.5h；然后使用15ml的30KD超滤管置换到偶联缓冲液中(50mM Tris，150mM NaCl，5mMEDTA，pH7.5)，共置换4次。然后检测样品蛋白浓度和自由巯基数。
根据蛋白质的量，向融合蛋白样品中加入5倍摩尔量的TM1，混匀后25℃反应2h，微型摇床震摇。偶联完后立刻通过阳离子层析进行纯化。
将纯化后的蛋白质命名为：HX008-TM1-3，并置换到暂存缓冲液中，以备下一步检测。
在本实施例中，参照实施例2的方法通过HIC-HPLC分析检测了在HX008-TM1-3分子中HX008和TM1的偶联比例(DAR)。
结果如图4所示，在HX008-TM1-3分子中，TM1偶联到HX008抗体的平均DAR值为：2.0。
实施例15.GLP-1-Fc-TM1与HSA蛋白结合活性
TM1中包含的脂肪酸链可以与血清白蛋白(HSA)结合，本实施例采用ELISA方法检测了融合物GLP-1-Fc-TM1与HSA结合的情况。用包被液(碳酸盐缓冲液)将HSA-his稀释至0.5ug/ml，包被3条酶标板，4℃过夜。PBST(含0.05％Tween20的PBS溶液)洗版3次，200ul/孔加入封闭液(1％BSA的PBST溶液)，37℃孵育1h。PBST洗板4次，用稀释液(1‰BSA的PBST溶液)将GLP-1-Fc-TM1样品稀释至1000nM作为起始浓度，再5倍梯度稀释7个稀释度，共8个梯度，100ul/孔分别加入酶标板孔中，37℃孵育1h。PBST洗板5次，100ul/孔加入HRP-Goat anti-Huamn 10000X工作液，37℃孵育1h。PBST洗板6次，100ul/孔加入新配显色液(5ml底物液，250ulTMB母液，16ul 0.75％H2O2)，37℃孵育10min。50ul/孔加入终止液 (2M H2SO4)，于450nm检测OD值。
结果见图5。结果显示偶联TM1后的样品GLP1-Fc-TM1能够很强的跟HSA-his结合。
实施例16.GLP-1-Fc-C18-叔丁醇酯与HSA蛋白结合活性
本实施例采用ELISA方法检测了融合物GLP-1-Fc-C18-叔丁醇酯与HSA结合的情况。用包被液将HSA-his稀释至0.5ug/ml，包被3条酶标板，4℃过夜。PBST(含0.05％Tween20的PBS溶液)洗版3次，200ul/孔加入封闭液，37℃孵育1h。PBST洗板4次，用稀释液将GLP-1-Fc-C18-叔丁醇酯样品稀释至1000nM作为起始浓度，再5倍梯度稀释7个稀释度，共8个梯度，100ul/孔分别加入酶标板孔中，37℃孵育1h。PBST洗板5次，100ul/孔加入HRP-Goat anti-Huamn 10000X工作液，37℃孵育1h。PBST洗板6次，100ul/孔加入新配显色液，37℃孵育10min。50ul/孔加入终止液(2M H2SO4)，于450nm检测OD值。
结果见图13。结果显示偶联C18-叔丁醇酯后的样品GLP1-Fc-C18-叔丁醇酯能够很强的跟HSA-his结合。
实施例17.HX006-TM1与HSA蛋白结合活性
本实施例采用ELISA方法检测了融合物HX006-TM1与HSA结合的情况。用包被液将HSA-his稀释至0.5ug/ml，包被8条酶标板，4℃过夜。PBST(0.05％Tween20inPBS)洗版3次，200ul/孔加入封闭液，37℃孵育1h。PBST洗板4次，用稀释液将HX006-TM1样品稀释至1000nM作为起始浓度，再5倍梯度稀释7个稀释度，共8个梯度，100ul/孔分别加入酶标板孔中，37℃孵育1h。PBST洗板5次，100ul/孔加入HRP-Goat anti-Huamn 10000X工作液，37℃孵育1h。PBST洗板6次，100ul/孔加入新配显色液，37℃孵育10min。50ul/孔加入终止液(2M H2SO4)，于450nm检测OD值。
结果见图6。结果显示偶联TM1后的样品HX006-TM1能够较强的跟HSA-his结合，且随DAR值增加，结合活性增强。
实施例18.HX006-C18-叔丁醇酯与HSA蛋白结合活性
本实施例采用ELISA方法检测了融合物HX006-C18-叔丁醇酯与HSA结合的情况。用包被液将HSA-his稀释至0.5ug/ml，包被8条酶标板，4℃过夜。PBST(0.05％Tween20inPBS)洗版3次，200ul/孔加入封闭液，37℃孵育1h。PBST洗板4次，用稀释液将HX006-TM1样品稀释至1000nM作为起始浓度，再5倍梯度稀释7个稀释度，共8个梯度，100ul/孔分别加入酶标板孔中，37℃孵育1h。PBST洗板5次，100ul/孔加入HRP-Goat anti-Huamn 10000X工 作液，37℃孵育1h。PBST洗板6次，100ul/孔加入新配显色液，37℃孵育10min。50ul/孔加入终止液(2M H2SO4)，于450nm检测OD值。
结果见图14。结果显示相比较于裸抗HX006-DS，偶联C18-叔丁醇酯后的样品HX006-C18-叔丁醇酯能够较强的跟HSA-his结合。
实施例19.HX008-TM1与HSA蛋白结合活性
本实施例采用ELISA方法检测了融合物HX008-TM1与HSA结合的情况。具体步骤参见实施例15，差别在于将测试样品换为HX008-TM1-3样品。结果如图7显示，相比较于裸抗HX008-DS，偶联TM1后的样品HX008-TM1能够较强的跟HSA-his结合，且随DAR增加，结合活性增强。
实施例20.GLP-1-Fc-TM1对HEK293-CRE-Luc-GLP1R细胞株的活性影响
在稳定表达人GLP-1受体(GLP-1R)和细胞内报告基因CRE-荧光素酶(CRE4-luciferase)的细胞HEK-293(HEK293-CRE-Luc-GLP1R，来源于南京金斯瑞生物科技有限公司)中，利用GLP-l-Fc能够与GLP-1R特异性结合产生cAMP，从而激活报告基因的方法来检测的生物学活性，从而评估受试物I(GLP-1-Fc-TM1)对人GLP-1受体的激活能力。HEK293-CRE-Luc-GLP1R复苏后，采用DMEM(含10％FBS,400μg/ml G418,200μg/ml HygromycinB)于37℃，5％的CO2条件下的培养箱中进行培养。收集对数生长期细胞，计数，用完全培养基重新悬浮细胞，调整细胞浓度至合适浓度，按2×103个细胞/孔，接种384孔板，每孔加20μl细胞悬液。细胞在37℃，100％相对湿度，5％CO2培养箱中孵育过夜。用培养基将待测化合物稀释至所设置的相应作用浓度，每孔30μl受试物溶液(待测化合物的作用终浓度及稀释梯度视具体要求而定)，共设置9个浓度梯度，每个浓度2个复孔。以Semaglutide(市售)、Dulaglutide(自制)为阳性对照。细胞置于37℃，100％相对湿度，5％CO2培养箱中孵育6h。弃上清，加入40μL/well的One-Glo检测溶液，震荡反应5min，在ENVISION 2104酶标仪上测定luminescience(RLU)。
结果如图16显示：激活报告基因CRE-荧光素酶的效力，受试物I(GLP-1-Fc-TM1)的EC50为8.4×10-3nM，Dulaglutide的EC50为4.4×10-3nM，Semaglutide的EC50为4.7×10-3nM，在体外报告基因检测条件下，受试物I(GLP-1-Fc-TM1)与Dulaglutide和Semaglutide的EC50处于同一数量级，受试物I(GLP-1-Fc-TM1)具有较强生物活性。
实施例21.表面等离子共振技术(SPR)测定受试物与人血清白蛋白的相互作用
将10mM N-(2-羟乙基)哌嗪-N-2磺酸(HEPES),150mM氯化钠(NaCl),3mM乙二胺四乙酸(EDTA),0.005％吐温-20(Tween-20),用pH调节至7.4，作为运行试剂。将鼠抗His抗体用固定试剂用固定试剂(10mM醋酸钠，pH 4.5)稀释到50μg/mL。首先，CM5芯片的表面用400mM EDC和100mM NHS以10μL/min的流速进行420s的活化。其次，将50μg/mL的鼠抗His抗体以10μL/min的流速注入到通道约420s，固定量约为7000-15000RU。最后，芯片用1M乙醇胺以10μL/min进行420s封闭。使用脱盐柱及相应运行试剂将人血清白蛋白(HSA)进行缓冲液置换，置换后的样品用SPECTROstarNano进行浓度测定。将配体(受试物GLP-1-Fc、GLP-1-Fc-TM1、HX006-DS、HX006-TM1-1、HX006-TM1-2、HX008-DS、HX008-TM1-2、HX008-TM1-3)用运行试剂稀释至5μg/mL并以10μL/min的流速注入到His捕获芯片实验通道(Fc2)约400RU。参比通道(Fc1)不需要进行配体(受试物)的捕获。将人血清白蛋白(HSA)用运行试剂进行2倍倍比稀释，共7个浓度，将稀释后的人血清白蛋白(HSA)依次以30μL/min的流速注入到实验通道与参比通道，结合(120s)和解离(300s)相应时间。结合解离步骤均在运行试剂中进行。每一个浓度分析后，芯片需要用pH值为1.5的甘氨酸盐酸，以20μL/min的流速再生30s，洗掉配体以及未解离的分析物。进行下一个浓度分析时，实验通道需要重新捕获相同量的配体(受试物)。使用Biacore 8K分析软件Biacore Insight Evaluation Software计算每个样品的KD值。参比通道(Fc1)用于背景的扣减。
结果显示：使用Biacore 8K检测的受试物与人血清白蛋白亲和力为，未修饰的GLP-1-Fc、HX006-DS、HX008-DS的Kd值为0，即不结合；GLP-1-Fc-TM1、HX006-TM1-1、HX006-TM1-2、HX008-TM1-2、HX008-TM1-3的Kd值分别为1.01×10-2nM、1.14×10-3nM，6.92×10-4nM、4.87×10-3nM、4.05×10-3nM，表现出与人血清白蛋白强的亲和力。
实施例22.GLP-1-Fc-TM1对II型糖尿病db/db小鼠多次给药的药效试验研究
12只m/m小鼠(正常对照)和60只雄性db/db小鼠购进后适应性饲养，待db/db小鼠血糖达标后(8-10周龄左右)选择合格的10m/m小鼠和50只db/db小鼠进行分组，分别为正常对照组(m/m小鼠)、模型组、阳性对照组(Dulaglutide)、受试物低剂量组、受试物中剂量组、受试物高剂量组，每组10只。分组后开始给药，阳性对照组给予10nmol/kg/dose剂量的TRULICITY(Dulaglutide)，皮下注射，2次/周，持续4周；受试物低、中、高剂量组给予3、10、30nmol/kg/dose剂量的受试物I(GLP-1-Fc-TM1)，皮下注射，2次/周，持续4周；db/db模型组和正常对照组给予相应的溶媒(PBS)，皮下注射，2次/周，持续4周。所有组别的给药体积为5ml/kg/dose。临床观察每日1次，给药结束检测血清胰岛素和糖化血红蛋白，随后进行OGTT测试。给药期间，每周两次监测体重、摄食量，首次给药和末次给药后，检测检 测0h(给药前0-60min)、0.5h、2h、4h、8h、24h、48h、72h的随机血糖，每周一次检测给药前空腹血糖(禁食4h)。给药期结束时进行随机血糖、空腹血糖及血清胰岛素检测。
结果显示：受试物I(GLP-1-Fc-TM1)与阳性对照药物(Dulaglutide)相比，在降低糖化血红蛋白，降低随机血糖、空腹血糖，刺激血清胰岛素分泌，降低体重和摄食量的作用效果整体更优；且低、中、高剂量的受试物I(GLP-1-Fc-TM1)的作用效果呈现疗效关系，表明受试物I(GLP-1-Fc-TM1)具有明显的降糖作用。
具体而言，受试物GLP-1-Fc-TM1可剂量依赖性地降低db/db小鼠4h空腹血糖(图19-1)、首次和末次给药后的随机血糖(图19-2和图19-3)以及末次给药后的OGTT测试的血糖AUC0-180min(图19-4和图19-5)，降低db/db小鼠糖化血红蛋白的含量(图19-6)，提升db/db小鼠胰岛素水平(图19-7)，减少db/db小鼠日均摄食(图19-8)；同等剂量下，受试物GLP-1-Fc-TM1对上述指标的作用优于阳性对照Dulaglutide。
实施例23.GLP-1-Fc-TM1对DIO模型小鼠体重影响的药效试验研究
72只雄性C57BL/6J小鼠购进后，除12只正常对照组小鼠(常规饲料)外，其余60只小鼠给予高脂饲料饲喂制备DIO模型小鼠，自由进食，连续饲喂约10周。造模期间，2次/周监测摄食量，2次/周体重监测，待体重超过正常小鼠的20％即达到DIO模型标准，随后开始分组。选择合格的10只正常对照组小鼠(常规饲料)和50只DIO模型小鼠进行分组，分别为正常对照组(常规饲料饲养小鼠)、模型组、阳性对照组(dulaglutide)、受试物低剂量组、受试物中剂量组、受试物高剂量组，每组10只。分组后开始给药，阳性对照组给予10nmol/kg/dose剂量的TRULICITY(dulaglutide)，皮下注射，2次/周，持续4周；受试物低、中、高剂量组给予3、10、30nmol/kg/dose剂量的受试物I(GLP-1-Fc-TM1)，皮下注射，2次/周，持续4周；肥胖DIO模型组和正常对照组给予相应的溶媒(PBS)，皮下注射，2次/周，持续4周。所有组别的给药体积为5ml/kg/dose。所有DIO小鼠在给药期间继续给予高脂饲料饲喂，持续整个实验周期。给药期间监测体重和摄食量，每周2次；给药前测定空腹血糖(禁食4h)，采血检测血脂四项、肝脏血生化，给药第一天记为D1，给药28天(D28)，给药期结束后进行空腹4h血糖检测，采血分离血清检测血脂四项、肝脏血生化、血清胰岛素水平。最后收集腹腔脂肪组织称重，摘取肝脏称重，计算其脏器系数，公式为脏器系数＝脏器重量(g)/小鼠体重(g)。摘取部分肝脏福尔马林固定，进行病理组织学检测(HE染色，油红-O染色)。
结果显示：受试物I(GLP-1-Fc-TM1)与阳性对照药物(dulaglutide)相比，在降低空腹血糖、改善血脂四项和肝脏血生化指标及肝脏病理，刺激血清胰岛素分泌，降低体重和摄食量的作用效果整体更优；且低、中、高剂量的受试物I(GLP-1-Fc-TM1)的作用效果呈现疗效关系， 受试物I(GLP-1-Fc-TM1)具有明显的降糖和减肥作用。
具体而言，GLP-1-Fc-TM1可剂量依赖性地减轻DIO小鼠体重(图20-1)，减少DIO小鼠摄食量(图20-2)，降低其体脂含量(图20-3)，降低空腹血糖(图20-4)；此外，受试物GLP-1-Fc-TM1还有可剂量依赖降低血脂含量(图20-5)、血清肝功指标ALT、AST水平(图20-6)，并有改善DIO小鼠肝脏组织气球样变和脂肪变性的作用(图20-7)。相同剂量剂量下，受试物GLP-1-Fc-TM1对肝脏脂肪病变的改善作用效果优于阳性对照Dulaglutide。
实施例24.GLP-1-Fc-TM1对大鼠葡萄糖耐量试验(IVGTT)研究
58只雄性SD大鼠(6-8周，体重180-220g)购进后适应性饲养一周后，选择合适的50只动物进行分组，分别为溶媒对照组、阳性对照组(Dulaglutide)、受试物低剂量组、受试物中剂量组、受试物高剂量组，每组10只。分组后开始给药，阳性对照组给予10nmol/kg剂量的TRULICITY(Dulaglutide)，皮下注射，单次给药；受试物低、中、高剂量组给予3、10、30nmol/kg剂量的受试物I(GLP-1-Fc-TM1)，皮下注射，单次给药；溶媒对照组给予相应的溶媒(PBS)，皮下注射，单次给药。所有组别的给药体积为5ml/kg/dose。大鼠禁食(不禁水)16h后皮下注射给药，24小时和72小时后静脉注射给予葡萄糖(0.5g/kg)刺激，分别于注射葡萄糖后2、4、6、10、20和30min采集血样，检测血糖浓度和胰岛素水平，并计算AUC。
结果显示：受试物I(GLP-1-Fc-TM1)与阳性对照药物(Dulaglutide)相比，在降低空腹血糖、刺激血清胰岛素分泌，作用效果相当或无明显差异；且低、中、高剂量的受试物I(GLP-1-Fc-TM1)的作用效果呈现一定的量效关系。
具体而言，以3、10、30nmol/kg剂量单次给予受试物GLP-1-Fc-TM1，给药后24h和72h均可剂量依赖性降低SD大鼠血糖AUC0-30min(图21-1、图21-2、图21-3和图21-4)以及升高血清胰岛素AUC0-30min(图21-5、图21-6、图21-7和图21-8)。
实施例25.SD大鼠单次皮下注射受试物的药代动力学研究
12只SD大鼠(6-8周，体重180-220g)，雌雄各半。适应后随机分为2组，分别为受试物I(GLP-1-Fc-TM1)组、阳性对照组(Dulaglutide，自制)，每组6只，雌雄各半。以0.1mg/kg给药剂量，4ml/kg给药体积，单次皮下注射相应的受试物I(GLP-1-Fc-TM1)和阳性对照物(Dulaglutide)。于给药前，给药后第24小时(±10分钟，第1天)、48小时(±10分钟，第2天)、72小时(±15分钟，第3天)、96小时(±15分钟，第4天)、144小时(±15分钟，第6天)、192小时(±30分钟，第8天)、240小时(±30分钟，第10天)、336小时(±30分钟，第14天)。经颈静脉采血约0.5mL，EDTA-K2抗凝。采用ELISA法检测各时间点血浆中受试 物I(GLP-1-Fc-TM1)和阳性对照物(Dulaglutide)的浓度。其中3个检测物质的标准曲线范围均为3.13～200ng/mL，采用Phoenix WinNonlin 8.2计算药代参数。
结果如图17和表1显示：SD大鼠单次皮下注射0.1mg/kg受试物I(GLP-1-Fc-TM1)和Dulaglutide后，血浆中受试物I(GLP-1-Fc-TM1)和Dulaglutide的平均Tmax均为24h，平均Cmax分别为225和191ng/mL，平均T1/2分别为45和28h，平均AUC0-t分别为15700和7540h*ng/mL，平均AUC0-∞分别为16100和7840h*ng/mL，平均Vz_F_obs分别为428和533ml/kg，平均Cl_F_obs分别为6.42和13.3mL/h/kg，MRT(0-t)分别为70和39h，MRT(0-∞)分别为77和44h。受试物I(GLP-1-Fc-TM1)的半衰期T1/2、Cmax、AUClast、AUC0-∞、Vz_F、Cl、MRT(0-t)、MRT(0-∞)分别为Dulaglutide的1.61、1.18、2.08、2.05、0.80、0.48、1.79、1.75倍，说明受试物I(GLP-1-Fc-TM1)可通过降低清除率延长半衰期。
实施例26.食蟹猴单次皮下注射受试物的药代动力学研究
4只食蟹猴(5-6周岁，体重约6.5kg)，雌雄各半。适应后随机分为2组，分别为参比制剂对照(Dulaglutide，市售药物Trulicity)组、受试物I(GLP-1-Fc-TM1)组，每组2只，雌雄各半。以摩尔剂量1.676nmol/kg给药(即Dulaglutide剂量为0.1mg/kg，GLP-1-Fc-TM1剂量为0.101mg/kg)，4ml/kg给药体积，单次皮下注射相应的参比制剂对照物Dulaglutide、受试物I(GLP-1-Fc-TM1)。于给药前，给药后4h、8h、24h、48h、96h、144h、240h、336h。经颈静脉采血约0.5mL，EDTA-K2抗凝。采用ELISA法检测各时间点血浆中参比制剂对照物Dulaglutide、受试物I(GLP-1-Fc-TM1)的浓度。其中检测物质的标准曲线检测限度LLOQ＝15.625ng/mL，采用Phoenix WinNonlin 8.2计算药代参数。
结果如图18和表2及下文显示：食蟹猴单次皮下注射.676nmol/kg受试物I(GLP-1-Fc-TM1) 和参比制剂对照物Dulaglutide，血浆中受试物I(GLP-1-Fc-TM1)和Dulaglutide的平均Tmax分别为16和8h，平均Cmax分别为760和602ng/mL，平均T1/2分别为97.9和48.2h，平均AUC0-t分别为98546和49118h*ng/mL，平均AUC0-∞分别为140678和50947h*ng/mL，平均Vz_F_obs分别为95.7和136.8ml/kg，平均Cl_F_obs分别为0.74和1.97mL/h/kg，MRT(0-t)分别为88和68h，MRT(0-∞)分别为163和77h。受试物I(GLP-1-Fc-TM1)的半衰期Tmax、Cmax、T1/2、AUC0-t、AUC0-∞、Vz-F、Cl、MRT(0-t)、MRT(0-∞)分别为Dulaglutide的2.00、1.26、2.03、2.01、2.76、0.70、0.38、1.29、2.12倍，说明受试物I(GLP-1-Fc-TM1)可通过降低清除率延长半衰期。

Claims (47)

  1. 一种结构为“活性分子-Fc-Cn”的缀合分子，其中活性分子选自对机体有益的任何分子，Fc是免疫球蛋白IgG Fc，Cn为包含C14-24脂肪酸链的修饰部分。
  2. 一种结构为“抗体-Cn”的缀合分子，其中Cn为包含C14-24脂肪酸链的修饰部分。
  3. 一种结构为“活性分子-融合蛋白-Cn”的缀合分子，其中活性分子选自对机体有益的任何分子，Cn为包含C14-24脂肪酸链的修饰部分。
  4. 权利要求1-3中任一项的缀合分子，其中所述Cn具有以下式(I)的结构：
    -Z-Y   (I)，
    其中
    Z具有以下结构：
    -Z1-Z2-Z3-Z4-，
    其中Z1为Fc中的硫原子或氮原子或氧原子，
    Z2为-C(＝O)-或5-10元杂环基，优选地含1或2个选自N、S和O的杂原子；
    Z3选自键、-C(＝O)-、-C1-C10亚烷基-C(＝O)-、-C3-C10亚炔基-C(＝O)-、-C3-C10亚烯基-C(＝O)-、-C1-C10亚杂烷基-C(＝O)-、-C3-C8亚环烷基-C(＝O)-、-O-C1-C8亚烷基-C(＝O)-、-亚芳基-C(＝O)-、-C1-C10亚烷基-亚芳基-C(＝O)-、-亚芳基-C1-C10亚烷基-C(＝O)-、-C1-C10亚烷基-C3-C8亚环烷基-C(＝O)-、-C3-C8亚环烷基-C1-C10亚烷基-C(＝O)-、-C3-C8亚杂环基-C(＝O)-、-C1-C10亚烷基-C3-C8亚杂环基-C(＝O)-、-C3-C8亚杂环基-C1-C10亚烷基-C(＝O)-，其中所述的亚烷基、亚炔基、亚烯基、亚杂烷基、亚环烷基、亚芳基和亚杂环基可任选地被取代；
    Z4是键或是下式表示的PEG单元，
    其中，R1选自C1-4亚烷基、-NH-、-NH-C1-4亚烷基-、-NH-C1-4亚烷基-杂芳基-，其中杂芳基为5元或6元的含氮杂芳基；R2为-C(＝O)-、-C1-4亚烷基、-C1-4亚烷基-C(＝O)-、-C1-4亚烷基-NH-C(＝O)-(CH2OCH2)p-C1-4亚烷基-、-C1-4亚烷基-C(＝O)-NH-(CH2OCH2)p-C1-4亚烷基-，其中m为2-6的整数，p为1-3的整数，
    Y是
    其中Y通过X与Z4连接，k是10-30的整数，
    其中R独立地表示氢、C1-6烷基、C1-6氨基烷基、C1-6卤代烷基、C1-6羟基烷基。
  5. 权利要求1-4中任一项的缀合分子，其中所述Z2为亚马来酰亚胺基，其中左侧波浪线表示与Z1连接的位置；右侧波浪线表示与Z3连接的位置。
  6. 权利要求1-5中任一项所述的缀合分子，其中所述Z3为-C1-C10亚烷基-C(＝O)-，其中所述的亚烷基任选地被取代并且其中Z3通过-C-(＝O)-与Z4连接。
  7. 权利要求1-6中任一项所述的缀合分子，其中所述Z2为亚马来酰亚胺基，Z3为-C1-6亚烷基-C(＝O)-。
  8. 权利要求1-7中任一项所述的缀合分子，其中所述Z4为键，Z3与式(I)中的Y直接连接。
  9. 权利要求1-8中任一项所述的缀合分子，其中所述Z4是下式表示的PEG单元，
    其中，R1选自-NH-和-NH-C1-4亚烷基-；R2为-C1-4亚烷基或-C1-4亚烷基-NH-C(＝O)-(CH2OCH2)p-C1-4亚烷基-，其中m为2-6的整数，p为1-3的整数。
  10. 权利要求1-9中任一项所述的缀合分子，其中所述Z4为包含2-6个PEG的单元。
  11. 权利要求1-10中任一项所述的缀合分子，其中所述Z4为
    其中m＝1-4，左侧的星号表示与Z3连接的位置；右侧的星号表示与式II中的Y连接的位置。
  12. 权利要求1-11中任一项所述的缀合分子，其中所述式(I)中的Z具有以下结构：
    其中RE是氢、C1-6烷基、C1-6氨基烷基、C1-6卤代烷基、C1-6羟基烷基，其中y＝0-4,m＝1-4，其中左侧的星号表示与Ab连接的位置，右侧的星号表示与Y连接的位置。
  13. 权利要求1-12中任一项所述的缀合分子，其中所述Y通过X与Z4连接，并且X是-NH-(C＝O)-或-(C＝O)-NH-。
  14. 权利要求1-13中任一项所述的缀合分子，其中所述Cn包含C16脂肪酸链、C18脂肪酸链或C20脂肪酸链。
  15. 权利要求1-14中任一项所述的缀合分子，其中所述Cn包含C18脂肪酸链且与Fc的游离巯基的硫原子偶联。
  16. 权利要求1-15中任一项所述的缀合分子，其中所述Cn选自

  17. 权利要求1-16中任一项所述的缀合分子，其中所述Fc是IgG1 Fc或IgG4 Fc，优选是人IgG1 Fc或人IgG4 Fc。
  18. 权利要求1-16中任一项所述的缀合分子，其中所述Fc区进一步包括免疫球蛋白IgG铰链区。
  19. 权利要求1-17中任一项所述的缀合分子，其中所述Fc区在选自第228，233，234，235，252，254，256，297，307，308，311，380，385，386，389，428，434和447位氨基酸处(按照EU编号)具有修饰。
  20. 权利要求19所述的缀合分子，其中所述修饰是将第254、308、434位氨基酸分别替换为Thr、Pro和Ala(按照EU编号)。
  21. 权利要求20所述的缀合分子，其中所述修饰是S228P，F234A和L235A，或S228P，F234A，L235A和447缺失(按照EU编号)。
  22. 权利要求1-21中任一项所述的缀合分子，其中所述Fc区包含SEQ ID NO:10，15或16所示的序列或由其组成。
  23. 权利要求1-22中任一项所述的缀合分子，其中所述活性分子是肽类活性分子，所述肽类活性分子直接或者通过肽接头与Fc区融合在一起。
  24. 权利要求1-23中任一项所述的缀合分子，其中所述肽类活性分子的C端和Fc区的N端融合到一起，或者所述肽类活性分子的N端和Fc区的C端融合到一起。
  25. 权利要求1-24中任一项所述的缀合分子，其中所述活性分子选自酶、酶抑制剂、抗原、抗体或抗体片段、激素、胰高血糖素样肽-1(GLP-1)、胰高血糖素、干扰素、细胞因子、生长因子和/或分化因子、参与细胞运动或迁移的因子、参与骨组织发生/再吸收的因子、趋化因子、血浆或间质粘连分子或细胞外基质、杀细菌或抗真菌因子。
  26. 权利要求1-25中任一项所述的缀合分子，其中所述活性分子选自GLP-1、抗体或其抗原结合片段。
  27. 权利要求1-26中任一项所述的缀合分子，其中所述活性分子选自抗PD-1的抗体或其抗原结合片段，抗VEGF的抗体或其抗原结合片段。
  28. 权利要求1-27中任一项所述的缀合分子，其中所述肽接头包含氨基酸序列(G4S)n，其中n是等于或大于1的整数，优选地，肽接头包括(G4S)3、(G4S)4、(G4S)6、GS(G4S)4、DAAALEAAALDAAAREAAARDAAAL、NVDHLPSNTLVDLA，(G3S)2、(G4S)2、(G3S)3、(G4S)3、(G3S)4、(G4S)4、(G3S)5、(G4S)5、(G3S)6、(G4S)6、GGG、DGGGS、TGEKP、GGRR、EGKSSGSGSESKVD、KESGSVSSEQLAQFRSLD、GGRRGGGS、LRQRDGERP、LRQKDGGGSERP和GSTSGSGK PGSGEGSTKG。
  29. 权利要求1-28中任一项所述的缀合分子，其中所述Fc包含SEQ ID NO:10,15或16所示的序列。
  30. 权利要求1-29中任一项所述的缀合分子，其是GLP-1-IgG4 Fc-TM1、GLP-1-IgG4 Fc-C18叔丁醇脂、GLP-1-IgG4 Fc-C16-NHS、GLP1-IgG4 Fc-C20-NHS。
  31. 权利要求30所述的缀合分子，其中IgG4 Fc包含SEQ ID NO:16所示的序列。
  32. 权利要求30或31所述的缀合分子，其中所述GLP-1-IgG4 Fc-TM1、GLP-1-IgG4 Fc-C18叔丁醇脂、GLP-1-IgG4 Fc-C16-NHS、GLP1-IgG4 Fc-C20-NHS缀合分子中的GLP-1-IgG4 Fc部分包含SEQ ID NO:1所示的氨基酸序列。
  33. 权利要求27所述的缀合分子，其中所述抗PD-1的抗体或其抗原结合片段包含SEQ ID NO:9所示的重链可变区和SEQ ID NO:11所示的轻链可变区。
  34. 权利要求33所述的缀合分子，其中所述抗PD-1抗体或其抗原结合片段包含SEQ ID NO:8所示的重链和SEQ ID NO:12所示的轻链。
  35. 权利要求33或34所述的缀合分子，其中所述Fc包含SEQ ID NO:10所示的序列。
  36. 权利要求35所述的缀合分子，其是HX008-TM1缀合分子，优选HX008-TM1-2缀合分子或HX008-TM1-3缀合分子。
  37. 权利要求27所述的缀合分子，其中所述抗VEGF抗体或其抗原结合片段包含SEQ ID NO:2,3和4所示的3个重链CDR和SEQ ID NO:5,6和7所示的3个轻链CDR。
  38. 权利要求37所述的缀合分子，其中所述抗VEGF抗体或其抗原结合片段包含SEQ ID NO:13所示的重链和SEQ ID NO:14所示的轻链。
  39. 权利要求37或38所述的缀合分子，其中，所述Fc包含SEQ ID NO:15所示的序列。
  40. 权利要求37-39中任一项所述的缀合分子，其是HX006-TM1缀合分子、HX006-C18-叔丁醇酯缀合分子、HX006-C16-NHS缀合分子和HX006-C20-NHS缀合分子。
  41. 一种制备结构为“活性分子-Fc-Cn”的缀合分子的方法，包括
    (a)将活性分子多肽连接至免疫球蛋白Fc区以制备“活性分子-Fc”融合物；和
    (b)将“活性分子-Fc”融合物与含有脂肪酸链的Cn在允许Fc区与Cn缀合的条件下发生偶联反应，产生“活性分子-Fc-Cn”缀合分子。
  42. 一种制备结构为“活性分子-Fc-Cn”的缀合分子的方法，包括
    (a)将抗体Fab片段连接至免疫球蛋白Fc区以制备“Fab-Fc”融合物，
    (b)将“Fab-Fc”融合物在允许Fc区与含有脂肪酸链的Cn缀合的条件下发生偶联反应产生“Fab–Fc-Cn”缀合分子的步骤。
  43. 一种制备结构为“活性分子-Fc-Cn”或结构为“抗体-Cn”的缀合分子的方法，其中包括将全抗体在允许Fc区与含有脂肪酸链的Cn缀合的条件下发生偶联反应产生“活性分子-Fc-Cn”缀合分子的步骤。
  44. 一种包含权利要求1-40中任一项所述的缀合分子的药物组合物。
  45. 一种有效延长活性分子血清半衰期的方法，包括根据权利要求41-43中任一项所述的方法将所述活性分子构建成结构为“活性分子-Fc-Cn”或结构为“抗体-Cn”的缀合分子的步骤，从而有效提高所述活性分子的血清半衰期。
  46. 权利要求1-40中任一项所述的缀合分子，或权利要求44所述的药物组合物在制备用于治疗人类疾病中的用途。
  47. 一种治疗人类疾病的方法，包括将有效量的权利要求1-40中任一项所述的缀合分子，或权利要求44所述的药物组合物施用给受试者。
PCT/CN2023/120731 2022-09-26 2023-09-22 包含Fc-高级脂肪酸链的超长效平台 WO2024067401A1 (zh)

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