WO2022017309A1 - 胰岛素-Fc融合蛋白及其应用 - Google Patents

胰岛素-Fc融合蛋白及其应用 Download PDF

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WO2022017309A1
WO2022017309A1 PCT/CN2021/107040 CN2021107040W WO2022017309A1 WO 2022017309 A1 WO2022017309 A1 WO 2022017309A1 CN 2021107040 W CN2021107040 W CN 2021107040W WO 2022017309 A1 WO2022017309 A1 WO 2022017309A1
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insulin
fusion protein
region
seq
chain
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French (fr)
Inventor
王亚里
陈宪
朱鹿燕
周婷婷
莫炜川
刘成亮
郝维维
王淑亚
姜召菊
任子甲
苏鸿声
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Jiangsu Gensciences Inc
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Jiangsu Gensciences Inc
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Priority to JP2023503514A priority Critical patent/JP7532638B2/ja
Priority to US18/016,714 priority patent/US20230272030A1/en
Priority to EP21845572.3A priority patent/EP4230216A4/en
Priority to CA3189527A priority patent/CA3189527A1/en
Publication of WO2022017309A1 publication Critical patent/WO2022017309A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • 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
    • A61K38/28—Insulins
    • 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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to the field of polypeptide drugs, in particular to an insulin-Fc fusion protein with enhanced insulin activity and prolonged in vivo half-life after being cleaved by site-specific protease, a preparation method and application thereof.
  • Type 1 diabetes mainly relies on exogenous insulin to control blood sugar; type 2 diabetes has become the main drug for blood sugar control as the disease progresses. Therefore, the use of insulin to treat diabetes has become a more effective way.
  • Insulin therapy is necessary for patients with abnormal insulin secretion (type I) or insulin resistance (type II), and blood glucose levels are normally regulated by insulin administration.
  • type I abnormal insulin secretion
  • type II insulin resistance
  • insulin has a very short half-life in vivo and thus suffers from the disadvantage of repeated administration.
  • Such frequent administration causes severe pain and discomfort to the patient.
  • many studies have been carried out on protein formulations and chemical conjugation (fatty acid conjugates, polyethylene polymer conjugates) in order to improve the quality of life by prolonging the in vivo half-life of proteins and reducing the frequency of administration.
  • long-acting insulins include insulin glargine (lantus, lasting about 20 hours to 22 hours), manufactured by Sanofi Aventis, and insulin detemir (levemir, lasting about 18 hours to 22 hours) and tresiba, manufactured by Novo Nordisk. Insulin degludec for about 40 hours).
  • These long-acting insulin formulations do not produce peak blood insulin concentrations, so they are suitable as basal insulins. However, since these formulations do not have a sufficiently long half-life, there is still the disadvantage of injecting once a day or every two to three days. Thus, there are limitations in achieving the intended goal of once-weekly dosing frequency to improve convenience for diabetic patients requiring chronic insulin administration.
  • Patent publication CN103509118B discloses a single-chain insulin fused to the Fc region of an antibody. Although in vitro experiments have shown that the insulin-Fc fusion protein has an improved half-life, the fusion insulin has low in vivo hypoglycemic activity and is not suitable for clinical use. apply.
  • the inventors provided an insulin-Fc fusion protein, which obtained enhanced insulin activity and prolonged in vivo half-life after being cleaved by site-specific proteases, and surprisingly found that the fusion protein has The in vivo hypoglycemic effect is more flat and stable, which can improve the safety of clinical medication and patient compliance, thereby better achieving blood sugar management and providing a better quality of life.
  • the present invention provides an insulin-Fc fusion protein having enhanced insulin activity and prolonged in vivo half-life upon site-specific protease cleavage, having the structure of formula (I):
  • X and Z are the B and A chains of insulin, respectively, and if X is the B chain, Z is the A chain; if X is the A chain, then Z is the B chain.
  • Y is an optional linking peptide; Y can be 1-100 amino acids or more in length, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100 or a value between any two numbers above; eg Y is insulin C-peptide or a variant or fragment thereof.
  • E1 and E2 are present and are a stretch of amino acids containing a site-specific protease cleavage site;
  • E1 and E2 can be 1-10 or more amino acids in length, such as 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10 amino acids; if present at the same time, E1 and E2 can be cleaved by the same or different site-specific proteases, e.g.
  • the site-specific protease cleavage site may be Kex2 and/or Furin
  • the cleavage site of a protease is, for example, the cleavage site of the Kex2 protease.
  • L is a linker (Linker) connecting Z and Fc;
  • L can be a polypeptide fragment, for example, L comprises a flexible unit of one, two or more amino acids selected from the group consisting of Ala, Thr, Gly and Ser (also referred to as flexible herein).
  • peptide segment more for example, a flexible unit composed of G and S;
  • L can also be a polypeptide fragment comprising a rigid unit (also referred to as a rigid peptide segment herein).
  • the rigid unit comprises or consists essentially of rigid amino acids including, but not limited to, V, P, I, K, and L.
  • the rigid unit comprises one or more PPPX1LP (SEQ ID NO: 125), wherein X1 is any amino acid;
  • the rigid unit comprises one or more of X2APPPX1LP (SEQ ID NO: 126), wherein X1 is any amino acid and X2 is K or V.
  • the rigid unit comprises a polypeptide fragment selected from the group consisting of:
  • PPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 127);
  • PPPALPAVAPPPALP (SEQ ID NO: 129).
  • the rigid unit comprises a polypeptide fragment selected from the group consisting of:
  • KAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 130);
  • VAPPPALPAPVRLPGP (SEQ ID NO: 131);
  • VAPPPALPAVAPPPALP (SEQ ID NO: 132).
  • L contains both rigid and flexible units, and may be more than two units.
  • Fc is the Fc region of an immunoglobulin; the Fc can be from a human immunoglobulin; the Fc region can be an Fc region from IgG, IgA, IgD, IgE or IgM; preferably, the Fc region is from IgG an Fc region, such as an Fc region from IgG1, IgG2, IgG3 or IgG4; further preferably, the Fc region is an Fc region from IgG2; wherein the Fc region may carry one or more Fc regions compared to the sequence from which it is derived Substitutions, additions and/or deletions while still retaining the ability to extend half-life, e.g.
  • the mutation may be selected from: N297A, G236R/L328R, L234A/L235A, N434A, M252Y/S254T/T256E, M428L/N434S, T250R/M428L; the Fc region may be glycosylated or unglycosylated.
  • the insulin is selected from human insulin, bovine insulin, or porcine insulin, preferably human insulin; eg, the A and B chains of the insulin are derived from human insulin.
  • Y, El and E2 are all present, or where Y is absent, one of El and E2 is present.
  • the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-72.
  • the present invention provides an insulin-Fc fusion protein having an Ins-L-Fc structure.
  • the fusion protein of the first aspect of the present invention can remove the C chain under the action of a specific protease to produce the fusion protein of the second aspect of the present invention.
  • the insulin-Fc fusion protein exists in the form of a homodimer, the schematic diagram of which is shown in FIG. 3 .
  • the insulin-Fc fusion protein has secondary and tertiary structures similar to native insulin.
  • Ins is the insulin moiety that provides insulin activity, which includes insulin A and B chains linked by a covalent bond, but the A and B chains are located in different peptide chains; the covalent bond is preferably a disulfide bond .
  • L is a linker linking Z to Fc;
  • L can be a polypeptide fragment (also referred to as a linker peptide in some embodiments herein), eg, L comprises one, two or more selected from Ala, Thr, Gly and Ser A flexible unit of amino acids;
  • L can also be a polypeptide fragment comprising a rigid unit.
  • the L comprises one or more rigid units comprising or consisting essentially of rigid amino acids including, but not limited to, V, P, I, K, and L.
  • the rigid unit comprises one or more PPPX1LP (SEQ ID NO: 125), wherein X1 is any amino acid.
  • the rigid unit comprises one or more of X2APPPX1LP (SEQ ID NO: 126), wherein X1 is any amino acid and X2 is K or V.
  • the rigid unit comprises a polypeptide fragment selected from the group consisting of:
  • PPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 127);
  • PPPALPAVAPPPALP (SEQ ID NO: 129).
  • the rigid unit comprises a polypeptide fragment selected from the group consisting of:
  • KAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 130);
  • VAPPPALPAPVRLPGP (SEQ ID NO: 131);
  • VAPPPALPAVAPPPALP (SEQ ID NO: 132).
  • Fc is the Fc region of an immunoglobulin; the Fc can be from a human immunoglobulin; the Fc region can be an Fc region from IgG, IgA, IgD, IgE or IgM; preferably, the Fc region is from IgG an Fc region, such as an Fc region from IgG1, IgG2, IgG3 or IgG4; further preferably, the Fc region is an Fc region from IgG2; wherein the Fc region may carry one or more Fc regions compared to the sequence from which it is derived Substitutions, additions and/or deletions while still retaining the ability to extend half-life, e.g.
  • the mutation may be selected from: N297A, G236R/L328R, L234A/L235A, N434A, M252Y/S254T/T256E, M428L/N434S, T250R/M428L; the Fc region may be glycosylated or unglycosylated.
  • the insulin is selected from human insulin, bovine insulin, or porcine insulin, preferably human insulin; eg, the A and B chains of the insulin are derived from human insulin.
  • the L comprises CTPs, eg, 1, 2, 3 or more CTPs.
  • the present invention provides a method of producing an insulin-Fc fusion protein with enhanced insulin activity and extended half-life, comprising combining the fusion protein of the first aspect of the present invention with a site capable of cleaving the said contact with a specific protease, preferably the site-specific protease is Kex2 and/or Furin protease.
  • the insulin-Fc fusion proteins of the present invention with enhanced insulin activity and prolonged half-life in vivo are obtained by the methods described above.
  • the present invention provides a polynucleotide encoding the fusion protein, preferably the polynucleotide is an expression vector capable of expressing the fusion protein.
  • the present invention provides cells capable of expressing an insulin-Fc fusion protein, comprising the above-described polynucleotide.
  • the present invention provides a method for producing an insulin-Fc fusion protein, which comprises culturing the cells described in the fifth aspect of the present invention under conditions of expressing the insulin-Fc fusion protein; preferably further comprising The insulin-Fc fusion protein is contacted with a specific protease capable of cleaving the site, wherein the incubation step and the contact cleavage step can be performed simultaneously or separately.
  • the method can also include a protein purification step to obtain the target fusion protein.
  • the present invention provides a method for characterizing the structure of an insulin-Fc fusion protein, comprising the detection of the deglycosylated molecular weight of the fusion protein and the characterization of disulfide bonds.
  • the present invention provides a pharmaceutical composition comprising the fusion protein described in the first and third aspects above, the polynucleotide described in the fourth aspect or the fifth aspect said cells.
  • the present invention provides a method for lowering blood sugar and/or treating diabetes, comprising combining the fusion protein described in the first and second aspects, the polynucleotide described in the fourth aspect Or the cells described in the fifth aspect are administered to a subject in need thereof, preferably the diabetes is Type I or Type II diabetes.
  • additional administration of appropriate site-specific proteases, or utilization of appropriate site-specific proteases present in vivo may also be considered.
  • the present invention also provides the use of the fusion protein, polynucleotide or cell in preparing a medicament for lowering blood sugar and/or treating diabetes.
  • the present invention also provides fusion proteins, polynucleotides or cells for lowering blood sugar and/or treating diabetes.
  • Fig. 1 shows a schematic diagram of the vector of the present invention used for the expression of insulin precursor fusion protein; wherein, Fig. 1A is a stable transfection expression vector, and Fig. 1B is a transient transfection expression vector.
  • Figure 2 shows the SDS-PAGE electropherogram of the insulin-Fc fusion protein captured in Example 3;
  • M denotes marker, different Ps are the target proteins collected in sections during the chromatography process, and P+DTT is the target band after protein reduction.
  • the marker size is marked next to the SDS map of the SS302-002 molecule, and the markers used in other maps are the same.
  • Figure 3 is a schematic diagram showing the structure of the insulin-Fc fusion protease of the present invention before (3A) and after (3B) cleavage.
  • Figure 4 shows the results of the efficacy of SS 302-002 molecules in normal Kunming mice before and after enzyme cleavage.
  • Figure 5 shows the results of hypoglycemic effect of different fusion proteins on normal C57 mice; 5A shows the results of SS302-012M, SS302-019M, SS302-029M and SS302-035M, 5B shows SS302-008M, SS302-014M, SS302 -015M and SS302-030M results.
  • Figure 6 shows a dose-response plot of SS302-035M in normal C57 mice.
  • Figure 7 shows the hypoglycemic effect of SS302-002M (7A) and SS302-004M (7B) in type I diabetes model mice.
  • 8A and 8B show the hypoglycemic effects of SS302-008M, SS302-012M and SS302-035M in type I diabetes model mice.
  • Figure 9 is a graph showing the efficacy results of SS302-008M and SS302-012M in normal SD rats.
  • Figure 10 shows the pharmacokinetic results of SS302-008M and SS302-012M in SD rats.
  • Figure 11 shows the hypoglycemic effect (10A) and the blood concentration curve (10B) of SS302-008M and SS302-012M in normal SD rats.
  • Insulin is a hormone secreted by pancreatic beta cells to promote glucose absorption and inhibit fat degradation, thereby controlling blood sugar levels.
  • the DNA of the insulin gene region on the short arm of the 11th pair of chromosomes is transcribed into mRNA, and the mRNA moves from the nucleus to the endoplasmic reticulum of the cytoplasm, and is translated into preproinsulin consisting of 106 amino acid residues.
  • preproinsulin consisting of 106 amino acid residues.
  • the signal peptide When preproinsulin passes through the endoplasmic reticulum membrane, the signal peptide is cleaved by signal peptidase to generate a long peptide chain composed of 86 amino acids-proinsulin.
  • Insulin acts by proteolytic enzymes in the Golgi apparatus to cleave two arginine residues at positions 31 and 32, lysine residues at 64 and arginine residues at position 65, and the chain scission generates as a connecting part.
  • C-peptide which simultaneously generates insulin, is secreted out of beta cells and enters the blood circulation.
  • a small part of proinsulin that has not been hydrolyzed by protease enters the blood circulation with insulin, and the proinsulin has almost no biological activity, and its biological activity value is only 5%-10% of that of insulin.
  • the "insulin" of the present invention includes not only naturally occurring insulin, but also functional variants of insulin, and the functional variants refer to modifications to the natural sequence and/or structure of insulin, such as additions, deletions and/or substitution of one or more amino acids, resulting in a polypeptide that still has insulin activity (regulating blood sugar levels in the body). Amino acid substitutions, additions or deletions can be in naturally occurring mutant forms or artificial modifications for specific purposes. It is well known to those of ordinary skill in the art that in practice functional variants of insulin are often also referred to as insulins. Another example is the insulin analogs disclosed in CN105636979B and CN 201480006998. With reference to this specification, this practice is also covered herein.
  • a functional variant of insulin means at least 80% (preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%) of native insulin , 98%, 99%, 100%) amino acid sequence homology and still have insulin activity.
  • chemical substitutions eg, alpha-methylation, alpha-hydroxylation
  • deletions eg, deaminations
  • modifications eg, N- methylation
  • insulin analogs that have been marketed such as insulin lispro (Lilly), insulin aspart (Novo Nordisk), insulin glulisine (Aventis), Insulin glargine (Sanofi), insulin detemir (Novo Nordisk) and insulin degludec (Novo Nordisk).
  • Insulin lispro replaces the proline at position 28 and lysine at position 29 on the B chain of human insulin, and the other amino acid sequence and structure remain unchanged.
  • the function of insulin has not changed, but the insulin that is easy to form dimers and hexamers is no longer easy to aggregate into dimers and hexamers, but exists in the form of monomers , so that it is easily absorbed after subcutaneous injection, resulting in a rapid onset of action.
  • Insulin aspart is also a fast-acting insulin, which replaces the proline at position B28 of human insulin with aspartic acid, so that the insulin analog is not easy to form a hexamer, making it easy to absorb subcutaneously. , which works quickly.
  • Insulin lysine uses lysine instead of asparagine at position B3 and glutamic acid instead of lysine at position B29 to achieve a rapid onset of action.
  • Insulin glargine insulin glargine, Lantus, Lantus
  • the difference from human insulin is: 1) The 21st aspartic acid in the A chain is replaced by glycine; 2) 2 arginines are added to the C-terminus of the B chain acid residue.
  • the result of this change can be changed as follows: the replacement of the A21 position by glycine results in a more stable binding of the hexamer, and in the neutral environment of the subcutaneous tissue, the solubility decreases and precipitates, resulting in a slow absorption, similar to the no peak secretion of basal insulin , suitable for long-acting treatment, if a small amount of zinc is added, its action time will be further extended; the result of adding 2 arginine residues to the C-terminal of the B chain changes the isoelectric point of insulin, which increases from the original pH4.5 To pH 6.7, micro-precipitates are formed in the neutral environment of subcutaneous tissue, which prolongs the decomposition, absorption and action time of insulin.
  • Insulin detemir (Levermir) was developed and produced by Novo Nordisk. The amino acid at position B30 was removed from the structure, and a 14-carbon N-16-alkanoic acid group was attached to the lysine position at position B29. free fatty acid chains. In the liquid with zinc ions, the insulin molecule still exists in the form of hexamers, the modification of the fatty acid chain makes the subcutaneous absorption slow, and the insulin detemir in the plasma will combine with the albumin in the plasma due to the presence of the fatty acid. , and only free insulin detemir can play a hypoglycemic effect, and also prolong the action time of insulin.
  • Insulin degludec removes the threonine at position B30, and at the lysine position at position B29, is linked to the 16-carbon fatty diacid side chain through a glutamic acid linker. Under the action of phenol and zinc ions, insulin degludec is polymerized into double hexamers in the preparation. After subcutaneous injection, with the diffusion of phenol and the slow release of zinc ions, insulin degludec monomer can be slowly and continuously released, and then the monomer can be absorbed into the blood. Based on the above characteristics, insulin degludec has an ultra-long action time in diabetic patients, and its half-life is about 25 hours.
  • the fusion protein described herein refers not only to a protein formed by connecting amino acids through peptide bonds, but also to a protein formed by connecting two or more peptide chains through disulfide bonds.
  • the "insulin-Fc fusion protein” in the present invention refers to a fusion protein formed by insulin (including its functional variants) and the Fc region of an immunoglobulin, and is sometimes simply referred to as "fusion protein” herein.
  • the fusion protein before enzyme cleavage is sometimes referred to as "insulin precursor-Fc fusion protein", and correspondingly the simultaneous occurrence of "insulin-Fc fusion protein”
  • Fc fusion protein refers to the fusion protein after removal of the linking peptide moiety by enzymatic cleavage.
  • fusion protein before and after cleavage is not specifically distinguished, in which case the fusion protein or insulin-Fc fusion protein encompasses its before and after cleavage.
  • fusion protein or insulin-Fc fusion protein are often used directly to refer to this form when it is clear from the context which form the fusion protein refers to.
  • the A chain sequence of native human insulin is:
  • the B chain sequence of native human insulin is:
  • the fusion proteins described herein may also comprise additional sequences that prolong half-life in vivo, for example, one or more selected from Fc, CTP (C-terminal peptide), XTEN, SABA (serum albumin binding adnectin) and PAS Piece.
  • the additional sequences may be located at termini, linkers or other positions in the fusion protein.
  • the structural formulae X-E1-Y-E2-Z-L-Fc and Ins-L-Fc used herein also encompass these situations where the additional sequence is located elsewhere.
  • the linking peptide linking the A and B chains of insulin is the C-peptide.
  • the C-peptide includes both its naturally-occurring sequence and a variant form with the same function formed by substitution, deletion or addition of one or more amino acids based on the naturally-occurring sequence.
  • sequence of the native form C-peptide of human insulin is:
  • the linking peptide is not limited to the C-peptide of natural insulin or its variant/fragment, but can also be any other suitable polypeptide that plays the role of linking the A chain and the B chain of insulin.
  • the linker peptide can be 1-100 amino acids or more in length, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 50, 60, 70, 80, 90, 100 amino acids, or a value between any two of the above.
  • sequence of the linker peptide is:
  • EAEDLQVGQVELGGGPGAGSLQPLALEGSL (SEQ ID NO: 4)
  • the linker peptide can be in the form of a polypeptide of any length:
  • Human immunoglobulin IgG consists of 4 polypeptides (two identical copies of the light and heavy chains) covalently linked by disulfide bonds. Proteolysis of IgG molecules by papain yields two Fab fragments and one Fc fragment. The Fc fragment consists of two polypeptides linked together by disulfide bonds. Each polypeptide, from N to C terminus, consists of a hinge region, a CH2 domain and a CH3 domain. The Fc fragment structure is nearly identical in all subtypes of human immunoglobulins. IgG is one of the most abundant proteins in human blood, making up 70 to 75% of the total immunoglobulins in human serum.
  • the Fc region of an immunoglobulin is safe for use as a drug carrier because it is a biodegradable polypeptide that is metabolized in the body, and in addition, the Fc region of an immunoglobulin has a relatively low molecular weight compared to the entire immunoglobulin molecule, so it can be used in fusions. It is advantageous in the preparation, purification and production of proteins. Since the immunoglobulin Fc region does not contain Fab fragments (the amino acid sequence of which varies according to antibody subclass and is therefore highly heterogeneous), the immunoglobulin Fc region can be expected to greatly increase the homogeneity of the substance and have low antigenicity .
  • Fc region of an immunoglobulin refers to the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin without the heavy chain constant region of an immunoglobulin. Protein segments of variable regions of chains and light chains. It may also comprise the hinge region at the heavy chain constant region. Furthermore, the immunoglobulin Fc region of the present invention may contain a part or all of the Fc region comprising the heavy chain constant region 1 (CH1) and/or the light chain constant region (CL) in addition to the variable regions of the heavy and light chains , as long as it has substantially similar or better physiological functions to natural proteins.
  • CH1 heavy chain constant region 1
  • CL light chain constant region
  • an immunoglobulin Fc region of the present invention may comprise 1) a CH1 domain, a CH2 domain and a CH3 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) a CH2 structure domain and a CH3 domain; 5) a CH1 domain, a CH2 domain, a CH3 or CL domain; 6) a combination of one or more constant region domains and an immunoglobulin hinge region (or portion thereof); or 7 ) dimers of the respective domains of the heavy chain constant region and the light chain constant region.
  • the immunoglobulin Fc region of the present invention refers to any form of Fc or variant thereof having one or more heavy/light chain constant region domains or variants thereof, capable of conferring a function of extending half-life in vivo to the fusion protein /derivatives such as single chain Fc, monomeric Fc.
  • the immunoglobulin Fc region of the present invention may include the native amino acid sequence and sequence variants (mutants) thereof.
  • Amino acid sequence derivatives have sequences that differ from the native amino acid sequence due to deletions, insertions, non-conservative or conservative substitutions of one or more amino acid residues, or a combination thereof.
  • amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 known to be important for binding can be used as suitable targets for modification.
  • deletions can occur at complement binding sites, such as the C1q binding site and ADCC site.
  • Fc region can enhance the affinity of Fc for FcRn and prolong serum half-life, such as the T250Q/M428L mutation (CN 1798767B), these mutant forms of the Fc region Also within the meaning of the Fc region of the present invention.
  • Amino acid substitutions in proteins and peptides that generally do not alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979).
  • the most frequently occurring substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, both ways.
  • the Fc region can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
  • Fc derivatives are derivatives having the same biological activity or improved structural stability (eg, structural stability to heat, pH, etc.) as the Fc region of the present invention.
  • these Fc regions may be obtained in native forms isolated from humans and other animals including cattle, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or may be derived from transformed animal cells or microorganisms in their native form. Recombinant or derivative.
  • they can be obtained from native immunoglobulins by isolating intact immunoglobulins from human or animal organisms and treating them with proteolytic enzymes. Papain digests native immunoglobulins into Fab and Fc regions, while pepsin treatment results in pFc' and F(ab')2 fragments. For example, these fragments can be subjected to size exclusion chromatography to isolate Fc or pFc'.
  • the immunoglobulin Fc region of the present invention may be in a form having natural sugar chains, increased sugar chains compared to the natural form, or reduced sugar chains compared to the natural form, or may be a deglycosylated form.
  • the increase, decrease or removal of immunoglobulin Fc sugar chains can be accomplished by methods commonly used in the art, such as chemical methods, enzymatic methods, using genetic engineering methods, or by mutating the N297 glycosylation site. Removal of sugar chains from the Fc fragment results in a marked reduction in binding affinity to complement (C1q) and reduction or loss of antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity, thereby not inducing unwanted in vivo immune responses.
  • the deglycosylated or unglycosylated form of the immunoglobulin Fc region may be more suitable for the purposes of the present invention for use as a medicament.
  • deglycosylation means the enzymatic removal of sugar moieties from the Fc region
  • aglycosylated means that the Fc region is in an aglycosylated form from a prokaryotic (preferably E. coli) ), or by mutating the N297 glycosylation site to G, A, or any other amino acid.
  • the immunoglobulin Fc region can be an Fc region derived from IgG, IgA, IgD, IgE and IgM, or prepared by a combination or hybridization thereof.
  • it is derived from IgG or IgM (which are among the most abundant proteins in human blood), most preferably from IgG (which is known to prolong the half-life of ligand-binding proteins).
  • dimer or multimer can be formed from two or more segments selected from the group consisting of: IgG Fc segment, IgA Fc segment, IgM Fc segment, IgD Fc segment, and IgE Fc segment.
  • Proinsulin is inactive or very low in activity.
  • the conventional process for preparing recombinant insulin in the prior art is to express it in Escherichia coli or yeast, and then use trypsin or trypsin plus carboxypeptidase B to process it into an active molecule.
  • the conventional preparation process cannot be used, because there are many trypsin cleavage sites on the Fc, which will cause the proinsulin to be processed into an active molecule. Cut to fail.
  • single-chain insulin is directly combined with the Fc region, but the inventors have found through research that the in vivo activity of the above-mentioned insulin is very low.
  • the inventors unexpectedly found that if the mature mechanism of insulin in vivo can be simulated, and the structure of insulin is closer to that of natural insulin (the A chain and the B chain in the mature molecule are connected by disulfide bonds), and the Fc region is connected to the insulin conjugate at the same time. It can greatly increase the activity of insulin.
  • the applicant found that if the fusion polypeptide of the structure of the present invention is prepared, an enzyme cleavage site such as Kex2 protease or furin (Furin) is introduced into it, and active long-acting insulin can be obtained by processing with the protease. Conjugate molecules.
  • the Kex2 protease described in the present invention is a calcium ion-dependent proteolytic enzyme, which can specifically recognize and cut the carboxyl-terminal peptide bonds of bibasic amino acids such as Arg-Arg and Lys-Arg. Unlike trypsin, Kex2 cannot recognize and cleave the carboxy-terminal peptide bond of a single basic amino acid, arginine or lysine.
  • the Kex2 protease is responsible for processing the precursors of killer toxin and ⁇ -factor in yeast. Kex2 protease activity is not inhibited by conventional serine protease inhibitors such as aprotinin, PMSF, TPCK.
  • the furin described in the present invention is an important endoprotease in eukaryotic cells, located in the network outside the Golgi body, and is a major protein converting enzyme in the exocrine pathway, which can Recognizing specific amino acid sequences, after two self-cleavage activation in the endoplasmic reticulum-Golgi apparatus, the precursors of many important polypeptides and proteins in the secretion pathway are sheared to make them biologically active. It is named after its encoding gene (fur) is located upstream of the proto-oncogene fes/feps.
  • furin catalyzes the cleavage of the Arg-Xaa-Yaa-Arg carboxy-terminal peptide bond in the original protein (Xaa is any amino acid, Yaa is Arg or Lys) to produce a mature protein.
  • the connecting peptide between the A chain and the B chain is cleaved, so that a disulfide bond is formed between the A chain and the B chain in a manner similar to natural insulin, for example, in A7(Cys)-B7
  • the sulfhydryl groups in the four cysteines (Cys), A20(Cys)-B19(Cys) form two disulfide bonds to connect the A and B chains.
  • the role of the linker L is to connect the insulin A chain or B chain and the Fc region, which can be a polypeptide or a non-peptide chain chemical structure.
  • the linker is a polypeptide comprising or consisting essentially of a flexible unit of A, T, G and/or S (a flexible peptide segment), more eg a stretch of a flexible unit consisting of G and S; the The length of the flexible unit can be 2-50 or more amino acids, eg 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids.
  • the linker is a polypeptide comprising a rigid unit (rigid peptide segment) consisting essentially of rigid amino acids including, but not limited to, V, P, I, K, and L.
  • Insulin-Fc fusion protein is fermented and secreted by CHO cells, and the fusion protein undergoes a series of processing after transcription and translation in CHO cells, including proline hydroxylation, O sugar, N sugar, C-terminal lysine deletion, etc. Modifications that occur at sequences other than the B and A chains of insulin.
  • the insulin-Fc fusion protein also formed disulfide bonds in the organelles of CHO cells to stabilize its structure.
  • the disulfide bond of the insulin-Fc fusion protein is formed between two cysteine (Cys) residues. Its disulfide bond can be divided into two parts according to the position, one part exists in insulin, and the other part exists in Fc.
  • the disulfide bonds of insulin are located in the B and A chains, and the amino acids of the B and A chains are represented by position (X), BX and AX, respectively, in order from the N-terminus to the C-terminus.
  • the disulfide bond CysA7-CysB7, CysA20-CysB19, CysA6-CysA11.
  • the Fc region is composed of two single chains with the same amino acid sequence.
  • each single chain has 2 pairs of disulfide bonds, and there are 2 pairs of interchain disulfide bonds between the two single chains. 6 pairs of disulfide bonds.
  • UPLC-QTOF is a conventional instrument for analyzing the structure of biological macromolecules. Its main functional modules are UPLC and QTOF. After the sample to be tested is separated by UPLC, it enters the ion source in the state of solution for ionization and becomes charged ions. Under the action of the accelerating electric field, it enters the mass analyzer QTOF. Then, the m/z of various ions are captured by triple quadrupole (Q) and time-of-flight mass spectrometry (TOF) mass analyzers, and the software calculates the precise molecular weight, and finally realizes the structure analysis of complex biological macromolecular proteins.
  • the present invention adopts UPLC-QTOF, a commonly used instrument method with high resolution and high sensitivity, as an ideal method for analyzing fusion proteins. The ratios were analyzed and characterized.
  • the molecular weight and disulfide bond of the insulin-Fc fusion protein are consistent with the theory, the mismatch rate is low, and there are proline hydroxylation, O glycosylation, N glycosylation, C-terminal Post-translational modifications such as lysine deletions.
  • insulin precursor fusion protein is sometimes also referred to as insulin fusion protein.
  • the molecular form of the insulin precursor fusion protein is: proINS-L-Fc. It can be secreted and expressed in yeast or eukaryotic cells (such as CHO, HEK293, etc.), and the expressed protein exists in the form of homodimer.
  • a signal peptide and/or propeptide can be added to the N-terminus of the protein.
  • Signal peptides include, but are not limited to, the sequences shown in Table 1 below.
  • proINS refers to natural insulin precursors or analogs thereof from human or other sources.
  • the analogs include insertion, deletion, truncated or mutated insulin precursors, such as A14E ⁇ B16E ⁇ B25H ⁇ desB30 variants, A14E ⁇ B16H ⁇ B25H ⁇ desB30 variants or A14E ⁇ desB30 variants, and the like.
  • the analog can reduce the immunogenicity of insulin, or reduce proteolysis to improve the stability of insulin, or reduce the affinity of insulin and insulin receptor (IR) to prolong the half-life in vivo, and the like. It can also be used for any other purpose.
  • the insulin precursor of this embodiment can be processed into mature insulin by proteases such as Kex2, Furin, pancreatin and the like.
  • proteases such as Kex2, Furin, pancreatin and the like.
  • the insulin precursor of this example can also promote the correct folding and processing of the protein through the optimized C-peptide.
  • the insulin precursor analogs selected in this example include but are not limited to those shown in Table 3 below.
  • L represents the linker between proINS and Fc, which can be composed of amino acids from 0 to any length, and can be either a flexible polypeptide or a rigid polypeptide. L can help each of the two insulin molecules linked to the Fc homodimer to form the correct spatial structure.
  • the L sequence includes but is not limited to the sequences shown in Table 4 and Table 5.
  • the Fc is preferably human IgG; more preferred is human IgG without ADCC and CDC activities and variants thereof, such as IgG2 and Igg4; more preferred is human IgG with extended half-life after mutation.
  • Fc can also be a fragment of Fc or a fusion of Fc to other proteins/protein fragments.
  • the Fc selected by the present invention includes, but is not limited to, the following sequences.
  • Fc1 Human IgG1Fc
  • Fc2 Human IgG2Fc, T250Q/P331S/M428L
  • Fc3 Human IgG4Fc, S228P
  • Fc4 Human IgG2Fc, T250Q/N297A/P331S/M428L
  • Fc5 Human IgG2Fc, M252Y/S254T/T256E/N297A
  • Fc6 Human IgG2 Fc, N297A/M428L/N434S
  • Fc7 Human IgG4Fc, S228P/F234A/L235A
  • Fc8 Human IgG4Fc, S228P/M252Y/S254T/T256E/N297A
  • Fc9 Human IgG4Fc, S228P/N297A/M428L/N434S
  • Fc15 Human IgG4 Fc, S228P/F234A/L235A
  • Fc16 Human IgG2Fc
  • Insulin precursor fusion proteins can be processed by proteases such as Kex2, Furin, trypsin, etc., to remove sequences such as C-peptide, and convert them into mature insulin fusion proteins.
  • proteases such as Kex2, Furin, trypsin, etc.
  • the name of the protein after enzyme digestion is to add the letter M (mature) at the end of the name of the precursor protein.
  • the mature protein is named SS302-002M.
  • the amino acid sequences of mature insulin fusion proteins obtained by protease processing of some insulin precursor fusion proteins shown in the present invention are as follows.
  • Each insulin precursor fusion protein sequence was codon optimized according to CHO cell codon preference.
  • the optimized DNA sequence was cloned into eukaryotic expression vector pFRL3.0 or pTS1 through HindIII and EcoRI sites after whole gene synthesis.
  • the pFRL3.0 vector contains the dihydrofolatereductase (DHFR) gene, which can achieve high-level protein expression through co-amplification of DHFR and the target gene.
  • DHFR dihydrofolatereductase
  • pTS1 is a transient transfection plasmid without pressurized selection marker, which can quickly obtain a small amount of insulin precursor fusion protein for early molecular identification.
  • a schematic diagram of the expression vector of the insulin precursor fusion protein is shown in Figures 1A and 1B.
  • Partially expressing the insulin precursor-Fc fusion protein plasmid prepared in Example 1 was transferred into human embryonic kidney cells HEK-293, and the target protein was transiently expressed.
  • the HEK-293 cells were recovered and placed in a cell culture shake flask for subculture at a subculture density of 1.0 ⁇ 10 6 cells/mL, the medium was OPM-293CD05Medium (Shanghai Opmax Biotechnology Co., Ltd.), and the culture condition was 37°C , 120rpm, CO 2 , passage every two days, and can be used for transient transfection after one week of culture. Adjust the cell density before transfection, so that the cell density on the day of transfection is about 4.0 ⁇ 10 6 cells/ml.
  • the plasmid was transiently transfected into HEK-293 cells using the FectoPRO kit (Polyplus Transfection).
  • the ratio of Reagent is 1:1 ( ⁇ g/ ⁇ L), 1 ⁇ g DNA is transfected per ml of cells, corresponding to 1 ⁇ L Reagent.
  • the plasmid was diluted with room temperature Opti-MEM (Gibco) at 10% of the total volume, and mixed by shaking. Add the plasmid diluent at one time to the Reagent in a centrifuge tube, mix immediately, and incubate at room temperature for 10 min.
  • the prepared plasmid and transfection reagent mixture to the adjusted HEK-293 cell suspension at one time, mix well, put the cell culture shaker into the incubator, and the culture conditions are 37°C, 5% CO 2 , shaker Speed 120rpm. After the cells were transfected and cultured for 4 hours, the Volume of Booster, add 0.5 ⁇ L per mL of cells. After culturing for 24 hours, the culture conditions were changed to a temperature of 31°C, 5% CO 2 , 120 rpm fermentation culture, and cultured for 3-5 days. When the cell viability was lower than 90%, the supernatant was harvested by centrifugation (3000 rpm) and the expression level was detected. One-step purification and obtain the target protein.
  • Example 1 Partially express the plasmid of insulin precursor-Fc fusion protein prepared in Example 1 (transferred into Chinese hamster ovary cells (CHO DG44) (Invitrogen), construct a stable expression cell line, and select high-yield cell lines from it for fed-batch batching. culture to prepare the protein of interest.
  • CHO DG44 Chinese hamster ovary cells
  • the host cell DG44 was recovered and cultured with complete medium.
  • the composition of the complete medium was CDM1N (Shanghai Opmax Biotechnology Co., Ltd.) plus 1% HT (Invitrogen), and the culture conditions were 37° C., 5% CO 2 , shaker The rotational speed is 120rpm.
  • CDM1N Dishanghai Opmax Biotechnology Co., Ltd.
  • HT Invitrogen
  • the rotational speed is 120rpm.
  • Sterile suction certain day count cell suspension the cells were subcultured when density reached 3 ⁇ 10 6 -4 ⁇ 10 6 cells / mL, the initial density of the cells after passage maintained at about 1 ⁇ 10 6 cells / mL. When the total amount of cells met the transfection requirements, the cells were collected for electrotransfection experiments.
  • Host cells (CHO DG44) were transfected by electroporation, using a Bio-Rad electroporator, and a 4 mm electroporation cup was used for electroporation. 1 x 10 7 cells per electroporation, 40 ⁇ g of plasmid, and a total volume of 0.8 mL. After electroporation, cells were transferred into recovery medium (CDM1N+1%HT) containing 15 mL, and cultured in cell culture dishes for 48 hours.
  • recovery medium CDM1N+1%HT
  • the cells were centrifuged and resuspended in screening medium (CDM1N+100nM MTX), the cells were diluted to about 1 ⁇ 10 4 cells/mL, and the cell dilution solution was seeded in a 96-well plate, 100 ⁇ L/well, and The cells were placed in an incubator for static culture at a temperature of 37°C and 5% CO 2 . The cells were cultured for 5 days and then supplemented with 50 ⁇ L of screening medium. When the clone confluence rate reached 80% or more, the expression level was analyzed by dot blotting.
  • the antibody was HRP-labeled goat anti-human IgG antibody.
  • the clones with high expression levels were transferred from 96-well plates to 24-wells for continued cultivation, and 1 mL of screening medium was added. The same method was used for screening and expansion of high-yielding clones in 12-well plates and 10 cm cell culture dishes.
  • the high-yielding clones were transferred to cell culture shake flasks for culture at a culture temperature of 37°C, 5% CO 2 , and a shaker rotation speed of 120 rpm. After the high-yielding cell clones grow to a certain number, a part of the cells are collected for cryopreservation, and the remaining cells are cultured in fed batches. In fed-batch, the seeding density was 1 ⁇ 10 6 cells/ml, and the cells were cultured in a cell culture shake flask at a culture temperature of 37° C., 5% CO 2 , and a shaker rotation speed of 120 rpm.
  • Example 2 The respective insulin precursor-Fc fusion proteins expressed in Example 2 (SS302-002, SS302-004, SS302-005, SS302-008, SS302-012, SS302-014, SS302-015, SS302-019, SS302-029 , SS302-030, SS302-035) cell fermentation broth was centrifuged and filtered with a 0.22 ⁇ m filter to remove cell debris and then captured by affinity chromatography.
  • the impurity protein was washed with washing buffer (20mM Tris, 1.5M NaCl, 2M Urea, pH 7.5) to the baseline, and finally with 20mM Na 2 HPO 4 -citric acid, 0.4M Arg, pH3.5 elution buffer for elution, collect according to the reading of the UV detector, collect in sections, start collecting when the UV280nm absorption value is higher than 0.15AU, and again lower than 0.20AU Stop collecting.
  • the collected samples were immediately added with 2.0 mol/L Tris-HCl buffer and stirred slowly to adjust the pH of the samples to 6.5-7.0. Samples were stored at -80°C and subjected to subsequent SDS-PAGE analysis (Figure 2) and structural identification (see Example 4).
  • the SDS-PAGE results are shown in Figure 2, where load represents the chromatographic loading sample, FT represents the flow through sample (flow through), wash represents the rinsing sample, and P1, P2, P3, etc. represent the chromatographically collected samples.
  • Target protein sample "P combined" indicates that the samples collected in sections are combined according to the volume ratio of the collection volume, NaOH indicates the sample collected by column cleaning, DTT indicates the target protein after reduction, and M indicates the molecular weight marker (maker); A: SS302-002, B: SS302-004, C: SS302-005, D: SS302-008, E: SS302-014, F: SS302-019, G: SS302-030, H: SS302-012, I: SS302- 015, J: SS302-029, K: SS302-035.
  • the SS302-002 protein has a distinct upper band (about 130KD) and lower band (between 95-130KD), and exists in a high molecular weight form (>170KD).
  • the yield of the upper band (130KD) with a purity greater than 90% is about 60%.
  • the SS302-004 protein has obvious upper band (95-130KD) and lower band (about 95KD), of which the lower band P1-4 combined sample and the upper band P13-15 combined sample were sent to mass spectrometry for structural identification (implementation Example 4), and most of this molecule is the lower band of 95KD in the captured protein, and the upper band with a purity greater than 90% between 95-130KD has a lower yield (about 15%) in the captured protein;
  • the -005 protein is between 72-95KD, and the electrophoresis band is wide and diffuse; the common feature of these molecules is that they all contain GS flexible linkers; other molecules such as SS302-008, SS302-012, SS302-015, etc.
  • the disulfide bond of insulin precursor-Fc fusion protein containing flexible linkers (such as GS) has a certain mismatch rate, and the proportion of correct band recovery is low; Insulin precursor-Fc fusion proteins with flexible linkers (eg GS) and insulin precursor-Fc fusion proteins with rigid linkers have a lower disulfide bond mismatch rate, and the resulting proteins have higher content of correctly folded insulin precursor proteins.
  • the protein captured in step 1 was exchanged with G25 buffer 50mM Tris, 150mM NaCl, pH 8.0. After the exchange, each protein was cleaved with Kex2 to cleave the C-peptide to obtain insulin-Fc fusion protein, SS302-002 and SS302-004 enzymes
  • the cutting conditions are: the final protein concentration is 1 mg/mL, the feeding ratio (mass ratio) is 200:1 (precursor: Kex2), the final concentration of CaCl 2 is 20 mM/L, the total reaction volume is 5 mL and 3 mL, and the enzyme is carried out in a 37 °C water bath for 6 h. cut.
  • the digestion conditions of the three proteins of SS302-008 and SS302-012 were as follows: the final protein concentration was 1 mg/mL, the feeding ratio (mass ratio) was 50:1 (precursor: Kex2), the final concentration of CaCl 2 was 20 mM/L, and the total reaction The volume was 190 mL, and the reaction was performed in a water bath at 37 °C for 6 h.
  • SS302-014, SS302-015, SS302-019, SS302-029, SS302-030, SS302-035 digestion conditions are the final protein concentration of 1 mg/mL, and the feeding ratio (mass ratio) is 1:25 (Kex2: precursor) , the final concentration of CaCl 2 is 20mM/L, the total reaction volume is 60-180mL (the reaction volume of different proteins is slightly different), and the water bath is 37 °C for 6h.
  • the insulin-Fc fusion proteins after each insulin precursor-Fc fusion protease cleavage were named S302-002M, SS302-004M, SS302-005M, SS302-008M, SS302-012M, SS302-014M, SS302-015M, SS302-019M, SS302 -029M, SS302-030M and SS302-035M.
  • SS302-004M and SS302-005M were digested with 10KD ultrafiltration tube to remove the protease and other impurities, and the higher purity insulin- Fc fusion proteins.
  • SS302-008M, SS302-012M, SS302-014M, SS302-015M, SS302-029M, SS302-030M were digested with hydrophobic chromatography to remove impurities.
  • the structure of the insulin fusion protein precursor is proINS-L-Fc
  • proINS is a human insulin precursor (including B-C-A)
  • L is a linker.
  • the schematic diagram is shown in Figure 3A.
  • the insulin fusion protein precursor is enzymatically hydrolyzed to form a mature protein, and its structure is insulin (B-A)-L-Fc, the schematic diagram of which is shown in Figure 3B.
  • the linkers used in insulin fusion proteins include flexible linkers (eg GS) or rigid linkers (eg CTP, C1).
  • SS302-002 (about 130KD), SS302-002 (between 95-130KD), SS302-008, SS302-008M, SS302-012, SS302-012M, SS302-014, SS302-014M, SS302-015, SS302- 015M, SS302-019, SS302-019M, SS302-029, SS302-029M, SS302-030, SS302-030M, SS302-035, SS302-035M were tested for complete deglycosylation and reduction of sugars, and SS302-004 (95 The molecular weight of insulin-Fc fusion protein is consistent with the theory.
  • the spatial structure of the insulin-Fc fusion protein is supported and stabilized by the formation of disulfide bonds between the sulfhydryl groups of two cysteine (Cys) residues. Its disulfide bond is divided into two parts, namely the disulfide bond of the insulin part and the disulfide bond of the Fc part. The disulfide bond of the insulin part is located in the B chain and the A chain.
  • the amino acids of the B chain and A chain are named according to the sequence from the N-terminus to the C-terminus, BX and AX respectively, where X is the sequence number of the amino acid, and the disulfide bond is CysA7 -CysB7, CysA20-CysB19, CysA6-CysA11.
  • Fc consists of two polypeptide chains with the same sequence, each polypeptide chain contains 2 pairs of disulfide bonds, the two polypeptide chains contain a total of 4 pairs of disulfide bonds, and there are 2 pairs of interchain disulfide bonds between the two polypeptide chains. , so Fc has a total of 6 pairs of disulfide bonds.
  • the disulfide bond of the insulin-Fc fusion protein is not affected by the kex2 enzymatic hydrolysis process.
  • the analysis of the disulfide bond structure of the insulin-Fc fusion protein was completed by non-reducing denaturation, medium exchange, restriction endonuclease digestion, and UNIFI software analysis. There are two pretreatment methods.
  • the two chains of the insulin-Fc fusion protein precursor were named as 1 chain and 2 chain, respectively, and the peptide segment formed by Glu-C enzymatic hydrolysis of pretreatment method 1 was named as 1: VN, 2: VN (see Table 8 to Table 11), the peptide segment formed by Glu-C enzyme and trypsin digestion of pretreatment method 2 is named 1: VTN, 2: VTN (see Table 15) by UNIFI software; insulin -The two B chains of the mature protein of Fc fusion protein are named as 1 and 3 chains, and the two A+Fc chains are named as 2 and 4 chains, respectively. They are digested by Glu-C enzyme and trypsin in pretreatment method 2.
  • SS302-002 (about 130KD), SS302-002 (between 95-130KD), SS302-004 (between 95-130KD), SS302-004 (about 95KD), SS302-005, using preprocessing method 1 Disulfide bond analysis.
  • Pretreatment method 1 The denaturation is incomplete, so the linker region is difficult to be enzymatically hydrolyzed, so that the disulfide bond on the insulin and the disulfide bond in the hinge region are connected together by the linking peptide, the molecular weight is large, and it is difficult to match, this method will lack the key disulfide bond Information, mainly used to compare the two belts of SS302-002 (about 130KD) and SS302-002 (between 95-130KD) and SS302-004 (between 95-130KD) and SS302-004 (about 95KD) Differences in disulfide mismatches between bands.
  • SS302-008, SS302-012, SS302-012M, SS302-014, SS302-014M, SS302-015, SS302-015M, SS302-019M, SS302-029M, SS302-030M, SS302-035, SS302-035M are pre-processed Method 2 performs disulfide bond analysis.
  • the 2 steps of the pretreatment method are as follows: take 40 ⁇ L of SS302 protein sample, add 120 ⁇ L of 8M guanidine hydrochloride, water bath at 60 °C for 1 h, cool to room temperature, add 3.2 ⁇ L of 1 M IAA, incubate at room temperature for 45 min in the dark, use a 0.5 mL 10 kD ultrafiltration tube Under the conditions of 12000rpm and 4°C, the liquid was changed three times to 50mM Tris-HCl (pH8) buffer, so that the sample concentration after the liquid change was about 0.62 mg/mL.
  • the disulfide bond mismatch is expressed by the total mismatch rate and insulin mismatch rate.
  • the ratio is the ratio of the total XIC peak area of the mismatched disulfide peptides to the total XIC peak area of all disulfide peptides
  • the insulin mismatch ratio is the total XIC peak area of the mismatched disulfide peptides of the insulin moiety and all the disulfide peptides. The ratio of the total XIC peak area of the segment.
  • Table 7 The mismatch rates of 030M, SS302-035, and SS302-035M are shown in Table 7.
  • SS302-005 has the highest mismatch rate of all molecules
  • SS302-004 has the highest mismatch rate in the target band (between 95-130KD) Relatively low, but it is not easy to separate from the components with high mismatch rate, so its yield is not high.
  • the target band was comparable to the total mismatch rate and insulin mismatch rate of the fusion protein containing the flexible linker (SS302-004), both of which were It contains components with high total mismatch rate and insulin mismatch rate, and is not easy to purify and isolate.
  • While the precursor and mature proteins containing rigid linkers (SS302-008, SS302-012, SS302-012M, SS302-014, SS302-014M, SS302-015, SS302-015M, SS302-019M, SS302-029M, SS302- 030M, SS302-035, SS302-035M) had a total mismatch rate and insulin mismatch rate of less than 8%.
  • the disulfide bond results of SS302-002, SS302-004, SS302-012M, SS302-019M, SS302-030M, SS302-035, and SS302-035M in Example 4 are described in detail, and the results are shown in Table 8-16.
  • the rigid linker has a great positive effect on the accuracy of the structural expression of insulin fusion protein in CHO cells, and the stronger the rigidity, the higher the accuracy of its molecular structural expression.
  • a means that the fusion protein contains a flexible linker
  • b means that the fusion protein contains a rigid linker
  • the total mismatch ratio is the ratio of the total XIC peak area of mismatched disulfide peptides to the total XIC peak area of all disulfide peptides;
  • the insulin mismatch ratio is the ratio of the total XIC peak area of the mismatched disulfide bonds of the insulin moiety to the total XIC peak area of all disulfide bond peptides.
  • this molecule can be purified to obtain a band of about 130KD and a band between 95-130KD.
  • Disulfide bond identification was performed on these two bands, respectively, and the total disulfide mismatch rate and insulin mismatch rate were estimated.
  • the results showed that the total mismatch rate and insulin mismatch rate of the band about 130KD were both 9%, and the total mismatch rate and insulin mismatch rate of the band between 95-130KD were both 29%.
  • the results of the disulfide bonds of the band of about 130KD are shown in Table 8, and the results of the disulfide bonds of the bands between 95 and 130KD are shown in Table 9.
  • the mismatched disulfide bonds are mainly reflected in the self-linking of the insulin B chain and the mismatch between the two B chains of insulin. .
  • the underline is the segment where the mismatched disulfide bond is located
  • the underline is the segment where the mismatched disulfide bond is located
  • the underline is the segment where the mismatched disulfide bond is located
  • the underline is the segment where the mismatched disulfide bond is located
  • the underline is the segment where the mismatched disulfide bond is located
  • the underline is the segment where the mismatched disulfide bond is located
  • the underline is the segment where the mismatched disulfide bond is located
  • the underline is the segment where the mismatched disulfide bond is located
  • the underline is the segment where the mismatched disulfide bond is located
  • mice 24 healthy male Kunming mice (22-28g) were randomly divided into 4 groups, 6 mice/group: (1) SS302-002M-24nmol/kg;
  • mice were not fasted, and were given sufficient water and food.
  • the effect of insulin glargine continued to 4h.
  • the SS302-002 group started to have obvious hypoglycemic effect 4h after administration, but the SS302-002 group was significantly weaker than the SS302-002M group in terms of hypoglycemic effect and duration of drug effect, SS302-002 group vs. SS302-002M group
  • the maximum hypoglycemic effect was (5.33vs.2.97mmol/L) and the duration of drug effect was (36h vs.72h).
  • the above data analysis showed that the insulin fusion protein after excision of C-peptide had higher titer and better hypoglycemic effect.
  • Example 6 Hypoglycemic effect of SS302-008M, SS302-012M, SS302-014M, SS302-015M, SS302-019M, SS302-029M, SS302-030M and SS302-035M on normal C57 mice
  • mice/group 50 healthy male C57 mice aged 8-10 weeks and weighing 22-28g were randomly divided into 10 groups, 5 mice/group, including SS302-008M, SS302-012M, SS302-014M, SS302-015M, SS302-019M , SS302-029M, SS302-030M, SS302-035M, insulin degludec and control group.
  • the samples to be tested were subcutaneously administered at 15 nmol/kg neck, and the dose of insulin degludec was 30 nmol/kg, and blood glucose values were detected at different time points before and after administration. Mice were not fasted during the experiment.
  • the experimental data were plotted using Graphpad prism 7.0, and differences were analyzed by Mann-whiteney statistics.
  • mice in the administration group had obvious hypoglycemic effect.
  • the efficacy of insulin degludec (30nmol/kg) lasted to 12h; at the dose of 15nmol/kg, the duration of efficacy of different insulin fusion proteins on normal C57 mice was as follows:
  • Example 7 Hypoglycemic effect of different doses of SS302-035M in normal C57 mice
  • mice/group Twenty-five healthy male C57 mice aged 8-10 weeks and weighing 22-28g were randomly divided into 5 groups, 5 mice/group.
  • SS302-035M was administered subcutaneously in the neck at 5, 7.5, 10, and 12.5 nmol/kg, respectively, and blood glucose levels were detected at 0, 4, 24, 48, 72, 96, and 120 h. Mice were not fasted during the experiment.
  • the experimental data were plotted with Graphpad prism 7.0, and differences were analyzed with Mann-whiteney statistics.
  • the hypoglycemic effect of SS302-035M had an obvious dose-dependent relationship.
  • the lowest blood glucose value was 4.3mmol/L, and the drug effect lasted for 72h;
  • the lowest blood sugar value was 3.2mmol/L, and the drug effect lasted for 72h;
  • SSS302- In the 035M-10nmol/kg group, the lowest blood glucose value was 2.8mmol/L, and the drug effect lasted for 96h; in the SSS302-035M-12.5nmol/kg group, the lowest blood sugar value was 2.5mmol/L, and the drug effect lasted for 96h.
  • Example 8 Hypoglycemic effect of SS302-004M and SS302-002M in diabetic model mice
  • C57BL/6j mice (8 weeks old, body weight 22-28 g) were injected intraperitoneally with a 0.4% streptozotocin (STZ) solution prepared in citric acid-sodium citrate buffer, 40 mg/kg for five consecutive days, once a day , the fasting blood glucose level was detected on the 7th to 10th day after the last administration.
  • a fasting blood glucose value >13.8 mmol/L (fasting time is 8:00a.m-14:00p.m) is a successful model.
  • mice Thirty-five STZ-induced type I diabetic mice were randomly divided into 7 groups according to their blood glucose values: 1-2: SS302-002M high-dose and low-dose groups; 3-4: SS302-004M high-dose and low-dose groups; 5-6: Insulin glargine (Glargine Insulin) high and low dose groups; (7) control group (20mM Tris+300mM NaCl).
  • the high and low dose groups of SS302-002M and SS302-004M were administered by subcutaneous injection of 12.5nmol/kg and 6.25nmol/kg in the neck, respectively, and the high and low dose groups of insulin glargine were administered by 25nmol/kg and 12.5nmol/kg, respectively. kg neck subcutaneous injection. Changes in blood glucose levels were monitored at different time points before and after administration. During the experiment, the mice were not fasted, and were given sufficient water and food.
  • Example 9 Hypoglycemic effect of SS302-008M, SS302-012M and SS302-035M in diabetic model mice
  • mice (12 weeks old, body weight 22-28 g) were injected intraperitoneally with a 0.4% streptozotocin (STZ) solution in citric acid-sodium citrate buffer, 40 mg/kg for five consecutive days, once a day , on the 7th to 10th day after the last administration, the fasting blood glucose value >13.8mmol/L (the fasting time is 8:00a.m-14:00p.m), the model was successfully established.
  • STZ streptozotocin
  • mice with successful STZ modeling were randomly divided into 8 groups according to their blood glucose values: (1) SS302-008M-7.5nmol/kg group; (2) SS302-012M-7.5nmol/kg group; (3) SS302-012M-7.5nmol/kg group; ) SS302-035M-7.5nmol/kg group; (4) SS302-008M-15nmol/kg group; (5) SS302-012M-15nmol/kg group; (6) SS302-035M-15nmol/kg group; (7) Insulin degludec-30nmol/kg; (8) buffer control group (20mM Tris+150mM NaCl). Blood glucose levels were detected at different time points before and after administration. Mice were not fasted during the experiment. The experimental results were plotted using Graphpad prism 7.0, and differences were analyzed by Mann-whiteney statistics.
  • the maintenance time of SS302-035M at the same dose was significantly better than that of SS302-008M and S302-012M, especially in the low dose 7.5nmol/kg group (144h vs. 72h).
  • Fig. 8B after administration of insulin degludec at 30 nmol/kg, the blood glucose level of the diabetic mice decreased and recovered rapidly, reaching the lowest point in about 1 h, and returning to the initial blood glucose value in 24 h. This suggests that SS302-008M, SS302-012M, and SS302-035M have a longer PD spectrum, and the duration of drug efficacy is much better than that of insulin degludec.
  • Mouse anti-insulin monoclonal antibody (abcam, ab8302) was diluted with PBS to 1ug/mL, 100 ⁇ L/well was added to the ELISA plate, and placed at 4°C overnight for coating; the coating solution was removed, and 4% BSA was added after washing the plate 4 times with PBST , 250 ⁇ l/well, blocked at 37°C for 2h; after removing the blocking solution, the plate was washed 4 times with PBST.
  • the SS302-008M/SS302-012M standard was diluted with 2% BSA, starting from 200ng/ml, and a total of 8 gradients of doubling dilution were used to establish a standard. Rat serum was diluted to various folds with 2% BSA.
  • the negative control was normal rat serum.
  • SD rats have obvious hypoglycemic effect after administration of SS302-008M and SS302-012M.
  • the efficacy of SS302-008M was maintained to 96h, while the efficacy of SS302-012M was maintained to 72h.
  • the pharmacokinetic results of SS302-008M and SS302-012M in SD rats are shown in FIG. 10 .
  • the half-lives (T1/2) of SS302-008M and SS302-012M in SD rats were 16.32 ⁇ 0.77h and 13.39 ⁇ 0.43h, respectively.
  • the specific PK parameters are shown in Table 17.
  • Pharmacodynamic and pharmacokinetic parameters were evaluated after a single subcutaneous administration of 2.5 nmol/kg SS302-035M to 8-12 kg male healthy general-grade beagle dogs (4 dogs). Blood samples were collected at different time points before and after administration, and the sampling sites were peripheral veins of extremities. About 1 mL of whole blood was collected at each time point, put into an anticoagulation tube containing EDTA-K2, and then centrifuged at 3000 g/min for 10 min at 4°C to collect plasma.
  • SS302-035M at a dose of 2.5nmol/kg can significantly reduce the random blood sugar of beagle dogs, the effect of reducing blood sugar lasts for 120h, and there is no obvious symptoms of hypoglycemia.
  • the PK results showed that at the dose of 2.5nmol/kg, the in vivo half-life of SS302-035M in normal beagle dogs was 37.65 ⁇ 7.36h.
  • PK parameter result AUC 0- ⁇ (ng*hr/mL) 14631.28 ⁇ 628.94 T1/2(hr) 37.65 ⁇ 7.36 Tmax (hr) 2 ⁇ 0 Cmax (ng/mL) 485.75 ⁇ 26.18 Vss(mL/kg) 498.53 ⁇ 55.90 CL(mL/hr/kg) 11.83 ⁇ 1.29 MRT(hr) 39.05 ⁇ 4.11

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Abstract

提供一种胰岛素与免疫球蛋白Fc区的融合蛋白。具体地，涉及具有延长的体内半衰期和稳定性的胰岛素融合蛋白、包含该融合蛋白的制剂及其制备方法和应用。

Description

胰岛素-Fc融合蛋白及其应用
本申请要求于2020年7月24日提交到中国专利局的发明名称为“胰岛素-Fc融合蛋白及其应用”的中国专利申请202010723972.9的优先权，其内容通过引用以整体并入本文。
技术领域
本发明涉及多肽类药物领域，尤其涉及一种在经过位点特异性蛋白酶切割后具有增强的胰岛素活性和延长的体内半衰期的胰岛素-Fc融合蛋白、其制备方法和应用。
背景技术
近年来，糖尿病的发病率呈逐年升高趋势，1型糖尿病主要依赖外源性的胰岛素控制血糖；2型糖尿病随着病情的恶化发展，胰岛素也成为其控制血糖的主要药物。因此，利用胰岛素治疗糖尿病已成为较有效的方式。
胰岛素疗法对于具有异常胰岛素分泌(I型)或胰岛素抗性(II型)的患者是必需的，而且通过胰岛素施用能正常调节血液葡萄糖水平。然而，像其它蛋白质和肽激素一样，胰岛素具有很短的体内半衰期，因而具有反复施用的缺点。此类频繁施用对患者引起严重的疼痛和不适。出于这个原因，为了通过延长蛋白质的体内半衰期及降低施用频率来改善生活质量，已经对蛋白质配制剂和化学缀合(脂肪酸缀合物，聚乙烯聚合物缀合物)进行了许多研究。商品化的长效胰岛素包括由Sanofi Aventis制造的甘精胰岛素(lantus，持续约20小时至22小时)，和由Novo Nordisk制造的地特胰岛素(levemir，持续约18小时至22小时)和tresiba(德谷胰岛素，持续约40小时)。这些长效胰岛素配制剂不产生血液胰岛素浓度峰，因而它们适合作为基础胰岛素。然而，因为这些配制剂不具有足够长的半衰期，所以仍然存在每天或者两到三天注射一次的缺点。因而，在实现每周一次给药频率，以改善需要长期施用胰岛素的糖尿病患者的便利性的预定目标方面存在限制。
专利公开CN103509118B中公开了一种与抗体Fc区融合的单链胰岛素，虽然体外实验中证明该胰岛素-Fc融合蛋白具有改善的半衰期，但是该融合胰岛素的体内降糖活性较低，不适于作为临床施用。
控制血糖病症的成功性与要治疗的患者的依从性高度相关，并且期望降低所需的注射频率。然而，现有的这些经修饰的胰岛素分子要么活性很低，不适于临床使用；要么活性很高，施用于患者后有快速降糖效应，导致低血糖的副作用。因此本领域迫切需要一种适合于临床施用的新型长效胰岛素。
发明内容
经过大量研究，发明人提供了一种胰岛素-Fc融合蛋白，该融合蛋白经过位点特异性蛋白酶切割后获得增强的胰岛素活性和延长的体内半衰期，并且令人惊奇地发现，所述融合蛋白的体内降血糖效应更为平坦和稳定，能够提高临床用药的安全性和患者依从性，从而更好地实现血糖管理并提供更好的生活质量。
在一个方面，本发明提供一种胰岛素-Fc融合蛋白，其经过位点特异性蛋白酶切割后具有增强的胰岛素活性和延长的体内半衰期，其具有式(I)的结构：
X-E1-Y-E2-Z-L-Fc   (I)，
其中，
X和Z分别是胰岛素的B链和A链，并且如果X是B链，则Z是A链；如果X是A链，则Z是B链。
Y为任选的连接肽；Y的长度可以为1-100个或更多个氨基酸，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、50、60、70、80、90、100个或以上任意两个数值之间的值；例如Y为胰岛素C肽或者其变体或片段。
E1和E2中的一个存在或两个都存在，是包含位点特异性蛋白酶切割位点的一段氨基酸；E1和E2的长度可以是1-10个或更多个氨基酸，例如1、2、3、4、5、6、7、8、9或10个氨基酸；如果同时存在，E1和E2可被相同或不同的位点特异性蛋白酶切割，例如可被相同的位点特异性蛋白酶切割；如果Y存在，则优选地E1和E2两个都存在；如果Y不存在，则优选地E1和E2中存在一个；所述位点特异性蛋白酶切割位点可以是Kex2和/或弗林(Furin)蛋白酶的切割位点，例如是Kex2蛋白酶的切割位点。
L为连接Z与Fc的接头(Linker)；L可以是多肽片段，例如，L包含选自Ala、Thr、Gly和Ser中一个、两个或更多个氨基酸的柔性单元(本文中也称柔性肽段)，更例如一段由G和S构成的柔性单元；L也可以是包含刚性单元(本文中也称刚性肽段)的多肽片段。
在一些实施方案中，所述刚性单元包含或主要由刚性氨基酸构成，所述刚性氨基酸包括但不限于V、P、I、K和L。
在一些实施例中，所述刚性单元包含一个或更多个PPPX1LP(SEQ ID NO:125)，其中X1为任意氨基酸；
在另一些实施例中，所述刚性单元包含一个或更多个X2APPPX1LP(SEQ ID NO:126)，其中X1为任意氨基酸，X2为K或V。
在另一些实施例中，所述刚性单元包含选自以下序列的多肽片段：
PPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:127)；
PPPALPAPVRLPGP(SEQ ID NO:128)；和
PPPALPAVAPPPALP(SEQ ID NO:129)。
在另一些实施例中，所述刚性单元包含选自以下序列的多肽片段：
KAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:130)；
VAPPPALPAPVRLPGP(SEQ ID NO:131)；和
VAPPPALPAVAPPPALP(SEQ ID NO:132)。
在一些实施方案中，L既包含刚性单元，也包含柔性单元，可以是多于两个单元。
Fc是免疫球蛋白的Fc区；所述Fc可以来自人免疫球蛋白；所述Fc区可以是来自IgG、IgA、IgD、IgE或IgM的Fc区；优选地，所述Fc区是来自IgG的Fc区，例如来自IgG1、IgG2、IgG3或IgG4的Fc区；再优选地，所述Fc区是来自IgG2的Fc区；其中与其来源序列相比，所述Fc区可以带有一个或更多个替换、添加和/或缺失，同时仍保留延长半衰期的能力，例如来自人IgG的Fc区并带有降低或消除与FcγR结合的突变和/或增强与FcRn结合的一个或更多个突变，所述突变可以选自：N297A、G236R/L328R、L234A/L235A、N434A、M252Y/S254T/T256E、M428L/N434S、T250R/M428L；所述Fc区可以是糖基化的或未糖基化的。
在一些实施方案中，对于本发明的融合蛋白来说，所述胰岛素选自人胰岛素、牛胰岛素或猪胰岛素，优选地是人胰岛素；例如所述胰岛素的A链和B链源自人胰岛素。
在一些实施例中，Y、E1和E2都存在，或者其中Y不存在，E1和E2中存在一个。
在另一些实施例中，所述融合蛋白具有选自SEQ ID NO:47-72的氨基酸序列。
在第二个方面中，本发明提供一种胰岛素-Fc融合蛋白，其具有Ins-L-Fc结构。在一些实施方案中，本发明第一个方面的融合蛋白在特异性蛋白酶的作用下，可以去除C链，产生本发明第二个方面所述的融合蛋白。在一些实施方案中，所述胰岛素-Fc融合蛋白以同源二聚体的形式存在，其结构示意图如图3所示。在一些实施方案中，所述胰岛素-Fc融合蛋白具有与天然胰岛素类似的二级及三级结构。
其中，Ins是提供胰岛素活性的胰岛素部分，其包括通过共价键连接的胰岛素A链和B链，但A链和B链位于不同的肽链中；所述共价键优选地是二硫键。
L为连接Z与Fc的接头；L可以是多肽片段(在本文一些实施方案中也可称为连接肽)，例如，L包含选自Ala、Thr、Gly和Ser中一个、两个或更多个氨基酸的柔性单元；L还可以是包含刚性单元的多肽片段。
在一些实施例中，所述L包含一个或更多个刚性单元，所述刚性单元包含或主要由刚性氨基酸构成，所述刚性氨基酸包括但不限于V、P、I、K和L。
在另一些实施例中，所述刚性单元包含一个或更多个PPPX1LP(SEQ ID NO:125)，其中X1为任意氨基酸。
在另一些实施例中，所述刚性单元包含一个或更多个X2APPPX1LP(SEQ ID NO:126)，其中X1为任意氨基酸，X2为K或V。
在另一些实施例中，所述刚性单元包含选自以下序列的多肽片段：
PPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:127)；
PPPALPAPVRLPGP(SEQ ID NO:128)；和
PPPALPAVAPPPALP(SEQ ID NO:129)。
在另一些实施例中，所述刚性单元包含选自以下序列的多肽片段：
KAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:130)；
VAPPPALPAPVRLPGP(SEQ ID NO:131)；和
VAPPPALPAVAPPPALP(SEQ ID NO:132)。
Fc是免疫球蛋白的Fc区；所述Fc可以来自人免疫球蛋白；所述Fc区可以是来自IgG、IgA、IgD、IgE或IgM的Fc区；优选地，所述Fc区是来自IgG的Fc区，例如来自IgG1、IgG2、IgG3或IgG4的Fc区；再优选地，所述Fc区是来自IgG2的Fc区；其中与其来源序列相比，所述Fc区可以带有一个或更多个替换、添加和/或缺失，同时仍保留延长半衰期的能力，例如来自人IgG的Fc区并带有降低或消除与FcγR结合的突变和/或增强与FcRn结合的一个或更多个突变，所述突变可以选自：N297A、G236R/L328R、L234A/L235A、N434A、M252Y/S254T/T256E、M428L/N434S、T250R/M428L；所述Fc区可以是糖基化的或未糖基化的。
在一些实施方案中，所述胰岛素选自人胰岛素、牛胰岛素或猪胰岛素，优选地是人胰岛素；例如所述胰岛素的A链和B链源自人胰岛素。
在另一些实施方案中，所述L包含CTP，例如包含1、2、3个或更多个CTP。
在第三个方面中，本发明提供一种生产具有增强胰岛素活性和延长半衰期的胰岛素-Fc融合蛋白的方法，其包括将本发明第一方面中所述的融合蛋白与能切割所述位点的特异性蛋白酶接触，优选地所述位点特异性蛋白酶是Kex2和/或弗林(Furin)蛋白酶。
在一些实施方案中，本发明的具有增强胰岛素活性和延长体内半衰期的胰岛素-Fc融合蛋白是由上述方法得到的。
在第四个方面中，本发明提供编码所述融合蛋白的多核苷酸，优选地所述多核苷酸是能表达所述融合蛋白的表达载体。
在第五个方面中，本发明提供能表达胰岛素-Fc融合蛋白的细胞，其包含上述的多核苷酸。
在第六个方面中，本发明提供一种生产胰岛素-Fc融合蛋白的方法，其包括在表达胰岛素-Fc融合蛋白的条件下培养本发明第五个方面中所述的细胞；优选地还包括使所述胰岛素-Fc融合蛋白与能切割所述位点的特异性蛋白酶相接触，其中所述培养步骤和接触酶切步骤可以同时进行或分开进行。所述方法还可包括蛋白纯化步骤，获得目的融合蛋白。
在第七个方面中，本发明提供能表征胰岛素-Fc融合蛋白结构的方法，其包含融合蛋白脱糖分子量的检测和二硫键的表征。
在第八个方面中，本发明提供一种药物组合物，其包含以上第一和第三个方面中所述的融合蛋白、第四个方面中所述的多核苷酸或者第五个方面中所述的细胞。
在第九个方面中，本发明提供一种降低血糖和/或治疗糖尿病的方法，其包括将第一和第二个方面中所述的融合蛋白、第四个方面中所述的多核苷酸或者第五个方面中所述的细胞施予有此需要的对象，优选地所述糖尿病为I型或II型糖尿病。此外，当施用本发明第一个方面中所述的融合蛋白时，还可以考虑额外施用适当的位点特异性蛋白酶，或者利用体内存在的适当的位点特异性蛋白酶。
对应于以上降低血糖和/或治疗糖尿病的方法，本发明还提供所述融合蛋白、多核苷酸或细胞在制备用于降低血糖和/或治疗糖尿病的药物中的用途。本发明还提供用于降低血糖和/或治疗糖尿病的融合蛋白、多核苷酸或细胞。
附图说明
图1示本发明用于胰岛素前体融合蛋白表达的载体示意图；其中，图1A为稳定转染表达载体，图1B为瞬时转染表达载体。
图2示实施例3捕获的胰岛素-Fc融合蛋白的SDS-PAGE电泳图；M表示marker，不同P为层析过程分段收集的目的蛋白，P+DTT为蛋白还原后的目的条带。SS302-002分子SDS图旁标注为marker大小，其它图谱中使用的marker相同。
图3示本发明胰岛素-Fc融合蛋白酶切前(3A)和酶切后(3B)结构示意图。
图4示酶切前后的SS 302-002分子在正常昆明小鼠中的药效结果图。
图5示不同融合蛋白对正常C57小鼠的降糖药效结果图；5A示SS302-012M、SS302-019M、SS302-029M和SS302-035M的结果，5B示SS302-008M、SS302-014M、SS302-015M和SS302-030M的结果。
图6示SS302-035M在正常C57小鼠中的剂量效应图。
图7示SS302-002M(7A)和SS302-004M(7B)在I型糖尿病模型小鼠中的降糖效果。
图8A和8B示SS302-008M、SS302-012M和SS302-035M在I型糖尿病模型小鼠中的降糖效果。
图9示SS302-008M和SS302-012M在正常SD大鼠中的药效结果图。
图10示SS302-008M和SS302-012M在SD大鼠中的药代动力学结果。
图11示SS302-008M和SS302-012M在正常SD大鼠中的降糖效果(10A)和血药浓度曲线图(10B)。
具体实施方式
接下来将结合实施例中对本发明进行更详细的描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
术语
胰岛素
胰岛素是由胰腺β细胞分泌以促进葡萄糖吸收和抑制脂肪降解的激素，从而起控制血糖水平的作用。在β细胞的细胞核中，第11对染色体短臂上胰岛素基因区DNA向mRNA 转录，mRNA从细胞核移向细胞浆的内质网，翻译成由106个氨基酸残基构成的前胰岛素原，在其N末端有20个残基左右的信号肽。前胰岛素原通过内质网膜时，被信号肽酶酶切去信号肽，生成86个氨基酸组成的长肽链——胰岛素原。胰岛素在高尔基体中经蛋白水解酶的作用，切去31位和32位两个精氨酸残基、64位赖氨酸残基和65位精氨酸残基，断链生成作为连接部分的C肽，同时生成胰岛素，分泌到β细胞外，进入血液循环中。未经过蛋白酶水解的胰岛素原，一小部分随着胰岛素进入血液循环，胰岛素原几乎没有生物活性，其生物活性值仅有胰岛素的5％-10％。
本发明所述的“胰岛素”除了包含天然存在的胰岛素之外，还包括胰岛素的功能性变体，所述功能性变体是指对胰岛素的天然序列和/或结构进行修饰，比如添加、缺失和/或替换一个或更多个氨基酸，得到的仍具有胰岛素活性(调节机体中血糖水平)的多肽。氨基酸的替换、添加或缺失可以是天然存在的突变形式，也可以是为了特定目的而进行人工修饰而导致的突变形式。本领域普通技术人员熟知，实践中经常会将胰岛素功能性变体也称为胰岛素。又如CN105636979B和CN 201480006998所公开的胰岛素类似物。参见本说明书，在本文中也符合这种实践。
从另一方面来说，胰岛素的功能性变体是指与天然胰岛素具有至少80％(优选85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％)的氨基酸序列同源性、并仍然具有胰岛素活性的多肽。对于一些功能性变体，在特定氨基酸残基的一些基团上可以进行化学替换(例如，α-甲基化、α-羟基化)、缺失(例如，脱氨)或修饰(例如，N-甲基化)。
本领域技术人员熟知胰岛素功能性变体的制备方法和效果检验方法，已经上市的胰岛素类似物例如赖脯胰岛素(礼来)、门冬胰岛素(诺和诺德)、赖谷胰岛素(Aventis)、甘精胰岛素(赛诺菲)、地特胰岛素(诺和诺德)和德谷胰岛素(诺和诺德)等。
赖脯胰岛素(insulin lispro)是将人胰岛素B链上的第28位的脯氨酸和第29位的赖氨酸位置互换，其他的氨基酸序列和结构没有变化。两个氨基酸顺序颠倒的结果，胰岛素的功能没有发生变化，但原来容易形成二聚体和六聚体的胰岛素，不再容易聚集成二聚体和六聚体，而是以单体的形式存在，这样其在皮下注射后容易吸收，从而实现快速起效。
门冬胰岛素(insulin aspart)同样是一种速效胰岛素，是将人胰岛素B28位的脯氨酸被天冬氨酸所取代，从而使该胰岛素类似物不易形成六聚体，使其在皮下容易吸收，迅速发挥作用。
赖谷胰岛素使用了用赖氨酸替代B3位的天冬酰胺，谷氨酸替代B29位赖氨酸，从而实现快速起效的作用。
甘精胰岛素(insulin glargine，来得时，Lantus)，与人胰岛素的区别在于:1)在A链的第21位天冬氨酸被甘氨酸替代；2)B链C-末端加上2个精氨酸残基。这样改变的结果可以发生如下变化：A21位被甘氨酸替换的结果使得六聚体的结合更加稳定，在皮下组织中性环境下，溶解度降低产生沉淀，从而缓慢吸收，类似于基础胰岛素的无峰值分泌，适合长效治疗，如果添加少量锌，则其作用时间会进一步延长；B链C-末端加上2个精氨酸残基的结果改变了胰岛素的等电点，由原来的pH4.5上升至pH6.7，使得在皮下组织中性环境中形成微沉淀物，使胰岛素的分解、吸收及作用时间延长。
地特胰岛素(insulin detemir，Levermir)由诺和诺德公司研制生产，结构上去除了B30位的氨基酸，在B29位赖氨酸位点上连接上1个N-16-烷酸基的14碳的游离脂肪酸链。在有锌离子存在的药液中，胰岛素分子仍以六聚体形式存在，脂肪酸链的修饰使其在皮下吸收缓慢，血浆中的地特胰岛素由于脂肪酸的存在而会与血浆中的白蛋白结合，而只有游离的地特胰岛素才能发挥降血糖作用，也延长了胰岛素的作用时间。
德谷胰岛素去掉B30位的苏氨酸，在B29位赖氨酸位点上，通过一个谷氨酸连接子，与16碳的脂肪二酸侧链相连。在苯酚和锌离子的作用下，德谷胰岛素在制剂中聚合成双六聚体。皮下注射后，随着苯酚的弥散和锌离子缓慢释放，可缓慢而持续地释放出德谷胰岛素单体，进而单体吸收入血。基于上述特征，德谷胰岛素在糖尿病患者体内具有超长作用时间，其半衰期为25小时左右。
融合蛋白
本文所述的融合蛋白，既指由氨基酸通过肽键连接而成的蛋白，也可以指由2条或更多条肽链通过二硫键连接而形成的蛋白。
本发明所述的“胰岛素-Fc融合蛋白”是指胰岛素(包括其功能性变体)与免疫球蛋白Fc区形成的融合蛋白，在本文中有时被直接简称为“融合蛋白”。此外，为了区分酶切去除连接肽部分前后的胰岛素-Fc融合蛋白，有时会将经酶切前的所述融合蛋白称为“胰岛素前体-Fc融合蛋白”，相应地同时出现的“胰岛素-Fc融合蛋白”则指经酶切去除连接肽部分后的所述融合蛋白。然而，在本文中更常见的是不特别区分酶切前后的所述融合蛋白，此时所述融合蛋白或胰岛素-Fc融合蛋白涵盖其酶切前后的形式。另外，当通过上下文就能理解所述融合蛋白是指哪种形式时，也经常直接使用“融合蛋白”或“胰岛素-Fc融合蛋白”来指代这种形式。
天然人胰岛素的A链序列为:
Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn(SEQ ID NO:1)
天然人胰岛素的B链序列为：
Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr(SEQ ID NO：2)
本文所述的融合蛋白还可以包含延长体内半衰期的附加序列，所述附加序列例如选自Fc，CTP(C-terminal peptide)、XTEN、SABA(serum albumin binding adnectin)和PAS中的一个或更多个。所述附加序列可以位于融合蛋白中的末端、接头或其他位置。为了简化起见，本文中使用的结构式X-E1-Y-E2-Z-L-Fc和Ins-L-Fc也涵盖了所述附加序列位于其他位置的这些情形。
连接肽(Linking Peptide)
在胰岛素体内加工过程中，连接胰岛素A链和B链的连接肽是C肽。所述C肽既包含其天然存在的序列，也包括在所述天然存在的序列基础上进行一个或多个氨基酸的替换、缺失或添加而形成的具有相同功能的变体形式。
作为参考，人胰岛素的天然形式C肽的序列为：
Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln(SEQ ID NO：3)
在本发明的胰岛素-Fc融合蛋白中，所述连接肽不限于天然胰岛素的C肽或其变体/片段，还可以是起到连接胰岛素A链和B链作用的任何其他合适的多肽。在一些实施方案中，所述连接肽的长度可以为1-100个或更多个氨基酸，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、50、60、70、80、90、100个氨基酸或以上任意两个数值之间的值。
在一些实施例中，连接肽的序列为：
EAEDLQVGQVELGGGPGAGSLQPLALEGSL(SEQ ID NO：4)
Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser(SEQ ID NO：5)，
Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly(SEQ ID NO：6)，或
EAEDLQVGQVELSLQPLAL(SEQ ID NO：7)。
在另一些实施例中，连接肽可以是任意不同长度多肽的形式：
EAED(SEQ ID NO：8)，
YPGDV(SEQ ID NO：9)，
AA(SEQ ID NO：10)，或
EW(SEQ ID NO：11)。
Fc区/Fc片段
人免疫球蛋白IgG由4个通过二硫键共价连接的多肽(轻链和重链的两个相同拷贝)组成。通过木瓜蛋白酶来蛋白水解IgG分子产生两个Fab片段和一个Fc片段。所述Fc片段由通过二硫键连接在一起的两个多肽组成。每个多肽，从N至C末端，由铰链区、CH2结构域和CH3结构域组成。所述Fc片段结构在所有亚型的人免疫球蛋白中几乎相同。IgG是人血液中最丰富的蛋白之一，其组成了人血清中总免疫球蛋白的70至75％。
免疫球蛋白的Fc区用作药物载体是安全的，因为它是体内代谢的可生物降解多肽，此外，与整个免疫球蛋白分子相比，免疫球蛋白Fc区具有相对低的分子量，因此在融合蛋白的制备、纯化和生产上是有利的。由于免疫球蛋白Fc区不包含Fab片段(其氨基酸序列根据抗体亚类而不同并且因此是高度不均一的)，因此可以预期免疫球蛋白Fc区可极大地增加物质的均一性并具有低抗原性。
本领域普通技术人员一般认为，术语“免疫球蛋白的Fc区”是指含有免疫球蛋白的重链恒定区2(CH2)和重链恒定区3(CH3)，而不含免疫球蛋白的重链和轻链的可变区的蛋白质区段。它还可包含重链恒定区处的铰链区。此外，本发明的免疫球蛋白Fc区可含有包含除了重链和轻链的可变区以外的重链恒定区1(CH1)和/或轻链恒定区(CL)的Fc区的一部分或全部，只要它具有与天然蛋白质基本相似或更好的生理功能即可。此外，它可以是在CH2和/或CH3的氨基酸序列的相对长的部分中具有缺失的片段。例如，本发明的免疫球蛋白Fc区可包含1)CH1结构域、CH2结构域和CH3结构域；2)CH1结构域和CH2结构域；3)CH1结构域和CH3结构域；4)CH2结构域和CH3结构域；5)CH1结构域、CH2结构域、CH3或CL结构域；6)一种或更多种恒定区结构域与免疫球蛋白铰链区(或其部分)的组合；或7)重链恒定区和轻链恒定区的各个结构域的二聚体。总之，本发明的免疫球蛋白Fc区是指具有一个或更多个重链/轻链恒定区结构域或其变体形式、能够赋予融合蛋白延长体内半衰期功能的任何形式的Fc或其变体/衍生物，例如单链Fc、单体Fc。
此外，本发明的免疫球蛋白Fc区可包括天然氨基酸序列及其序列变体(突变体)。由于一个或更多个氨基酸残基的缺失、插入、非保守或保守替换或其组合，氨基酸序列衍生物 具有与天然氨基酸序列不同的序列。例如，在IgG Fc中，第214至238、297至299、318至322或327至331位处的已知对于结合重要的氨基酸残基可用作修饰的适合靶点。此外，其它多种衍生物也可以，包括其中缺失了能够形成二硫键的区域的、缺失了天然Fc形式N-端处数个氨基酸残基的或者向天然Fc形式的N-端添加了甲硫氨酸残基的衍生物。此外，为了去除效应子功能，缺失可发生于补体结合位点，如C1q结合位点和ADCC位点。制备这样的免疫球蛋白Fc区的序列衍生物的技术公开于WO97/34631和WO96/32478中，其作为参考全文并入本文。此外，本领域普通技术人员熟知，对Fc区中一个或更多个氨基酸进行突变可以增强Fc与FcRn的亲和力，延长血清半衰期，比如T250Q/M428L突变(CN 1798767B)，这些突变体形式的Fc区也在本发明Fc区含义范围内。
蛋白质和肽中一般不改变分子活性的氨基酸替换是本领域已知的(H.Neurath，R.L.Hill，The Proteins，Academic Press，New York，1979)。最常发生的替换是Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Thy/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/Glu和Asp/Gly，双向皆可。
如果需要，Fc区可以通过磷酸化、硫酸化、丙烯酸酯化、糖基化、甲基化、法尼基化(farnesylation)、乙酰化、酰胺化等进行修饰。
上述Fc衍生物是与本发明的Fc区具有相同生物活性或者改善的结构稳定性(例如对热、pH等的结构稳定性)的衍生物。
此外，这些Fc区可以得自从人和包括牛、山羊、猪、小鼠、兔、仓鼠、大鼠和豚鼠的其他动物中分离的天然形式，或者可以是得自转化的动物细胞或微生物的其重组体或衍生物。在本文中，它们可通过从人或动物有机体分离完整的免疫球蛋白并用蛋白水解酶对它们进行处理而从天然免疫球蛋白获得。木瓜蛋白酶将天然免疫球蛋白消化成Fab区和Fc区，而胃蛋白酶处理则导致产生pFc’和F(ab’)2片段。例如，可对这些片段进行尺寸排阻层析以分离Fc或pFc’。
此外，本发明的免疫球蛋白Fc区可以是具有天然糖链、与天然形式相比糖链增加或与天然形式相比糖链减少的形式，或者可以是去糖基化的形式。免疫球蛋白Fc糖链的增加、减少或去除可以通过本领域中常用方法完成，如化学法、酶促法、利用遗传工程方法，或者通过将N297糖基化位点突变的方法。从Fc片段去除糖链导致与补体(C1q)的结合亲和力明显降低和抗体依赖性细胞介导的细胞毒性或补体依赖性细胞毒性的降低或丧失，从而不会诱导不必要的体内免疫应答。鉴于此，去糖基化或未糖基化形式的免疫球蛋白Fc区可更适于本发明的目的以作为药物使用。
本发明所使用的术语“去糖基化”意指从Fc区酶促地去除糖部分，而术语“未糖基化”意指Fc区是以无糖基化形式由原核动物(优选大肠杆菌)产生，或者通过将N297糖基化位点突变为G、A或任意其它氨基酸的方法产生的。
此外，免疫球蛋白Fc区可以是衍生自IgG、IgA、IgD、IgE和IgM的Fc区，或者通过其组合或杂合制备而成。优选地，其衍生自IgG或IgM(它们是人类血液中最丰富的蛋白质之一)，最优选衍生自IgG(已知其延长配体结合蛋白质的半衰期)。
本发明所使用的术语“组合”意指编码相同来源的单链免疫球蛋白Fc区的多肽与不同来源的单链多肽连接从而形成二聚体或多聚体。即，二聚体或多聚体可以由选自以下的两种或更多种区段形成：IgG Fc区段、IgA Fc区段、IgM Fc区段、IgD Fc区段和IgE Fc区段。
蛋白酶酶解
胰岛素原是没有活性的或活性很低，现有技术中制备重组胰岛素的常规工艺是用大肠杆菌或酵母菌进行表达，然后用胰酶或胰酶加羧肽酶B来加工成活性分子。然而当使用抗体的Fc区与胰岛素形成缀合物时，常规制备工艺是不能使用的，这是因为Fc上有很多胰酶的酶切位点，在胰岛素原的加工成活性分子的过程中会被切割失效。现有技术中为了规避上述问题，直接将单链胰岛素与Fc区结合，不过发明人经过研究发现，上述胰岛素的体内活性非常低。
发明人意外的发现，如果能够模拟胰岛素体内的成熟机制，并制备出与天然胰岛素结构更加接近(成熟分子中的A链和B链通过二硫键连接)、同时连接有Fc区的胰岛素缀合物，则可以大大提高胰岛素的活性。申请人经过大量的筛选，发现如果制备本发明结构的融合多肽，其中引入Kex2蛋白酶或弗林蛋白酶(Furin)等酶切位点，利用所述蛋白酶进行加工即可获得有活性的长效的胰岛素缀合物分子。
本发明中所述的Kex2蛋白酶是一种钙离子依赖性蛋白水解酶，能特异性识别和切割Arg-Arg、Lys-Arg等双碱基氨基酸羧基端肽键。与胰蛋白酶不同，Kex2不能识别和切割单一碱性氨基酸即精氨酸或赖氨酸的羧基端肽键。在酵母体内Kex2蛋白酶负责加工killer toxin和α-factor的前体。Kex2蛋白酶活性不受常规的丝氨酸蛋白酶抑制剂如抑肽酶、PMSF、TPCK所抑制。
本发明中所述的弗林蛋白酶(Furin)是真核生物细胞中一种重要的内切蛋白酶，定位于高尔基体外侧的网络中，是一种外泌途径中主要的蛋白质转化酶，其能够识别特定的氨基酸序列，在内质网-高尔基体中经两次自剪切活化后，对分泌途径中许多重要的多肽和蛋白 的前体进行剪切加工，使之具有生物活性。因其编码基因(fur)位于原癌基因fes/feps的上游而得名。具体而言，弗林蛋白酶催化切割原蛋白中的Arg-Xaa-Yaa-Arg羧基端肽键(Xaa为任意一种氨基酸，Yaa为Arg或Lys)产生成熟的蛋白质。
本发明的融合多肽经过蛋白酶处理后，A链和B链之间的连接肽被切除，于是A链和B链之间通过类似天然胰岛素的方式形成二硫键，例如在A7(Cys)-B7(Cys)、A20(Cys)-B19(Cys)四个半胱氨酸中的巯基形成两个二硫键，使A、B两链连接起来。另外A链内部的A6(Cys)与A11(Cys)之间也优选存在一个二硫键。发明人惊奇的发现，即使A链或B链与Fc区存在连接，并不影响A链和B链之间形成正确的二硫键连接和空间折叠，形成与Fc区融合的胰岛素，进而完成了本发明。
接头(Linker)
在本发明融合蛋白中，接头L的作用在于连接胰岛素A链或B链与Fc区可以是多肽，也可以是非肽链的化学结构。
在一些实施方案中，接头是多肽，所述多肽包含或主要由A、T、G和/或S构成的柔性单元(柔性肽段)，更例如一段由G和S构成的柔性单元；所述柔性单元的长度可以是2-50个或更多个氨基酸，例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45或50个氨基酸。
在另一些实施方案中，接头是多肽，所述多肽包含主要由刚性氨基酸构成的刚性单元(刚性肽段)，所述刚性氨基酸包括但不限于V、P、I、K和L。
结构表征
胰岛素-Fc融合蛋白由CHO细胞发酵分泌，融合蛋白在CHO细胞中转录翻译后又经历了一系列的加工，包括脯氨酸羟基化、O糖、N糖、C端的赖氨酸缺失等翻译后修饰，这些修饰发生在胰岛素B链和A链之外的序列上。除此之外，胰岛素-Fc融合蛋白在CHO细胞的细胞器中还形成了二硫键以稳定其结构。
胰岛素-Fc融合蛋白的二硫键在两个半胱氨酸(Cys)残基间形成。其二硫键根据位置可分为两部分，一部分存在于胰岛素，一部分存在于Fc。胰岛素的二硫键位于B链和A链，按照从N端到C端的顺序对B链和A链的氨基酸以位置(X)表示，分别为BX和AX，在一些实施例中，二硫键为CysA7-CysB7、CysA20-CysB19、CysA6-CysA11。Fc区由两条氨 基酸序列一样的单链组成，在一些实施例中，每条单链内均有2对二硫键，两条单链之间有2对链间二硫键，Fc上共有6对二硫键。
UPLC-QTOF是一种解析生物大分子结构的常规仪器。其主要功能模块为UPLC和QTOF，待测样品经UPLC分离后以溶液的状态进入离子源发生电离，变成带电离子，在加速电场的作用下，进入质量分析器QTOF，在电场和磁场的作用下，经三重串联四级杆(Q)和飞行时间质谱(TOF)两种质量分析器捕获得到各种离子的m/z，软件计算出精确分子量，最终实现复杂生物大分子蛋白的结构解析。本发明采用了UPLC-QTOF这种常用的具有高分辨率、高灵敏度的仪器方法作为解析融合蛋白的理想手段，主要对融合蛋白的去糖及去糖还原分子量、二硫键及二硫键错配率进行了分析表征。
结果表明，在一些实施例中，胰岛素-Fc融合蛋白的分子量和二硫键均与理论一致，错配率低，且存在脯氨酸羟基化、O糖基化、N糖基化、C端的赖氨酸缺失等翻译后修饰。
实施例1：胰岛素前体融合蛋白表达载体的构建
本实施例主要记载胰岛素前体融合蛋白分子构建方法，本文中，胰岛素前体融合蛋白有时也称为胰岛素融合蛋白。该胰岛素前体融合蛋白的分子形式为：proINS-L-Fc。可在酵母或真核细胞(如CHO、HEK293等)分泌表达，表达后的蛋白以同源二聚体的形式存在。为帮助蛋白分泌表达，可在蛋白的N端加入信号肽和/或前肽。信号肽包括但不限于如下表1所示序列。
表1.信号肽序列
信号肽名称 序列
NS MALWMRLLPLLALLALWGPDPAAA(SEQ ID NO: 12)
LS MRSLGALLLLLSACLAVSA(SEQ ID NO: 13)
HMM+38 MWWRLWWLLLLLLLLWPMVWA(SEQ ID NO: 14)
Exendin-4 MKIILWLCVFGLFLATLFPISWQ(SEQ ID NO: 15)
proINS指来自人或其它的天然胰岛素前体或其类似物。所述类似物包括插入、缺失、截短或是突变的胰岛素前体，例如A14E\B16E\B25H\desB30变体，A14E\B16H\B25H\desB30变体或者A14E\desB30变体等。所述类似物可以是降低胰岛素的免疫原性、或者是降低蛋白酶解以提高胰岛素的稳定性、或者是降低胰岛素与胰岛素受体(IR)的亲和力以延长体内半衰期等。也可以是其它任何用途。
本实施例的胰岛素前体可通过蛋白酶如Kex2、Furin、胰酶等加工成成熟胰岛素。本实施例的胰岛素前体也可通过优化的C肽，促进蛋白的正确折叠和加工。本实施例中选取的胰岛素前体类似物包括但不限于下表3所示。
表3.胰岛素前体或类似物序列
Figure PCTCN2021107040-appb-000001
Figure PCTCN2021107040-appb-000002
L表示proINS和Fc之间的接头，可以是0到任意长度的氨基酸组成，既可以是柔性多肽，也可以是刚性多肽。L可以帮助与Fc的同源二聚体相连的两个胰岛素分子各自形成正确的空间结构。优选的，所述L序列包括但不限于如表4、表5所示的序列。
表4.柔性接头序列
L名称 L序列
GS-(G 4S) 5 GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:23)
(G 4S) 5 GGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:24)
(G 4S) 3 GGGGSGGGGSGGGGS(SEQ ID NO:25)
表5.刚性接头序列
Figure PCTCN2021107040-appb-000003
Figure PCTCN2021107040-appb-000004
Fc优选人的IgG；更优选的是不含ADCC和CDC活性的人IgG及其变体，如IgG2和IGg4；更优选的是突变后延长半衰期的人IgG。Fc也可以是Fc的片段或是Fc与其它蛋白/蛋白片段的融合。本发明所选择的Fc包括但不限于以下序列。
Fc1：人IgG1Fc
Figure PCTCN2021107040-appb-000005
Fc2：人IgG2Fc，T250Q/P331S/M428L
Figure PCTCN2021107040-appb-000006
Fc3：人IgG4Fc，S228P
Figure PCTCN2021107040-appb-000007
Fc4：人IgG2Fc，T250Q/N297A/P331S/M428L
Figure PCTCN2021107040-appb-000008
Fc5：人IgG2Fc，M252Y/S254T/T256E/N297A
Figure PCTCN2021107040-appb-000009
Fc6：人IgG2Fc，N297A/M428L/N434S
Figure PCTCN2021107040-appb-000010
Fc7：人IgG4Fc,S228P/F234A/L235A
Figure PCTCN2021107040-appb-000011
Fc8：人IgG4Fc，S228P/M252Y/S254T/T256E/N297A
Figure PCTCN2021107040-appb-000012
Fc9：人IgG4Fc，S228P/N297A/M428L/N434S
Figure PCTCN2021107040-appb-000013
Figure PCTCN2021107040-appb-000014
Fc15：人IgG4Fc，S228P/F234A/L235A
Figure PCTCN2021107040-appb-000015
Fc16：人IgG2Fc
Figure PCTCN2021107040-appb-000016
本发明所构建的部分胰岛素前体融合蛋白的氨基酸序列特征如表6所示。
表6.胰岛素前体融合蛋白(proINS-L-Fc)序列特征
Figure PCTCN2021107040-appb-000017
Figure PCTCN2021107040-appb-000018
胰岛素前体融合蛋白可通过蛋白酶如Kex2、Furin、胰酶等加工，去除C肽等序列，转变为成熟的胰岛素融合蛋白。在本专利的所有实施例中，酶切加工后蛋白的命名均为在前体蛋白名称的末端添加字母M(mature)。例如胰岛素前体融合蛋白SS302-002通过蛋白酶Kex2加工后，成熟蛋白命名为SS302-002M。本发明所示的一些胰岛素前体融合蛋白经蛋白酶加工后得到的成熟胰岛素融合蛋白的氨基酸序列如下。
SS302-001M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:73)；
A链-L-Fc1：
Figure PCTCN2021107040-appb-000019
SS302-002M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:75)；
A链-L-Fc2：
Figure PCTCN2021107040-appb-000020
SS302-003M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:77)；
A链-L-Fc3：
Figure PCTCN2021107040-appb-000021
SS302-004M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:79)；
A链-L-Fc2：
Figure PCTCN2021107040-appb-000022
SS302-005M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:81)；
A链-L-Fc4：
Figure PCTCN2021107040-appb-000023
Figure PCTCN2021107040-appb-000024
SS302-006M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:83)；
A链-L-Fc16：
Figure PCTCN2021107040-appb-000025
SS302-007M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:85)；
A链-L-Fc16：
Figure PCTCN2021107040-appb-000026
SS302-008M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:87)；
A链-L-Fc16：
Figure PCTCN2021107040-appb-000027
SS302-009M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:89)；
A链-L-Fc16：
Figure PCTCN2021107040-appb-000028
SS302-011M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:91)；
A链-L-Fc16：
Figure PCTCN2021107040-appb-000029
SS302-012M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:93)；
A链-L-Fc16：
Figure PCTCN2021107040-appb-000030
SS302-013M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:95)；
A链-L-Fc16：
Figure PCTCN2021107040-appb-000031
Figure PCTCN2021107040-appb-000032
SS302-014M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:97)；
A链-L-Fc5：
Figure PCTCN2021107040-appb-000033
SS302-015M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:99)；
A链-L-Fc6：
Figure PCTCN2021107040-appb-000034
SS302-016M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:101)；
A链-L-Fc7：
Figure PCTCN2021107040-appb-000035
SS302-017M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:103)；
A链-L-Fc8：
Figure PCTCN2021107040-appb-000036
SS302-018M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:105)；
A链-L-Fc9：
Figure PCTCN2021107040-appb-000037
SS302-019M
B链：FVNQHLCGSHLVEALELVCGERGFHYTPKTRR(SEQ ID NO:107)；
A链-L-Fc7：
Figure PCTCN2021107040-appb-000038
SS302-022M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTKRIKR(SEQ ID NO:109)；
A链-L-Fc16：
Figure PCTCN2021107040-appb-000039
Figure PCTCN2021107040-appb-000040
SS302-023M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTDDDDK(SEQ ID NO:111)；
A链-L-Fc16：
Figure PCTCN2021107040-appb-000041
SS302-029M
B链：FVNQHLCGSHLVEALELVCGERGFHYTPKTRR(SEQ ID NO:113)；
A链-L-Fc8：
Figure PCTCN2021107040-appb-000042
SS302-030M
B链：FVNQHLCGSHLVEALELVCGERGFHYTPKTRR(SEQ ID NO:115)；
A链-L-Fc9：
Figure PCTCN2021107040-appb-000043
SS302-035M
B链：FVNQHLCGSHLVEALHLVCGERGFHYTPKR(SEQ ID NO:117)；
A链-L-Fc15：
Figure PCTCN2021107040-appb-000044
SS302-036M
B链：FVNQHLCGSHLVEALELVCGERGFHYTPKR(SEQ ID NO:119)；
A链-L-Fc15：
Figure PCTCN2021107040-appb-000045
SS302-037M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKR(SEQ ID NO:121)；
A链-L-Fc15：
Figure PCTCN2021107040-appb-000046
SS302-038M
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR(SEQ ID NO:123)；
A链-L-Fc15：
Figure PCTCN2021107040-appb-000047
Figure PCTCN2021107040-appb-000048
根据《分子克隆实验指南(第三版)》记载的方法，进行胰岛素前体融合蛋白的表达载体构建。
根据CHO细胞密码子偏好性对各胰岛素前体融合蛋白序列进行密码子优化。
优化后的DNA序列经全基因合成后通过HindIII和EcoRI位点克隆到真核表达载体pFRL3.0或pTS1上。pFRL3.0载体包含dihydrofolatereductase(DHFR)基因，可通过DHFR和目的基因的共扩增来实现蛋白的高水平表达，该载体转染CHO细胞后通过MTX加压筛选建立稳定表达细胞系。pTS1为瞬时转染质粒，不含加压筛选标记，可以快速获得少量胰岛素前体融合蛋白用于早期分子鉴定。胰岛素前体融合蛋白的表达载体示意图如图1A和1B所示。
实施例2：胰岛素融合蛋白的表达
1.瞬时转染制备目的蛋白
将实施例1制备的部分表达胰岛素前体-Fc融合蛋白的质粒转入人胚胎肾细胞HEK-293中，瞬时表达目的蛋白。复苏HEK-293细胞，置于细胞培养摇瓶中传代培养，传代接种密度为1.0×10 6个细胞/mL，培养基为OPM-293CD05Medium(上海奥浦迈生物科技有限公司)，培养条件为37℃，120rpm，CO 2，每隔两天传代一次，培养一周后可用于瞬时转染。转染前调整细胞密度，使转染当天细胞密度在4.0×10 6个细胞/ml左右。采用FectoPRO试剂盒(Polyplus Transfection)将质粒瞬时转染HEK-293细胞中，DNA与
Figure PCTCN2021107040-appb-000049
Reagent比例为1：1(μg/μL)，每毫升细胞转染1μgDNA，对应1μL
Figure PCTCN2021107040-appb-000050
Reagent。用瞬转总体积10％的室温Opti-MEM(Gibco)稀释质粒，震荡混匀。将质粒稀释液一次性加入到
Figure PCTCN2021107040-appb-000051
Reagent的离心管中，立即混匀，室温孵育10min。将制备好的质粒与转染试剂混合物一次性加入调整好的HEK-293细胞悬液中，混匀，细胞培养摇瓶放入培养箱，培养条件为温度37℃，5％CO 2，摇床转速120rpm。细胞转染培养4h后，加入Volume of
Figure PCTCN2021107040-appb-000052
Booster，每毫升细胞加入0.5μL。培养24小时后，培养条件转换为温度31℃，5％CO 2，120rpm发酵培养，培养3-5天，细胞活力低于90％时，离心收获上清(3000rpm)并检测表达量，进行下一步纯化并获得目的蛋白。
2.稳定表达细胞株的转染和高表达细胞株的筛选
将实施例1制备的部分表达胰岛素前体-Fc融合蛋白的质粒(转入中国仓鼠卵巢细胞(CHO DG44)(Invitrogen)中，构建稳定表达细胞系，从中筛选高产的细胞株进行补料分批培养以制备目的蛋白。
用完全培养基复苏宿主细胞DG44并进行培养，完全培养基的组成是CDM1N(上海奥浦迈生物科技有限公司)加上1％HT(Invitrogen)，培养条件为37℃，5％CO 2，摇床转速为120rpm。每天无菌吸取一定的细胞悬液计数，当细胞密度达到3×10 6-4×10 6个细胞/mL时进行传代培养，传代后细胞初始密度保持在1×10 6个细胞/mL左右。当细胞总量满足转染需求时，收集细胞进行电转染试验。采用电穿孔方法转染宿主细胞(CHO DG44)，使用Bio-Rad电转仪，电转时选用4mm的电转杯，具体电转参数：电压为290V、脉冲长度为20毫秒，电转次数为1次。每次电击1×10 7个细胞，质粒40μg，总体积为0.8mL。电转后将细胞转入含有15mL的恢复培养基(CDM1N+1％HT)中，在细胞培养皿中静置培养48小时。48小时后，离心细胞并重悬于筛选培养基(CDM1N+100nM MTX)中，将细胞稀释到1×10 4个细胞/mL左右，接细胞稀释液种于96孔板中，100μL/孔，将细胞置于培养箱中静置培养，培养温度37℃，5％CO 2。细胞培养5天后补50μL的筛选培养基，待克隆汇合率达到80％或以上时，采用点印迹(Dot Blotting)方法分析表达量，抗体为HRP标记羊抗人IgG抗体，筛选出表达量相对高的克隆，将表达量高的克隆由96孔板转入24孔中继续培养，并补加1mL筛选培养基。采用同样的方法进行12孔板和10cm细胞培养皿高产克隆筛选和扩增。
为了提高融合蛋白产量，采用递增MTX浓度的加压方法培养细胞。通过MTX对DHFR基因的抑制，实现DHFR基因和融合蛋白基因的共扩增。筛选过程采用了本领域普通技术人员公知的方法，例如具体细节可参见：1.杨薇,王迪,陈克清,等.质粒电穿孔转染条件的选择[J].华中科技大学学报,2009,38(6):858-860.；2.谷欣，黎燕.电转染哺乳类动物细胞DG44-CHO的方法探讨[J].生物技术通讯，2008，19(1):87；3.Jun,Kim,Baik,Hwang,Lee:Selection strategies for the establishment of recombinant Chinese hamster ovary cell line with dihydrofolate reductase-mediated gene amplification.Appl Microbiol Biotechnol.2005,69(2):162-169.10.1007/s00253-005-1972-8.
10cm细胞培养皿中的克隆池经过筛选后，将高产克隆转入细胞培养摇瓶中培养，培养温度37℃，5％CO 2，摇床转速120rpm。高产细胞克隆长到一定数目后，收集一部分细胞进行冻存保种，剩余细胞进行补料分批培养。分批补料时接种密度为1×10 6个细胞/ml，细胞置于细胞培养摇瓶中培养，培养温度37℃，5％CO 2，摇床转速120rpm。接种后每天取样计数，记录细胞密度和活力。从培养第3天开始补料，每天补料1次。第3～8天，补料量分别为初始体积的2％、3％、4％、3％、3％、3％，第9天后补料量均为2％，总补料比例在20％～30％ 之间。每天补充一次葡萄糖，维持培养体系内葡萄糖浓度在3～4g/L。培养周期为12-14天，培养结束后离心(3000rpm)收获上清液并检测表达量，进行下一步纯化以获得目的蛋白。
实施例3：胰岛素-Fc融合蛋白的纯化
1.胰岛素前体-Fc融合蛋白的捕获
实施例2中表达的各个胰岛素前体-Fc融合蛋白(SS302-002、SS302-004、SS302-005、SS302-008、SS302-012、SS302-014、SS302-015、SS302-019、SS302-029、SS302-030、SS302-035)的细胞发酵液经离心和0.22μm的滤膜过滤除去细胞碎片后用亲和层析进行捕获。博格隆proteinA作为亲和介质，用3-5倍柱体积缓冲液(20mM Na 2HPO4-柠檬酸，pH7.5)平衡protein A层析柱淋洗至基线平稳，再将处理过的发酵液上清进行上样(载量3-8g/L)，上样完毕后，用清洗缓冲液(20mM Tris,1.5M NaCl,2M Urea,pH7.5)冲洗杂蛋白至基线，最后用20mM Na 2HPO 4-柠檬酸，0.4M Arg,pH3.5洗脱缓冲液进行洗脱，根据紫外检测器的示数收集，分段收集，当UV280nm吸收值高于0.15AU开始收集，再次低于0.20AU停止收集。收集样品立即加入2.0mol/L Tris-HCl缓冲液，并缓慢搅动，调整样品至pH6.5-7.0。样品储存于-80℃，并进行后续SDS-PAGE分析(图2)及结构鉴定(见实施例4)。
SDS-PAGE结果如图2所示，其中，load表示层析上样样品，FT表示流穿样品(flow through)，wash表示淋洗样品，P1、P2、P3等表示层析过程分段收集的目的蛋白样品，“P合”表示分段收集的样品按照收集体积的体积比合并后样品，NaOH表示柱清洗收集的样品，DTT表示还原后的目的蛋白，M表示分子量标记(maker)；A:SS302-002，B:SS302-004，C:SS302-005，D:SS302-008，E:SS302-014，F:SS302-019，G:SS302-030，H:SS302-012，I:SS302-015，J:SS302-029，K:SS302-035。从图2可以看出，SS302-002蛋白有明显的上条带(约130KD)和下条带(95-130KD之间)，且存在高分子量形式(＞170KD)。纯度大于90％的上条带(130KD)收率约60％。SS302-004蛋白具有明显的上条带(95-130KD)和下条带(约95KD)，其中下条带P1-4合并样品和上条带P13-15合并样品分别送质谱进行结构鉴定(实施例4)，且此分子在捕获蛋白中大部分为95KD的下条带，95-130KD之间纯度大于90％的上条带在捕获收集的蛋白中收率较低(15％左右)；SS302-005蛋白在72-95KD之间，电泳条带较宽且称弥散状；这几个分子的共同特征是均含GS柔性接头；而其他分子如SS302-008、SS302-012、SS302-015等，基本都为单一条带，这些分子的共同特征是含CTP、C1等刚性接头。质谱鉴定结果进一步表明(见实施例4)：含柔性接头(例如GS)的胰岛素前体-Fc融合蛋白二硫键具有一定的错配率，正确条带回收率占比较低；而相对于含柔性接头 (例如GS)的胰岛素前体-Fc融合蛋白，含刚性接头的胰岛素前体-Fc融合蛋白二硫键错配率较低，得到的蛋白中正确折叠的胰岛素前体蛋白含量较高。
2.胰岛素前体-Fc融合蛋白酶切
步骤1中捕获的蛋白利用G25使用缓冲液50mMTris,150mM NaCl,pH8.0进行换液，换液后各蛋白用Kex2酶切切除C肽得到胰岛素-Fc融合蛋白，SS302-002和SS302-004酶切条件为:蛋白终浓度1mg/mL，投料比(质量比)为200:1(前体：Kex2)，CaCl 2终浓度20mM/L,总反应体积为5mL和3mL，37℃水浴6h进行酶切。SS302-008、SS302-012三个蛋白的酶切条件为：蛋白终浓度1mg/mL，投料比(质量比)为50:1(前体：Kex2)，CaCl 2终浓度20mM/L,总反应体积为190mL，37℃水浴反应6h。SS302-014、SS302-015、SS302-019、SS302-029、SS302-030、SS302-035酶切条件为蛋白终浓度1mg/mL，投料比(质量比)为1:25(Kex2：前体)，CaCl 2终浓度20mM/L,总反应体积为60-180mL(不同蛋白反应体积略有差异)，37℃水浴6h。各胰岛素前体-Fc融合蛋白酶切后胰岛素-Fc融合蛋白命名为S302-002M、SS302-004M、SS302-005M、SS302-008M、SS302-012M、SS302-014M、SS302-015M、SS302-019M、SS302-029M、SS302-030M和SS302-035M。
3.酶切后的胰岛素-Fc融合蛋白纯化
为除去蛋白酶及反应后杂质，得到纯度较高且正确折叠的胰岛素-Fc融合蛋白，SS302-004M和SS302-005M酶切后用10KD超滤管去除蛋白酶及其它杂质，得到纯度较高的胰岛素-Fc融合蛋白。SS302-008M、SS302-012M、SS302-014M、SS302-015M、SS302-029M、SS302-030M酶切后利用疏水层析去除杂质。用3-5个柱体积的20mM Tris,1M(NH 4) 2SO 4，pH7.5缓冲液平衡疏水层析介质Butyl HP(博格隆)，平衡完成后开始上样(载量3-8g/L)，上样完毕后，用20mM Tris,pH7.5缓冲液线性洗脱(0-100％，20个柱体积)，根据紫外检测器的示数分段收集，并取样检测。SS302-019M、SS302-035M酶切后分两步进行杂质去除，第一步采用阴离子层析，首先用3-5个柱体积的20mM Tris，pH8.5缓冲液平衡阴离子层析介质Q HP(博格隆)，平衡完成后开始上样(载量为5g/L)，上样完毕后，用20mM Tris，0.5M NaCl，pH8.5缓冲液以3ml/min的流速0-60％B 15CV线性洗脱，根据紫外检测器的示数分段收集(方法同上)，并取样检测，将纯度较高样品合并用于下一步疏水层析。用3-5个柱体积的20mM Tris，1M NaCl，pH8.0缓冲液平衡疏水层析介质Butyl HP(博格隆)，平衡完成后开始上样，上样载量3-8g/L，上样完毕后，用20mM Tris，pH8.0缓冲液以1ml/min的流速0-100％B 15CV线性洗脱，根据紫外检测器的示数分段收集(方法同上)，并取样进行结构解析-采用UPLC-QTOF表征其分子量和二硫键，详见实施例4。
实施例4：融合蛋白分子结构解析
胰岛素融合蛋白前体的结构为proINS-L-Fc，proINS为人胰岛素前体(包括B-C-A)，L为接头(linker)，其示意图见图3A。胰岛素融合蛋白前体经酶解后会形成成熟蛋白，其结构为胰岛素(B-A)-L-Fc，其示意图见图3B。胰岛素融合蛋白采用的接头有柔性接头(例如GS)或刚性接头(例如CTP、C1)。在CHO细胞中发酵时，前肽、刚性接头(例如CTP、C1)上的S和T都可能发生O糖基化，C1这种接头上的P可能产生脯氨酸羟基化，而GS这种柔性接头则几乎不会发生翻译后修饰。本结构解析采用UPLC-QTOF表征其分子量和二硫键。胰岛素-Fc融合蛋白分子(含糖修饰)经去糖及去糖还原后，得到容易分析的无糖修饰分子。对SS302-002(约130KD)、SS302-002(95-130KD之间)、SS302-008、SS302-008M、SS302-012、SS302-012M、SS302-014、SS302-014M、SS302-015、SS302-015M、SS302-019、SS302-019M、SS302-029、SS302-029M、SS302-030、SS302-030M、SS302-035、SS302-035M进行去糖完整、去糖还原分子检测，对SS302-004(95-130KD之间)、SS302-004(约95KD)、SS302-005进行完整、还原分子检测，结果表明，胰岛素-Fc融合蛋白的分子量均与理论一致。
胰岛素-Fc融合蛋白的空间结构由两个半胱氨酸(Cys)残基的巯基间形成二硫键支撑稳定。其二硫键分为两部分，即胰岛素部分的二硫键和Fc部分的二硫键。胰岛素部分的二硫键位于B链和A链，按照从N端到C端的顺序对B链和A链的氨基酸命名，分别为BX和AX，其中X为氨基酸的序列号，二硫键为CysA7-CysB7、CysA20-CysB19、CysA6-CysA11。Fc由两条序列相同的多肽链组成，每条多肽链上含有2对二硫键，两条多肽链共含有4对二硫键，且两条多肽链之间有2对链间二硫键，故Fc共6对二硫键。理论上，胰岛素-Fc融合蛋白的二硫键不受kex2酶解过程影响。胰岛素-Fc融合蛋白的二硫键结构解析经由非还原变性换液，以限制性内切酶酶切处理，结合UNIFI软件分析完成。前处理方法一共有两种。经UNIFI软件分析时，胰岛素-Fc融合蛋白前体的两条链分别命名为1链和2链，经前处理方法1的Glu-C酶解后形成的肽段UNIFI软件命名为1：VN、2：VN(见表8～表11)，经前处理方法2的Glu-C酶和胰酶酶解后形成的肽段UNIFI软件命名为1：VTN、2：VTN(见表15)；胰岛素-Fc融合蛋白成熟蛋白的两条B链分别命名为1链和3链，两条A+Fc链分别命名为2链和4链，经前处理方法2的Glu-C酶和胰酶酶解后形成的肽段UNIFI软件命名为1：VTN、2：VTN、3：VTN、4：VTN(见表12～表14、表16)；其中N为蛋白酶解后肽段的软件编号，编号规则为从N端至C端依次为1、2、3、……等等；同时，UNIFI软件里的二硫键以＝表示，链间二硫键位于两个肽段之间，链内二硫键位于肽段的右侧。
SS302-002(约130KD)、SS302-002(95-130KD之间)、SS302-004(95-130KD之间)、SS302-004(约95KD)、SS302-005，采用了前处理方法1进行了二硫键解析。前处理方法1 步骤如下：取SS302蛋白样品至0.5mL 10kD超滤管中，在4℃、12000rpm的条件下，浓缩至5mg/mL，取30μL上述浓缩样品，加入8M盐酸胍18μL(pH7.5)和1M IAA(碘乙酰胺)0.48μL，涡旋混匀后于室温避光孵育40min，取1.8μL上述样品，加入50mM Tris-HCl23μL(pH8)缓冲液稀释样品，并按照蛋白：酶＝25:1(μg：μg)加入0.1mg/mL的Glu-C酶2.25μL，37℃水浴过夜，次日加入3μL的10％FA(甲酸)终止反应，用UPLC-QTOF检测。前处理方法1变性不完全，故linker区域难以酶解，使得胰岛素上的二硫键和铰链区的二硫键通过连接肽连接在一起，分子量大，难以匹配，此方法会缺失关键二硫键信息，主要用于对比SS302-002(约130KD)与SS302-002(95-130KD之间)这两条带及SS302-004(95-130KD之间)与SS302-004(约95KD)这两条带之间的二硫键错配情况的差异。
SS302-008、SS302-012、SS302-012M、SS302-014、SS302-014M、SS302-015、SS302-015M、SS302-019M、SS302-029M、SS302-030M、SS302-035、SS302-035M采用前处理方法2进行二硫键解析。前处理方法2步骤如下：取40μL SS302蛋白样品，加入8M盐酸胍120μL，于60℃水浴1h，冷却至室温后，加入1M IAA3.2μL，于室温避光孵育45min，用0.5mL 10kD超滤管在12000rpm、4℃条件下换液3次换至50mM Tris-HCl(pH8)缓冲液中，使得换液后样品浓度约为0.62mg/mL。取40μL上述样品，按照蛋白：酶＝25:1(μg：μg)加入2μL Glu-C酶(0.5mg/mL)和2μLTrypsin酶(0.5mg/mL)，37℃水浴过夜，次日加入5μL的10％FA终止反应，用UPLC-QTOF检测。前处理方法2采用胰酶和Glu-C酶一起酶解，上述SS302分子均实现linker区域的酶解，正确二硫键得以匹配，此方法错配二硫键的计算结果更真实。
结合UNIFI软件分析UPLC-QTOF的二硫键检测结果，可解析正确二硫键和错配二硫键，其二硫键错配情况以总错配率和胰岛素错配率表示，其中总错配率为错配二硫键肽段总XIC峰面积与所有二硫键肽段总XIC峰面积之比，胰岛素错配率为胰岛素部分的错配二硫键总XIC峰面积与所有二硫键肽段总XIC峰面积之比。SS302-002、SS302-004、SS302-005、SS302-008、SS302-012、SS302-012M、SS302-014、SS302-014M、SS302-015、SS302-015M、SS302-019M、SS302-029M、SS302-030M、SS302-035、SS302-035M的错配率见表7。
就包含柔性接头的融合蛋白(SS302-004、SS302-005)而言，SS302-005的错配率是所有分子中最高的，SS302-004的目标带(95-130KD之间)的错配率比较低，但是不易与错配率高的组分分离，故其收率不高。对于接头中既包含柔性部分又包含刚性部分的融合蛋白(SS302-002)，其目标带与包含柔性接头的融合蛋白(SS302-004)的总错配率及胰岛素错配率相当，二者均含有总错配率及胰岛素错配率高的组分，且不易于纯化分离。而包含刚性接头的前体蛋白和成熟蛋白(SS302-008、SS302-012、SS302-012M、SS302-014、SS302-014M、SS302-015、SS302-015M、SS302-019M、SS302-029M、SS302-030M、SS302-035、SS302-035M)的总错配率和胰岛素错配率均低于8％。对实施例4中SS302-002、SS302-004、SS302-012M、 SS302-019M、SS302-030M、SS302-035、SS302-035M的二硫键结果做详细说明，结果见表8-16。
综上表明：刚性接头对胰岛素融合蛋白在CHO细胞中结构表达的准确性有很大的积极作用，且刚性越强，其分子结构表达的准确性越高。
表7.融合蛋白中二硫键的错配率
Figure PCTCN2021107040-appb-000053
注： a表示该融合蛋白包含柔性接头， b表示该融合蛋白包含刚性接头；
总错配率为错配二硫键肽段总XIC峰面积与所有二硫键肽段总XIC峰面积之比；
胰岛素错配率为胰岛素部分的错配二硫键总XIC峰面积与所有二硫键肽段总XIC峰面积之比。
1.SS302-002
结合SDS-PAGE技术，此分子可纯化得到约130KD条带和95-130KD之间条带。对这两条带分别进行二硫键鉴定，估算二硫键总错配率和胰岛素错配率。结果显示约130KD条带的总错配率和胰岛素错配率均为9％，95-130KD之间条带的总错配率和胰岛素错配率均为29％。约130KD条带的二硫键结果见表8，95-130KD之间条带的二硫键结果见表9，错配二硫键主要体现为胰岛素B链自连和胰岛素两条B链错配。
表8.胰岛素前体-Fc融合蛋白(SS302-002)约130KD位置条带的二硫键检测结果
Figure PCTCN2021107040-appb-000054
注：下划线为错配二硫键所在区段
表9.胰岛素前体-Fc融合蛋白(SS302-002)95-130KD之间条带二硫键检测结果
Figure PCTCN2021107040-appb-000055
注：下划线为错配二硫键所在区段
2.SS302-004
此分子纯化得到95-130KD之间条带(P1-4合并样品)和约95KD条带(P13-15合并样品)。对这两条带分别进行二硫键鉴定，结果显示95-130KD之间条带的总错配率和胰岛素错配率均为4％，约95KD条带的总错配率和胰岛素错配率均为37％。95-130KD之间条带的二硫键结果见表10，约95KD条带的二硫键结果见表11，错配二硫键主要体现为胰岛素B链自连和胰岛素两条B链错配。
表10.胰岛素前体-Fc融合蛋白(SS302-004)95-130KD之间条带二硫键检测结果
Figure PCTCN2021107040-appb-000056
注：下划线为错配二硫键所在区段
表11.胰岛素前体-Fc融合蛋白(SS302-004)约95KD位置条带二硫键检测结果
Figure PCTCN2021107040-appb-000057
注：下划线为错配二硫键所在区段
3.SS302-012M
此分子的二硫键与理论一致，总错配率为2.9％，胰岛素错配率为2.2％，其二硫键结果见表12。
表12.胰岛素前体-Fc融合蛋白(SS302-012M)的二硫键检测结果
Figure PCTCN2021107040-appb-000058
注：下划线为错配二硫键所在区段
4.SS302-019M
此分子的二硫键与理论一致，总错配率为2.8％，胰岛素错配率为1.2％，其二硫键结果见表13。
表13.胰岛素前体-Fc融合蛋白(SS302-019M)的二硫键检测结果
Figure PCTCN2021107040-appb-000059
注：下划线为错配二硫键所在区段
5.SS302-030M
此分子的二硫键与理论一致，总错配率为1.7％，胰岛素错配率为0％，其二硫键结果见表14。
表14.胰岛素前体-Fc融合蛋白(SS302-030M)的二硫键检测结果
Figure PCTCN2021107040-appb-000060
注：下划线为错配二硫键所在区段
6.SS302-035
此分子的二硫键与理论一致，总错配率为4.3％，胰岛素错配率为2.2％，其二硫键结果见表15。
表15.胰岛素前体-Fc融合蛋白(SS302-035)的二硫键检测结果
Figure PCTCN2021107040-appb-000061
注：下划线为错配二硫键所在区段
7.SS302-035M
此分子的二硫键与理论一致，总错配率为2.5％，胰岛素错配率为2.0％，其二硫键结果见表16。
表16.胰岛素前体-Fc融合蛋白(SS302-035M)的二硫键检测结果
Figure PCTCN2021107040-appb-000062
注：下划线为错配二硫键所在区段
实施例5：SS302-002和SS302-002M对昆明小鼠的降糖效果
24只健康雄性昆明小鼠(22-28g)随机分成4组，6只/组：(1)SS302-002M-24nmol/kg；
(2)SS302-002-24nmol/kg；(3)甘精胰岛素-48nmol/kg；(4)阴性对照组。颈部皮下注射给药。分别在0、1、2、4、6、8、10、12、24、36、48、60、72、96h进行血糖值检测。实验过程中小鼠不禁食，给予充足的水和食物。
如图4所示，甘精胰岛素药效持续到4h。SS302-002组在给药后4h开始出现明显降糖效果，但SS302-002组就降糖效果及药效持续时间方面均明显弱于SS302-002M组，SS302-002组vs.SS302-002M组最大降糖效果依次为(5.33vs.2.97mmol/L)和药效持续时间依次为(36h vs.72h)。上述数据分析表明切除C肽后的胰岛素融合蛋白效价更高，降糖效果更优。
实施例6：SS302-008M、SS302-012M、SS302-014M、SS302-015M、SS302-019M、SS302-029M、SS302-030M和SS302-035M对正常C57小鼠的降糖效果
将50只8-10周龄，体重22-28g健康雄性C57小鼠，随机分为10组，5只/组，包括SS302-008M、SS302-012M、SS302-014M、SS302-015M、SS302-019M、SS302-029M、SS302-030M、SS302-035M、德谷胰岛素和对照组。待测样品按15nmol/kg颈部皮下给药，德谷胰岛素剂量为30nmol/kg，分别于给药前和给药后不同时间点检测血糖值。实验过程中小鼠不禁食。实验数据采用Graphpad prism7.0作图，Mann-whiteney统计分析差异性。
如图5A和5B所示，与对照组相比，给药组小鼠均有明显的降血糖效果。德谷胰岛素(30nmol/kg)药效持续到12h；在15nmol/kg剂量下，不同胰岛素融合蛋白在正常C57小鼠上的药效持续时间如下：
SS302-035M/SS302-030M/SS302-019M/SS302-008M(96h)>SS302-012M(72h)>SS302-015M(48h)>SS302-029M/SS302-014M(24h)。
实施例7：不同剂量SS302-035M在正常C57小鼠中的降糖效果
将25只8-10周龄，体重22-28g健康雄性C57小鼠，随机分为5组，5只/组。SS302-035M分别按5、7.5、10、12.5nmol/kg颈部皮下给药，并在0、4、24、48、72、96、120h进行血糖值检测。实验过程中小鼠不禁食。实验结果数据用Graphpad prism7.0进行作图，并用Mann-whiteney统计分析差异性。
如图6所示，在正常C57小鼠中，SS302-035M的降糖效果具有明显的剂量依赖关系。SS302-035M-5nmol/kg组，血糖最低值4.3mmol/L，药效持续至72h；SSS302-035M-7.5nmol/kg组，血糖最低值至3.2mmol/L，药效持续至72h；SSS302-035M-10nmol/kg组，血糖最低值至2.8mmol/L，药效持续至96h；SSS302-035M-12.5nmol/kg组，血糖最低值至2.5mmol/L，药效持续至96h。
实施例8：SS302-004M和SS302-002M在糖尿病模型小鼠中的降糖效果
1.STZ诱导I型糖尿病小鼠模型
C57BL/6j小鼠(8周龄,体重22-28g)腹腔注射柠檬酸-柠檬酸钠缓冲液配制的0.4％链脲佐菌素(STZ)溶液，40mg/kg连续给药五天，每天一次，末次给药后第7-10天检测空腹血糖值。空腹血糖值>13.8mmol/L(空腹时间为8:00a.m-14:00p.m)即为造模成功。
2.体内活性和长效性检测
取35只STZ诱导的I型糖尿病小鼠，按血糖值随机分成7组：1～2：SS302-002M高、低剂量组；3～4：SS302-004M高、低剂量组；5～6：甘精胰岛素(Glargine Insulin)高、低剂量组；(7)对照组(20mM Tris+300mM NaCl)。其中，SS302-002M和SS302-004M的高、低剂量组分别按12.5nmol/kg和6.25nmol/kg颈部皮下注射给药，甘精胰岛素高、低剂量组分别按25nmol/kg和12.5nmol/kg颈部皮下注射给药。分别于给药前和给药后不同时间点监测血糖值变化。实验过程中小鼠不禁食，给予充足的水和食物。
结果如图7A(SS302-002M)和7B(SS302-004M)所示。在STZ诱导的I型糖尿病小鼠中给予SS302-002M、SS302-004M后均有明显的降血糖药效，S302-002M低剂量组药效维持至120h，高剂量组药效维持至192h；S302-004M低剂量组药效维持至84h，高剂量组药效维持至144h。
值得注意的是，在相同摩尔的胰岛素，即25nmol/kg给药剂量下，相比SS302-002M和SS302-004M组，甘精胰岛素组的血糖值下降和恢复程度更为迅速，在给药后1h左右即降到血糖最低点(约5mmol/L)，低于正常C57血糖值(约8mmol/L)，随后又快速回升，6h回升至初始血糖值。这提示SS302-002M、SS302-004M具有更为平坦和稳定PD谱，临床安全性更高。
实施例9：SS302-008M、SS302-012M和SS302-035M在糖尿病模型小鼠中的降糖效果
1.STZ诱导小鼠I型糖尿病模型
C57BL/6j小鼠(12周龄，体重22-28g)腹腔注射柠檬酸-柠檬酸钠缓冲液配制的0.4％链脲佐菌素(STZ)溶液，40mg/kg连续给药五天，每天一次，在最后一次给药后第7-10天检测空腹血糖值>13.8mmol/L(空腹时间为8:00a.m-14:00p.m)，即为造模成功。
2.体内活性和长效性检测
取40只STZ造模成功的I型糖尿病小鼠，按血糖值随机分成8组：(1)SS302-008M-7.5nmol/kg组；(2)SS302-012M-7.5nmol/kg组；(3)SS302-035M-7.5nmol/kg组；(4)SS302-008M-15nmol/kg组；(5)SS302-012M-15nmol/kg组；(6)SS302-035M-15nmol/kg组；(7)德谷胰岛素-30nmol/kg；(8)缓冲液对照组(20mM Tris+150mM NaCl)。分别在给药前和给药后不同时间点进行血糖值检测。实验过程中小鼠不禁食。实验结果采用Graphpad prism7.0进行作图，Mann-whiteney统计分析差异性。
如图8A和8B所示，相同剂量下SS302-035M的药效维持时间明显优于SS302-008M和S302-012M，尤其在低剂量7.5nmol/kg组(144h vs.72h)。图8B中德谷胰岛素在30nmol/kg给药后，糖尿病小鼠的血糖值迅速下降和恢复，1h左右即降到最低点，24h回升至初始血糖值。这提示SS302-008M、SS302-012M、SS302-035M具有更长的PD谱，药效维持时间远优于德谷胰岛素。
实施例10：SS302-008M和SS302-012M在SD大鼠的药效动力学
(PD)和药代动力学(PK)实验
10只SD大鼠(8-10周龄，体重250-350g)随机分为2组，每组3♂/2♀，分别按20nmol/kg颈部皮下给与SS302-008M、SS302-012M。在给药前和给药后的不同时间点检测血糖值，并采集全血，分离血清用于PK检测。实验过程中不禁食，给予充足的水和食物。全部数据用Graphpad prism7.0进行作图，并用Mann-whiteney统计分析差异性。
2、ELISA检测
鼠抗胰岛素单克隆抗体(abcam，ab8302)用PBS稀释至1ug/mL，100μL/孔加入酶标板，放置于4℃过夜包被；去除包被液，PBST洗板4次后加入4％BSA，250μl/孔，37℃封闭2h；去除封闭液后用PBST洗板4次。SS302-008M/SS302-012M标准品用2％BSA稀释，从200ng/ml起始，倍比稀释共8个梯度建立标曲。大鼠血清用2％BSA稀释为不同的倍数。阴性对照为正常大鼠血清。将上述样品100μl/孔加入酶标板，于37℃孵育1h；PBST洗板4次，加入2％BSA稀释的二抗(Mouse monoclonal Anti-Human IgG2Fc(HRP)，1:3000) (abcam，ab99779)，100μL/孔，37℃孵育1h；PBST洗板4次后加入TMB显色液，100μl/孔，37℃避光显色10min；加入2M H 2SO4，50μL/孔，终止反应，酶标仪检测OD450/630值。
3、药效动力学实验结果
如图9所示：给与SS302-008M和SS302-012M后SD大鼠均有明显的降血糖效果。SS302-008M药效维持至96h，而SS302-012M药效维持至72h。
4、药代动力学结果和分析
SS302-008M和SS302-012M在SD大鼠中的药代动力学结果如图10所示。SS302-008M和SS302-012M在SD大鼠中的半衰期(T1/2)分别为16.32±0.77h和13.39±0.43h，具体PK参数见表17。
表17.SS302-008M和SS302-012M的PK参数
组别 SS3302-008M SS3302-0012M
T1/2(hr) 16.32±0.77 13.39±0.43
Tmax(hr) 24.00±0 24.00±0
Cmax(nmol/L) 82.71±7.77 74.72±8.66
AUC(hr*nmol/L) 3217.73±326.15 2664.67±208.28
Vss(L/kg) 0.289±0.039 0.289±0.031
Cl(L/hr/kg) 0.012±0.001 0.015±0.001
MRT(hr) 34.41±2.23 25.60±2.23
实施例11：SS302-035M在比格犬中药效动力学(PD)和药代动力学(PK)实验
8-12kg雄性健康普通级比格犬(4只)单次皮下给与2.5nmol/kg SS302-035M后评估药效动力学和药代动力学参数。分别于给药前和给药后不同时间点采集血样，采样部位为四肢外周静脉。每个时间点采集约1mL全血，放入含EDTA-K2的抗凝管中，随后于4℃条件下3000g/min离心10min收集血浆。在给药前0h和给药后1、2、3、4、6、24、48、72、96、120、144及168h时间点取一滴全血用快速血糖仪(罗氏ACCU-CHEK Performa)和血糖试纸(罗氏ACCU-CHEK Performa)测定动物的血糖值。药效动力学(PD)结果如图10A所示；药代动力学(PK)结果如图10B所示。实验过程中0-6h内禁食，之后动物自由进食进 水。使用WinNonlin 8.2软件计算药代动力学参数(非房室模型)，相关的PK参数显示在表18中。PD结果显示：SS302-035M在2.5nmol/kg剂量下能显著性地降低比格犬随机血糖，降糖的效果持续到120h，并且未出现明显的低血糖的症状。PK结果显示：在2.5nmol/kg剂量下，SS302-035M在正常比格犬的体内半衰期为37.65±7.36h。
表18.SS302-035M的PK参数
PK参数 结果
AUC 0-∞(ng*hr/mL) 14631.28±628.94
T1/2(hr) 37.65±7.36
T max(hr) 2±0
C max(ng/mL) 485.75±26.18
Vss(mL/kg) 498.53±55.90
CL(mL/hr/kg) 11.83±1.29
MRT(hr) 39.05±4.11
本发明实施例中构建的融合蛋白前体全长序列如下：
1)胰岛素前体融合蛋白SS302-001SEQ ID NO:47
Figure PCTCN2021107040-appb-000063
2)胰岛素前体融合蛋白SS302-002SEQ ID NO:48
Figure PCTCN2021107040-appb-000064
3)胰岛素前体融合蛋白SS302-003SEQ ID NO:49
Figure PCTCN2021107040-appb-000065
4)胰岛素前体融合蛋白SS302-004SEQ ID NO:50
Figure PCTCN2021107040-appb-000066
5)胰岛素前体融合蛋白SS302-005SEQ ID NO:51
Figure PCTCN2021107040-appb-000067
6)胰岛素前体融合蛋白SS302-006SEQ ID NO:52
Figure PCTCN2021107040-appb-000068
7)胰岛素前体融合蛋白SS302-007SEQ ID NO:53
Figure PCTCN2021107040-appb-000069
Figure PCTCN2021107040-appb-000070
8)胰岛素前体融合蛋白SS302-008SEQ ID NO:54
Figure PCTCN2021107040-appb-000071
9)胰岛素前体融合蛋白SS302-009SEQ ID NO:55
Figure PCTCN2021107040-appb-000072
10)胰岛素前体融合蛋白SS302-011SEQ ID NO:56
Figure PCTCN2021107040-appb-000073
11)胰岛素前体融合蛋白SS302-012SEQ ID NO:57
Figure PCTCN2021107040-appb-000074
12)胰岛素前体融合蛋白SS302-013SEQ ID NO:58
Figure PCTCN2021107040-appb-000075
13)胰岛素前体融合蛋白SS302-014SEQ ID NO:59
Figure PCTCN2021107040-appb-000076
14)胰岛素前体融合蛋白SS302-015SEQ ID NO:60
Figure PCTCN2021107040-appb-000077
15)胰岛素前体融合蛋白SS302-016SEQ ID NO:61
Figure PCTCN2021107040-appb-000078
16)胰岛素前体融合蛋白SS302-017SEQ ID NO:62
Figure PCTCN2021107040-appb-000079
17)胰岛素前体融合蛋白SS302-018SEQ ID NO:63
Figure PCTCN2021107040-appb-000080
18)胰岛素前体融合蛋白SS302-019SEQ ID NO:64
Figure PCTCN2021107040-appb-000081
19)胰岛素前体融合蛋白SS302-022SEQ ID NO:65
Figure PCTCN2021107040-appb-000082
20)胰岛素前体融合蛋白SS302-023SEQ ID NO:66
Figure PCTCN2021107040-appb-000083
21)胰岛素前体融合蛋白SS302-029SEQ ID NO:67
Figure PCTCN2021107040-appb-000084
Figure PCTCN2021107040-appb-000085
22)胰岛素前体融合蛋白SS302-030SEQ ID NO:68
Figure PCTCN2021107040-appb-000086
23)胰岛素前体融合蛋白SS302-035SEQ ID NO:69
Figure PCTCN2021107040-appb-000087
24)胰岛素前体融合蛋白SS302-036SEQ ID NO:70
Figure PCTCN2021107040-appb-000088
25)胰岛素前体融合蛋白SS302-037SEQ ID NO:71
Figure PCTCN2021107040-appb-000089
26)胰岛素前体融合蛋白SS302-038SEQ ID NO:72
Figure PCTCN2021107040-appb-000090

Claims (20)

  1. 一种胰岛素-Fc融合蛋白，其包含第一部分和第二部分，所述第一部分是提供胰岛素活性的胰岛素部分，所述第二部分是具有延长所述第一部分体内半衰期作用的Fc部分，所述第一部分与所述第二部分通过共价键相连，所述胰岛素-Fc融合蛋白切割后具有胰岛素活性。
  2. 如权利要求1所述的胰岛素-Fc融合蛋白，其具有式(I)的结构：
    X-E1-Y-E2-Z-L-Fc  (I)，
    其中，
    X和Z分别是胰岛素的B链和A链，并且如果X是B链，则Z是A链；如果X是A链，则Z是B链；
    Y为任选的连接肽；Y的长度可以为1-100个或更多个氨基酸，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、50、60、70、80、90、100个或以上任意两个数值之间的值；例如Y为胰岛素C肽或者其变体或片段；
    E1和E2中的一个存在或两个都存在，是包含位点特异性蛋白酶切割位点的一段氨基酸；E1和E2的长度可以是1-10个或更多个氨基酸，例如1、2、3、4、5、6、7、8、9或10个氨基酸；如果同时存在，E1和E2可被相同或不同的位点特异性蛋白酶切割，例如可被相同的位点特异性蛋白酶切割；如果Y存在，则优选地E1和E2两个都存在；如果Y不存在，则优选地E1和E2中存在一个；所述位点特异性蛋白酶切割位点可以是Kex2和/或弗林(Furin)蛋白酶的切割位点，例如是Kex2蛋白酶的切割位点；
    L为连接Z与Fc的接头，可以是氨基酸片段，也可以是非肽链的化学结构；以及
    Fc是免疫球蛋白的Fc区；所述Fc可以来自人免疫球蛋白；所述Fc区可以是来自IgG、IgA、IgD、IgE或IgM的Fc区；优选地，所述Fc区是来自IgG的Fc区，例如来自IgG1、IgG2、IgG3或IgG4的Fc区；再优选地，所述Fc区是来自IgG2的Fc区；其中与其来源序列相比，所述Fc区可以带有一个或更多个替换、添加和/或缺失，同时仍保留延长半衰期的能力，例如来自人IgG的Fc区并带有降低或消除与FcγR结合的突变和/或增强与FcRn结合的一个或更多个突变，所述突变可以选自：N297A、G236R/L328R、L234A/L235A、N434A、M252Y/S254T/T256E、M428L/N434S、T250R/M428L；所述Fc区可以是糖基化的或未糖基化的。
  3. 根据权利要求1或2所述的融合蛋白，其中所述L为多肽片段，
    优选地，所述L包含选自Ala、Thr、Gly和Ser中一个、两个或更多个氨基酸的柔性肽段，更例如一段由G和S构成的柔性肽段；所述柔性肽段的长度可以是2-50个或更多个氨基酸，例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45或50个氨基酸；
    优选地，所述L包含一个或更多个刚性单元，所述刚性单元包含或主要由刚性氨基酸构成，所述刚性氨基酸包括但不限于V、P、I、K和L；
    更优选地，所述刚性单元包含一个或更多个PPPX 1LP(SEQ ID NO:125)，其中X 1为任意氨基酸；
    更优选地，所述刚性单元包含一个或更多个X 2APPPX 1LP(SEQ ID NO:126)，其中X 1为任意氨基酸，X 2为K或V。
  4. 根据权利要求3所述的融合蛋白，其中所述刚性单元包含选自以下序列的多肽片段：
    PPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:127)；
    PPPALPAPVRLPGP(SEQ ID NO:128)；
    PPPALPAVAPPPALP(SEQ ID NO:129)；
    优选地，所述刚性单元包含选自以下序列的多肽片段：
    KAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:130)；
    VAPPPALPAPVRLPGP(SEQ ID NO:131)；和
    VAPPPALPAVAPPPALP(SEQ ID NO:132)。
  5. 根据权利要求1至4所述的融合蛋白，其中所述L包含选自以下序列的多肽片段：
    Figure PCTCN2021107040-appb-100001
    Figure PCTCN2021107040-appb-100002
  6. 根据权利要求1至5任一项所述的融合蛋白，其中所述胰岛素选自人胰岛素、牛胰岛素或猪胰岛素，优选地是人胰岛素；例如所述胰岛素的A链和B链源自人胰岛素。
  7. 根据权利要求1至6任一项所述的融合蛋白，其中Y、E1和E2都存在，或者其中Y不存在，E1和E2中存在一个。
  8. 根据权利要求1至7中任一项所述的融合蛋白，其具有选自SEQ ID NO:47-72的氨基酸序列。
  9. 一种生产具有增强胰岛素活性和延长半衰期的胰岛素-Fc融合蛋白的方法，其包括将权利要求1至8中任一项所述的融合蛋白与能切割所述位点特异性蛋白酶切割位点的位点特异性蛋白酶接触，优选地所述位点特异性蛋白酶是Kex2和/或Furin蛋白酶。
  10. 一种胰岛素-Fc融合蛋白，其是由权利要求9所述的方法得到。
  11. 一种胰岛素-Fc融合蛋白，其具有Ins-L-Fc结构，其中
    Ins是提供胰岛素活性的胰岛素部分，其包括通过共价键连接的胰岛素A链和B链，但A链和B链位于不同的肽链中；所述共价键优选地是二硫键；
    L为连接Z与Fc的接头，可以是氨基酸片段，也可以是非肽链的化学结构；以及
    Fc是免疫球蛋白的Fc区；所述Fc可以来自人免疫球蛋白；所述Fc区可以是来自IgG、IgA、IgD、IgE或IgM的Fc区；优选地，所述Fc区是来自IgG的Fc区，例如来自IgG1、IgG2、IgG3或IgG4的Fc区；再优选地，所述Fc区是来自IgG2的Fc区；其中与其来源序列相比，所述Fc区可以带有一个或更多个替换、添加和/或缺失，同时仍保留延长半衰期的能力，例如来自人IgG的Fc区并带有降低或消除与FcγR结合的突变和/或增强与FcRn结合的一个或更多个突变，所述突变可以选自：N297A、G236R/L328R、L234A/L235A、N434A、 M252Y/S254T/T256E、M428L/N434S、T250R/M428L；所述Fc区可以是糖基化的或未糖基化的。
  12. 根据权利要求11所述的融合蛋白，其中所述胰岛素选自人胰岛素、牛胰岛素或猪胰岛素，优选地是人胰岛素；例如所述胰岛素的A链和B链源自人胰岛素。
  13. 根据权利要求11或12所述的融合蛋白，其中所述L为多肽片段，
    优选地，所述L包含选自Ala、Thr、Gly和Ser中一个、两个或更多个氨基酸的柔性肽段，更例如一段由G和S构成的柔性肽段；所述柔性肽段的长度可以是2-50个或更多个氨基酸，例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45或50个氨基酸；
    优选地，所述L包含一个或更多个刚性单元，所述刚性单元包含或主要由刚性氨基酸构成，所述刚性氨基酸包括但不限于V、P、I、K和L；
    更优选地，所述刚性单元包含一个或更多个PPPX 1LP(SEQ ID NO:125)，其中X 1为任意氨基酸；
    更优选地，所述刚性单元包含一个或更多个X 2APPPX 1LP(SEQ ID NO:126)，其中X 1为任意氨基酸，X2为K或V。
  14. 根据权利要求13所述的融合蛋白，其中所述刚性单元包含选自以下序列的多肽片段：
    PPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:127)；
    PPPALPAPVRLPGP(SEQ ID NO:128)；和
    PPPALPAVAPPPALP(SEQ ID NO:129)；
    优选地，所述刚性单元包含选自以下序列的多肽片段：
    KAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:130)；
    VAPPPALPAPVRLPGP(SEQ ID NO:131)；和
    VAPPPALPAVAPPPALP(SEQ ID NO:132)。
  15. 根据权利要求11至14任一项所述的融合蛋白，其中所述L包含选自以下序列的多肽片段：
    Figure PCTCN2021107040-appb-100003
    Figure PCTCN2021107040-appb-100004
  16. 编码权利要求1至8中任一项所述融合蛋白的多核苷酸。
  17. 表达胰岛素-Fc融合蛋白的细胞，其包含权利要求16所述的多核苷酸，优选地，所述细胞为CHO细胞。
  18. 一种生产胰岛素-Fc融合蛋白的方法，其包括在表达胰岛素-Fc融合蛋白的条件下培养权利要求17所述的细胞；优选地还包括使所述胰岛素-Fc融合蛋白与能切割所述位点特异性蛋白酶切割位点的位点特异性蛋白酶相接触，其中所述培养步骤和接触步骤可以同时进行或分开进行。
  19. 一种药物组合物，其包含权利要求1至8中任一项所述的融合蛋白或权利要求10至15中任一项所述的融合蛋白。
  20. 一种降低血糖和/或治疗糖尿病的方法，其包括将权利要求1至8中任一项所述的融合蛋白或者权利要求10至15中任一项所述的融合蛋白施予有此需要的对象，优选地所述糖尿病为I型或II型糖尿病。
PCT/CN2021/107040 2020-07-24 2021-07-19 胰岛素-Fc融合蛋白及其应用 Ceased WO2022017309A1 (zh)

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WO2023247640A1 (en) * 2022-06-23 2023-12-28 Sanofi Single chain insulins and fc conjugates thereof

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