WO2018032785A1 - 人成纤维细胞生长因子21融合蛋白及其制备方法与用途 - Google Patents

人成纤维细胞生长因子21融合蛋白及其制备方法与用途 Download PDF

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WO2018032785A1
WO2018032785A1 PCT/CN2017/079871 CN2017079871W WO2018032785A1 WO 2018032785 A1 WO2018032785 A1 WO 2018032785A1 CN 2017079871 W CN2017079871 W CN 2017079871W WO 2018032785 A1 WO2018032785 A1 WO 2018032785A1
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fusion protein
hfgf21
amino acid
protein according
human
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English (en)
French (fr)
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董炤
周驰
冯雄
李子瑞
李媛丽
李强
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安源医药科技（上海）有限公司
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Priority to MX2019012081A priority Critical patent/MX2019012081A/es
Priority to CA3059662A priority patent/CA3059662C/en
Priority to CN201780000366.0A priority patent/CN107995914A/zh
Priority to US16/604,088 priority patent/US20210380654A1/en
Priority to EP17840761.5A priority patent/EP3620474A4/en
Publication of WO2018032785A1 publication Critical patent/WO2018032785A1/zh

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Definitions

  • the present invention relates to a human fibroblast growth factor 21 (hFGF21) fusion protein, and to the use of the fusion protein for the preparation of a medicament for the treatment of diabetes, obesity, dyslipidemia and/or metabolic syndrome.
  • hFGF21 human fibroblast growth factor 21
  • Fibroblast growth factors (FGFs) family is a kind of polypeptide growth factor with various physiological functions.
  • the mammalian FGF family has been found to have 22 members, which are divided into 7 subfamilies.
  • Fibroblast growth factor 21 (FGF21) belongs to the FGF19/21/23 subfamily and is mainly expressed in the liver. It is also expressed in tissue cells related to glycolipid metabolism, such as adipose tissue, islet ⁇ cells, and muscle tissue. . Unlike other FGF members, this subfamily functions in an endocrine manner, involved in the regulation of energy and bile acid homeostasis, glucose and lipid metabolism, and homeostasis of phosphate and vitamin D (Moore DD et al., Science, 2007).
  • the FGF21 mature protein consists of 181 amino acids. Unlike most members of the FGF family, FGF21 does not specifically bind to heparin to promote cell growth and differentiation.
  • the C-terminus of FGF21 is closely related to its biological activity. The C-terminus of FGF21 directly binds to the cofactor ⁇ -Klotho, and then activates the FGFR receptor and downstream related signaling molecules, which in turn exerts its biological effects (Yie J et al., FEBS Lett, 2009, 583). (1): 19-24; and Micanovic R et al, J Cell Physiol, 2009, 219(2): 227-234).
  • FGF21 regulates the expression of glucose transporter 1 (GLUT1) and promotes the uptake of glucose in 3T3-L1 cells and human primary adipocytes.
  • GLUT1 glucose transporter 1
  • FGF21 significantly reduces mouse body weight and blood glucose levels in genetically controlled or diet-induced obese mice, while also reducing triglyceride levels in the liver and serum, and also has an important effect on mouse insulin sensitivity ( Kharitonenkov A et al, Journal of clinical Investigation, 2005, 115(6): 1627-1635; Coskun T et al, Endocrinology, 2008, 149(12): 6018-6027; Xu J et al, Diabetes, 2009, 58(1): 250-259).
  • Exogenous FGF21 in diet-induced obese mice and ob/ob mice can reverse liver hepatic degeneration and enhance hepatic insulin sensitivity (including reducing hepatic glucose production and increasing hepatic glycogen content), thereby improving systemic glucose intolerance Receptivity and insulin resistance (Xu J et al, Diabetes, 2009, 58(1): 250-259; Berglund ED et al, Endocrinology, 2009, 150(9): 4084-4093; Xu J et al, Am J Physiol Endocrinol Metab, 2009, 297(5)E1105-1104).
  • FGF21 has the same metabolic regulation in diabetic monkeys, while also reducing LDL cholesterol and increasing HDL cholesterol concentration (Kharitonenkov A et al, Endocrinology, 2007, 148(2): 774-781).
  • FGF21 has a very significant beneficial metabolic regulation effect on obese rodent and non-human primates.
  • FGF21 which does not rely on insulin to regulate blood glucose levels, does not cause hypoglycemia when administered as a hypoglycemic agent in large doses.
  • FGF21 is the only cytokine currently found in the FGF family that does not have mitogenic effects, thereby greatly reducing the risk of side effects in clinical use.
  • FGF21 is a potential target for the treatment of obesity, type 2 diabetes and hyperlipidemia.
  • natural FGF21 is difficult to be developed into clinical therapeutic biological agents. The main reasons include: 1. FGF21 protein has poor stability and is susceptible to protease hydrolysis or enzymatic degradation; 2. FGF21 conformation is very unstable It is prone to aggregation, low stability also increases the difficulty of FGF21 in large-scale production; 3.
  • Natural FGF21 half-life is very short, human FGF21 has a half-life of 0.5-1 hour in mice, and half-life in cynomolgus monkeys 2-3 Hours (Kharitonenkov A et al, Journal of clinical Investigation, 2005, 115: 1627-1635). A variety of protein long-acting techniques have been reported to extend the in vivo half-life of recombinant FGF21.
  • FGF21 is linked to a PEG molecule, increasing molecular weight, reducing glomerular filtration rate, and prolonging in vivo retention time (see patents WO2005/091944, WO2006/050247, WO2008/121563 and WO2012/066075); FGF21 and fatty acid long chain fusion ( Ability to bind serum albumin) (see WO2010/084169 and WO2012/010553); or to prepare an agonist antibody that specifically binds to the FGFR or FGFR/ ⁇ -klotho complex to mimic the mechanism of action of FGF21 to activate the FGF/FGFR signaling pathway ( See WO2011/071783, WO2011/130417, WO2012/158704 and WO2012/170438); or FGF21 half-life can be improved by fusion with Fc fragments (see WO2004/110472, WO2005/113606, WO2009/149171, WO2010/042747, WO2010/ 129503, WO2010/129600,
  • Fc fusion technology In the field of protein drug long-acting technology, Fc fusion technology is the most widely used because it has fewer clinical side effects (for example, it is not easy to induce an allergic reaction or increase the toxicity of the drug due to prolonged half-life).
  • the key to the development of FGF21/Fc long-acting fusion protein drugs lies in the following points: First, can we maintain FGF21 Biological activity.
  • the C-terminus of FGF21 contains a binding site for ⁇ -Klotho, and thus fusion of Fc to the C-terminus of FGF21 results in a significant decrease in its activity, while fusion at the N-terminus retains its binding affinity to ⁇ -Klotho to the utmost extent.
  • the Fc fragment is mostly fused to the N-terminus of FGF21 (Fc-FGF21); secondly, whether the fusion of Fc can significantly improve the pharmacokinetic properties of FGF21, and effectively extend its half-life to meet clinically twice a week or even once per week.
  • the half-life improvement was not significant because the C-terminus of FGF21 in the fusion protein (especially between Pro171 and Ser172) was not protected by Fc and thus susceptible to Degradation occurs by protease attack (Hecht, R et al, PLoS One, 2012, 7: e49345).
  • the ideal FGF21Fc fusion protein on the one hand, the anti-proteolytic ability of the C-terminus of FGF21 is enhanced, and a significantly extended half-life is obtained; on the other hand, the binding affinity of the C-terminus of FGF21 to ⁇ -Klotho is not due to the steric hindrance effect of Fc.
  • the significant decrease leads to a significant decrease in its activity; at the same time, the introduction of fusion ligands and linker peptides does not increase its immunogenicity and can improve its stability.
  • CTP human chorionic gonadotropin
  • CTP acts as a linker between the beta subunit and the alpha subunit of follicle stimulating hormone; among the fusion proteins disclosed in WO2005058953A2, CTP is used as a linker for Linking the beta and alpha subunits of the glycoprotein hormone.
  • the present inventors did not use CTP as a linker or as an extended half-life part according to the prior art, but instead linked it to a flexible peptide linker (for example, (GGGGS)n) to form a new linker sequence, which was placed between FGF21 and the extended half-life portion (
  • a flexible peptide linker for example, (GGGGS)n
  • an immunoglobulin Fc fragment but not including the CTP suggested by the prior art, constitutes a novel FGF21 fusion protein, thereby further extending the half-life and maintaining good biological activity and function.
  • the object of the present invention is to solve the problems of short half-life, poor stability of FGF21, and to provide a highly glycosylated human FGF21 fusion protein with improved pharmaceutical properties, for example, enhanced proteolytic resistance, prolonged in vivo half-life and reduced aggregation.
  • the present invention provides a hyperglycosylated human fibroblast growth factor 21 (hFGF21) fusion protein (hereinafter referred to as hFGF21 fusion protein), which comprises wild type human fibrils from N-terminal to C-terminal.
  • hFGF21 hyperglycosylated human fibroblast growth factor 21
  • hFGF21 fusion protein which comprises wild type human fibrils from N-terminal to C-terminal.
  • Cell growth factor 21 or an analog thereof denoted as hFGF21
  • L flexible peptide linker
  • CTP carboxy terminal peptide rigid unit of at least one human chorionic gonadotropin beta subunit
  • CTP human chorionic gonadotropin beta subunit
  • fusion ligands eg, immunoglobulin and its Fc fragment, albumin or transferrin.
  • the fusion proteins disclosed herein comprise a hFGF21 wild-type polypeptide, wherein the wild-type hFGF21 polypeptide comprises the sequence set forth in SEQ ID NO: 1 with amino acid leader 1-28 removed; or comprises 1- The amino acid leader peptide of 28 and having the equivalent sequence of SEQ ID NO: 1 substituted with G141S or L174P.
  • the fusion proteins disclosed herein comprise an hFGF21 analog, eg, having one or more amino acid deletions, insertions, additions or substitutions relative to its wild-type sequence, and deletion of the N-terminus or C-terminus a truncated form of one or more amino acids; preferably, the hFGF21 analog is at least 70% homologous to the amino acid sequence of wild-type hFGF21; more preferably, the hFGF21 analog is at least 80% identical to the wild-type hFGF21 amino acid sequence More preferably, the hFGF21 analog is at least 90% homologous to the amino acid sequence of wild-type hFGF21. Most preferably, the hFGF21 analog is at least 95% homologous to the amino acid sequence of wild-type hFGF21.
  • the flexible peptide linker is preferably non-immunogenic and produces sufficient distance between hFGF21 and the fusion ligand to minimize steric effects between each other.
  • a flexible peptide linker comprising two or more amino acids is used and is selected from the group consisting of Gly (G), Ser (S), Ala (A) and Thr (T).
  • the flexible peptide linker comprises G and S residues.
  • the length of the linker peptide is very important for the activity of the fusion protein.
  • the flexible peptide linker amino acid composition has the structural formula of (GS)a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are greater than or equal to 0. An integer, and a+b+c+d ⁇ 1.
  • the peptide linker is selected from the group consisting of:
  • the CTP rigid unit is selected from the full length or truncated sequence consisting of amino acids 113 to 145 of the carboxy terminus of human chorionic gonadotropin ⁇ subunit, in particular, the CTP rigid unit comprises SEQ ID NO: 2 or its truncated sequence.
  • the CTP rigid unit comprises at least 2 glycosylation sites; for example, in a preferred embodiment of the invention, the CTP rigid unit comprises 2 glycosylation sites, exemplarily, the CTP The rigid unit comprises 10 amino acids of the N-terminus of SEQ ID NO: 2, ie SSSS*KAPPPS*; or the CTP rigid unit comprises 14 amino acids of the C-terminus of SEQ ID NO: 2, ie S*RLPGPS*DTPILPQ; as another example Wherein the CTP rigid unit comprises 3 glycosylation sites, exemplarily, the CTP rigid unit comprises 16 amino acids of the N-terminus of SEQ ID NO: 2, ie SSSS*KAPPPS*LPSPS*R; In an embodiment, the CTP rigid unit comprises four glycosylation sites, exemplarily, the CTP rigid unit comprises 28, 29, 30, 31, 32 or 33 amino acids and begins with human chorionic gonadotropin The 113th, 114th, 115th, 116th, 117th or 118th position of
  • the CTP rigid unit comprises 28 amino acids of the N-terminus of SEQ ID NO: 2, namely SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ.
  • * represents a glycosylation site.
  • the CTP rigid units provided herein are at least 70% homologous to the native CTP amino acid sequence; in other embodiments, the CTP rigid units provided herein are at least 80% homologous to the native CTP amino acid sequence; In other embodiments, the CTP rigid units provided herein are at least 90% homologous to the native CTP amino acid sequence; in other embodiments, the CTP rigid units provided herein are at least 95% homologous to the native CTP amino acid sequence.
  • the CTP rigid unit of the present invention may preferably comprise the following sequence:
  • CTP1 SSSSKAPPPSLPSPSRLPGPSDTPILPQ;
  • CTP2 PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ;
  • the fusion protein comprises one of the above CTP rigid units.
  • the fusion protein of the present invention may further comprise one or more of the above CTP rigid units, preferably comprising 2, 3, 4 or 5 of the above CTP rigid units.
  • the fusion protein comprises three CTP3 rigid units: SSSSKAPPPSSSSSKAPPPSSSSSKAPPPS (CTP3-CTP3-CTP3, or expressed as (CTP3) 3 ); in another embodiment of the invention, the fusion The protein comprises two CTP5 rigid units: SSSSKAPPPSLPSPSRSSSSKAPPPSLPSPSR (CTP5-CTP5, or expressed as (CTP5) 2 ).
  • the fusion ligand is preferably an immunoglobulin Fc fragment; more preferably, the Fc fragment is preferably an Fc fragment from human immunoglobulin IgG, IgM, IgA and variants thereof; more preferably from human IgG1, IgG2, IgG3 or IgG4 and A variant Fc fragment, wherein the human IgG Fc variant (denoted as vFc) comprises at least one amino acid modification in wild-type human IgG Fc, and the Fc variant is non-lytic and exhibits minimal Fc - mediated adverse side effects (ADCC and CDC effects) and/or enhanced binding affinity to the FcRn receptor.
  • ADCC and CDC effects minimal Fc - mediated adverse side effects
  • human IgG Fc variant may be selected from the group consisting of:
  • vFc ⁇ 1 human IgG1 hinge region, CH2 and CH3 regions containing the Leu234Val, Leu235Ala and Pro331Ser mutations (such as the amino acid sequence set forth in SEQ ID NO: 3);
  • vFc ⁇ 2-1 human IgG2 hinge region, CH2 and CH3 region containing the Pro331Ser mutation (such as the amino acid sequence shown in SEQ ID NO: 4);
  • vFc ⁇ 2-2 human IgG2 hinge region, CH2 and CH3 region containing the Thr250Gln and Met428Leu mutations (such as the amino acid sequence shown in SEQ ID NO: 5);
  • vFc ⁇ 2-3 human IgG2 hinge region, CH2 and CH3 regions (such as the amino acid sequence shown in SEQ ID NO: 6) containing the Pro331Ser, Thr250Gln and Met428Leu mutations.
  • vFc ⁇ 4 human IgG4 hinge region, CH2 and CH3 region containing the Ser228Pro and Leu235Ala mutations (such as the amino acid sequence shown in SEQ ID NO: 7).
  • the IgG Fc variants provided by the present invention include, but are not limited to, the five variants described in (i) to (v), and may also be Combination or superposition of two types of functional variant mutation sites between IgG isotypes, as described in (iv) above, which is a new one obtained by superimposing the mutation sites in (ii) and (iii) A combined variant of IgG2Fc.
  • An Fc variant (vFc) in a fusion protein of the invention which comprises a hinge region, a CH2 and a CH3 region of human IgG such as human IgG1, IgG2 and IgG4.
  • This CH2 region contains amino acid mutations at positions 228, 234, 235 and 331 (as determined by the EU counting system). It is believed that these amino acid mutations reduce the effector function of Fc.
  • Human IgG2 does not bind to FcyR but shows very weak complement activity.
  • An Fc[gamma]2 variant with a Pro331Ser mutation should have a lower complement activity than native Fc[gamma]2 and is still an Fc[gamma]R non-binding element.
  • IgG4Fc is defective in the activation of the complement cascade and its binding affinity to Fc ⁇ R is about an order of magnitude lower than that of IgG1.
  • the Fc ⁇ 4 variant with the Ser228Pro and Leu235Ala mutations should exhibit minimal effector function compared to native Fc ⁇ 4.
  • Fc ⁇ 1 with Leu234Val, Leu235Ala and Pro331Ser mutations also showed reduced effector function compared to native Fc ⁇ 1.
  • These Fc variants are more suitable for the preparation of hFGF21 and its analog fusion proteins than native human IgG Fc.
  • the 250 and 428 positions contain amino acid mutations that increase the binding affinity of the Fc region to the neonatal receptor FcRn, thereby further extending the half-life (Paul R et al, J Biol Chem, 2004, 279: 6213). – 6216); variants of the above two types of functions are combined or superimposed to obtain new combined variants, which reduce the effector function while prolonging its half-life.
  • the Fc variants of the invention comprise, but are not limited to, mutations at several of the above sites, and substitutions at other sites may be introduced such that the Fc has reduced effector function and/or enhanced binding to the FcRn receptor, while still It does not cause a decrease in Fc variant function/activity or cause a poor conformational change.
  • Common mutation sites can be found in Shields RL et al, J Biol Chem, 2001, 276(9):6591-604.
  • amino acid sequence of the fusion protein is set forth in SEQ ID NO: 8.
  • the fusion protein of the present invention is glycosylated; preferably, the fusion protein is glycosylated by expression in mammalian cells; more preferably, the fusion protein is passed through a Chinese hamster ovary cell Glycosylation is achieved by expression.
  • a DNA encoding the above fusion protein is provided.
  • the DNA sequence of the fusion protein is set forth in SEQ ID NO: 9.
  • a carrier is provided.
  • This vector contains the above DNA.
  • a host cell comprises the vector described above or is transfected with the vector described above.
  • the host cell is a CHO derived cell line DXB-11.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and an effective amount of the above fusion protein.
  • fusion protein for the preparation of a medicament for treating a condition associated with FGF21, for example, obesity, type 1 or type 2 diabetes, pancreatitis, dyslipidemia, non-alcoholic fatty liver disease (NAFLD) Non-alcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arteries Disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, conditions associated with severe inactivation or mutation of the insulin receptor.
  • a condition associated with FGF21 for example, obesity, type 1 or type 2 diabetes, pancreatitis, dyslipidemia, non-alcoholic fatty liver disease (NAFLD)
  • NASH Non-alcoholic steatohepatitis
  • insulin resistance hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral
  • a mammalian cell line such as a CHO-derived cell line
  • the mammalian cell in step (a) is a CHO cell; more preferably the CHO-derived cell line DXB-11.
  • the hFGF21 fusion protein of the present invention has the following outstanding advantages:
  • the hFGF21 fusion protein has an extended half-life of in vivo function.
  • the hFGF21 fusion protein was administered for 16 hours.
  • the medium and high dose groups significantly improved the glucose utilization level of obese mice.
  • the high dose group could significantly improve the glucose of obese mice.
  • the hFGF21 fusion protein has long-lasting effect on improving glucose utilization.
  • this also demonstrates that the hFGF21 fusion protein has a significantly enhanced anti-proteolytic ability, which is not easily degraded in vivo and reduces or loses activity.
  • the fusion protein constructed by the present invention is fused to the C-terminus of FGF21, and the protective effect of Fc reduces the susceptibility to proteolytic enzyme, so the N-terminal Fc fusion protein (Fc-FGF21) reported in the prior art is compared.
  • the half-life has been greatly extended.
  • the CTP rigid unit contains a glycosyl group, and the negatively charged, highly sialylated CTP rigid unit is able to counteract the clearance of the kidney and further prolong the half-life of the fusion protein.
  • the CTP-containing hFGF21 fusion protein has a longer in vivo circulating half-life and higher bioavailability than the CTP-free hFGF21 fusion protein.
  • the single-dose pharmacokinetic data showed that the circulating half-life T 1/2 of the 3F/kg hFGF21 fusion protein FP4I in SD rats was 29.81 ⁇ 1.56h, whereas the CTP-free hFGF21 fusion protein.
  • the T 1/2 of FP4J is only 22.43 ⁇ 1.45h, which is greatly prolonged compared with the natural half-life of hFGF21 (the circulating half-life in rats is 1-1.5h), which greatly reduces the frequency of administration.
  • the C-terminal fusion Fc (FGF21-Fc) of FGF21 often leads to a significant decrease in its activity
  • the C-terminal Fc fusion protein constructed by the present invention utilizes a novel linker peptide, the linker peptide Consisting of a flexible peptide and a CTP rigid peptide
  • the CTP rigid unit contains a plurality of O-glycosyl side chains, which can form a relatively stable stereo conformation, and can effectively isolate hFGF21 from Fc, thereby minimizing the Fc-induced
  • the steric effect allows FGF21 to maintain better biological activity.
  • the hFGF21 fusion protein can effectively control the body mass of obese mice induced by high fat diet, improve insulin resistance and hepatic steatosis, and exhibit significant inhibition of obesity.
  • CTP is derived from natural human hCG, is non-immunogenic, and is more suitable as a linker peptide than a non-naturally encoded amino acid sequence.
  • Fc variants with enhanced binding affinity to neonatal receptor (FcRn) can further extend the half-life of the fusion protein.
  • the hFGF21 fusion protein constructed by the present invention exhibits stronger thermal stability during preparation and storage, and has a higher expression level. At the same time, in the highly concentrated preparation, the hFGF21 fusion protein is also less likely to aggregate.
  • human FGF21 and hFGF21 refers to a wild type human FGF21 polypeptide and analogs thereof.
  • the sequence of the wild-type hFGF21 protein is available from the UNIPERT database under accession number Q9NSA1.
  • the precursor protein consists of 209 amino acids, including the signal peptide (amino acids 1-28) and the mature protein (amino acids 29-209).
  • a wild-type hFGF21 isoform or allelic form having Pro instead of Leu (position 174 of SEQ ID NO: 1 of the present invention) in a mature protein is known, in particular, from US2001012628A1.
  • Another wild type hFGF21 isoform Gly was substituted with Ser (in position 141 of SEQ ID NO: 1 of the present invention).
  • Another isoform with a shorter signal peptide (in the present invention, Leu at position 23 of SEQ ID NO: 1) is known from WO 2003/011213 (see SEQ ID NO: 2 in the publication of WO 2003/011213, with 27 amino acid residues) Base signal peptide).
  • the wild-type hFGF21 comprises the mature protein partial sequence (amino acids 29-209) shown by SEQ ID NO: 1 and L174P or G141S in place of the isoform-removed leader peptide; in addition, the above-mentioned 27 or 28 is added before these sequences are included.
  • the full length sequence of the precursor protein of the amino acid signal peptide is included.
  • the hFGF21 analog according to the invention refers to a polypeptide which is or can be deduced or derived from wild type human FGF21, in particular or can be derived from SEQ ID NO: 1, ie by its amino acid sequence modification. Such modifications, modifications, or alterations can include substitutions, deletions, and/or additions of one or more amino acids.
  • the term "mutein” or “mutant” is sometimes used herein in place of the term “analog”.
  • an amino acid may be added and/or deleted at the C-terminus, N-terminus or internal to the amino acid sequence (the amino acid added inside the sequence may be referred to as an insertion).
  • Amino acids are preferably added and/or deleted at the C-terminus and/or the N-terminus, more preferably at the N-terminus.
  • An amino acid sequence having a C-terminal or N-terminal deletion may also be referred to as a truncated sequence, which is capable of providing a similar activity to the un truncated form of the mature hFGF21 polypeptide, in some cases better activity This is known in the art and includes forms of N-terminal truncation or C-terminal truncation.
  • the N-terminally truncated hFGF21 analog comprises 1, 2, 3, 4, 5, 6, 7 or 8 amino acid residues selected from the N-terminus of the deleted mature hFGF21 polypeptide.
  • Peptide. N-terminally truncated hFGF21 polypeptide with less than 9 amino acid residues retains mature hFGF21 polypeptide Reduce the ability of an individual's blood sugar.
  • the invention encompasses a truncated form of mature hFGF21 polypeptide or hFGF21 protein having an N-terminal truncation of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues. body.
  • the C-terminally truncated hFGF21 analog comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 selected from the C-terminus of the mature hFGF21 polypeptide. Or a polypeptide of 12 amino acid residues.
  • C-terminal truncated truncated FGF21 polypeptide with less than 13 amino acid residues in an in vitro ELK-luciferase assay (Yie J. et al., FEBS Letts, 2009, 583: 19-24)
  • the efficacy is at least 50% of the efficacy of wild-type hFGF21, suggesting that these hFGF21 mutants retain the ability of mature hFGF21 polypeptide to lower blood glucose in an individual.
  • the invention encompasses a truncated form having a C-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues Mature hFGF21 polypeptide or hFGF21 protein variant.
  • an hFGF21 analog is a truncated form of wild-type mature hFGF21 in which the four N-terminal amino acid residues (HPIP) of the mature protein are removed, as disclosed, for example, in WO2006/065582.
  • This truncated form is believed to stimulate glucose uptake by mouse 3T3-L1 adipocytes at the same level as wild-type hFGF21.
  • the truncated form of hFGF21 has the amino acid sequence shown in SEQ ID NO: 1 at 33-209, designated hFGF21 (HPIP - ).
  • hFGF21 is a SEQ ID NO: 1 polypeptide (also referred to as "Met-hFGF21”) having an N-terminal Met.
  • the N-terminal Met is added when hFGF21 is expressed in E. coli, see, for example, Table 6 in WO2006/050247.
  • hFGF21 analog is the substitution/replacement of one or more amino acids in the hFGF21 wild-type precursor protein set forth in SEQ ID NO: 1, including but not limited to: Q55C, A109T, L126R, G148C , K150R, P158S, S195A, P199G, G202A.
  • amino acid sequences containing other modified hFGF21 analogs are disclosed in, for example, WO2003/061712, WO2005/091944, WO2006/028595, WO2006/028714, WO2006/065582, and WO2008/121563.
  • CTP is a short peptide derived from the carboxy terminus of the ⁇ -subunit of human chorionic gonadotropin (hCG).
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • TSH thyrotropin
  • hCG chorionic gonadotropin
  • the native CTP contains 37 amino acid residues with four O-glycosylation sites and a terminal sialic acid residue. Negatively charged, highly sialylated CTP is resistant to the clearance of the kidneys, thereby prolonging the half-life of the protein in the body.
  • the inventors creatively linked at least one CTP polypeptide to a flexible linker of appropriate length, collectively as a linker peptide, for ligation of hFGF21 with a fusion ligand (eg, an immunoglobulin Fc fragment).
  • hFGF21 N-terminal and C-terminal sequences of hFGF21 are critical for their function, and the spatial conformation of hFGF21 is complex and fragile, making it poorly stable, easily degradable and easily polymerized, and then linked to the fusion ligand, and its steric hindrance. The effect can interfere with its correct folding, causing its activity to drop significantly or even be lost, or to produce aggregates more easily.
  • a CTP rigid unit between hFGF21 and the Fc variant an equivalent of a rigid linker peptide is added.
  • the N-terminally fused hFGF21 does not affect the binding site of the Fc variant and FcRn, thereby affecting the half-life; in addition, the Protein A binding site of Fc is important for the purification step in the preparation process, and the CTP rigid unit is connected to ensure The N-terminally fused hFGF21 also does not "cover" its binding site to protein A.
  • the addition of a CTP rigid unit also allows the Fc fragment of about 25 kD size to not interfere with the correct folding of the N-terminally fused hFGF21, resulting in a decrease or loss of its biological activity/function.
  • CTP glycosyl side chain can reduce the sensitivity of the linker peptide to proteases, making the fusion protein less susceptible to degradation in the junction region.
  • CTP is derived from native human hCG and is not immunogenic, and thus is more suitable for use as a linker peptide than a non-naturally encoded amino acid sequence.
  • the Fc element is derived from the constant region Fc fragment of immunoglobulin IgG, which plays an important role in eradicating the immune defense of pathogens.
  • the effector function of Fc-mediated IgG is exerted through two mechanisms: (1) binding to cell surface Fc receptors (Fc ⁇ Rs), digestion of pathogens by phagocytosis or cleavage or killer cells via antibody-dependent cellular cytotoxicity (ADCC) pathway , or (2) binding to C1q of the first complement component C1, eliciting a complement-dependent cytotoxicity (CDC) pathway, thereby lysing the pathogen.
  • Fc ⁇ Rs cell surface Fc receptors
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • IgG1 and IgG3 efficiently bind to Fc ⁇ Rs, and the binding affinity of IgG4 to Fc ⁇ Rs is low, and the binding of IgG2 to Fc ⁇ Rs is too low to be determined, so human IgG2 has almost no ADCC effect.
  • human IgG1 and IgG3 can also efficiently bind to C1q to activate the complement cascade.
  • Human IgG2 binds relatively weakly to C1q, whereas IgG4 does not bind to C1q (Jefferis R et al, Immunol Rev, 1998, 163: 59-76), so the human IgG2 CDC effect is also weak.
  • the most effective method is to mutate the complement and receptor binding domain of the Fc fragment, modulate the binding affinity of Fc to related receptors, reduce or eliminate ADCC and CDC effects, and retain only Fc. Long cycle half-life characteristics without cytotoxicity.
  • the mutation sites can be found in Shields RL et al, J Biol Chem, 2001, 276(9): 6591-604 or Chinese invention patent CN 201280031137.2.
  • Fc variant with enhanced binding affinity to neonatal receptor (FcRn)
  • the plasma half-life of IgG depends on its binding to FcRn, which generally binds at pH 6.0 and dissociates at pH 7.4 (plasma pH). By studying the binding sites of the two, the site of binding to FcRn on IgG was engineered to increase the binding ability at pH 6.0. Mutations in some residues of the human Fc ⁇ domain important for binding to FcRn have been shown to increase serum half-life. Mutations in T250, M252, S254, T256, V308, E380, M428 and N434 have been reported to increase or decrease FcRn binding affinity (Roopenian et al, Nat. Rview Immunology 7: 715-725, 2007). Korean Patent No.
  • KR10-1027427 discloses variants of trastuzumab (Herceptin, Genentech) having increased FcRn binding affinity, and these variants are selected from the group consisting of 257C, 257M, 257L, 257N, 257Y, 279Q, 279Y.
  • Korean Patent Publication No. KR2010-0099179 provides variants of bevacizumab (Avastin, Genentech) and these variants include Amino acid modifications of N434S, M252Y/M428L, M252Y/N434S, and M428L/N434S, showing increased in vivo half-life.
  • a condition associated with a severe inactivating mutation of the insulin receptor describes a condition in a subject having a mutation in the insulin receptor (or a protein directly downstream thereof) that causes severe insulin resistance, but usually does not have type 2 Obesity common in diabetes.
  • subjects with these conditions exhibit a syndrome of type 1 diabetes and type 2 diabetes. Therefore, the affected subjects are basically divided into several categories according to the severity, including: type A diabetes resistance, type C insulin resistance (AKA HAIR-AN syndrome), Rabson-Mendenhall syndrome, Donohue's syndrome or dwarf disease ( Leprechaunism). These conditions are associated with very high levels of endogenous insulin, leading to elevated blood glucose levels.
  • the affected subjects also have a variety of clinical features associated with "insulin toxicity", including androgen excess, polycystic ovary syndrome (PCOS), hirsutism and acanthosis nigricans (skin fold overgrowth and pigmentation).
  • PCOS polycystic ovary syndrome
  • hirsutism hirsutism
  • acanthosis nigricans skin fold overgrowth and pigmentation
  • Diabetes complications are dysfunctions in other parts of the body caused by chronic hyperglycemia such as diabetic nephropathy, diabetic neuropathy, diabetic foot (foot ulcer and hypotonic), and ocular lesions (retinopathy). Diabetes also increases the risk of heart disease and bone and joint disorders. Other long-term complications of diabetes include skin problems, digestive problems, sexual dysfunction, and tooth and gum problems.
  • metabolic syndrome is a pathological condition in which a variety of metabolic components are abnormally aggregated, including: (1) abdominal obesity or overweight; (2) atherosclerosis dyslipidemia, such as high triglyceride (TG) Hypertension and high-density lipoprotein cholesterol (HDL-C) are low; (3) hypertension; (4) insulin resistance and/or impaired glucose tolerance. Some criteria also include microalbuminuria, hyperuricemia, and pro-inflammatory state (C-reactive protein CRP) and thrombotic state (fibrinogen elevation and plasminogen inhibitor-1, PAI-1) Increase.
  • C-reactive protein CRP pro-inflammatory state
  • thrombotic state fibrinogen elevation and plasminogen inhibitor-1, PAI-1
  • Dyslipidemia is a condition of lipoprotein metabolism, including lipoprotein overproduction or defects. Dyslipidemia can be manifested by an increase in blood total cholesterol, low-density lipoprotein (LDL) cholesterol and triglyceride concentrations, and a decrease in high-density lipoprotein (HDL) cholesterol.
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • Non-alcoholic fatty liver disease is a liver disease unrelated to alcohol consumption and is characterized by hepatic steatosis.
  • Non-alcoholic steatohepatitis is a liver disease that is not associated with alcohol consumption and is characterized by hepatic steatosis accompanied by inflammation and fibrosis in the lobule.
  • Figure 1 shows SpeI/EcoRI in a PCDNA3.1 expression vector according to an embodiment of the present invention.
  • the nucleotide sequence of the fragmented hFGF21 fusion protein and the deduced amino acid sequence are indicated by the ⁇ 1 microglobulin leader peptide (1-19, Marked), hFGF21 (20-200, P substituted L), flexible peptide linker (201-227, Mark), CTP 1 (228-255, to Marked) and vFc (256-478) constitute.
  • FIG. 2-a hFGF21 fusion protein FP4I reduction and non-reduction SDS-PAGE electrophoresis results, from left to right band in order: NR (non-reducing); M (protein Marker); R (reductive).
  • Figure 2-b Results of FP-HPLC detection of hFGF21 fusion protein FP4I.
  • FIG 7, hFGF21 FP4I fusion protein in liver function in mice fed the high fat diet conditions (means ⁇ SEM, n 8 ); statistically different tag Note: Compared with low-fat diet group, # P ⁇ 0.05, # # P ⁇ 0.01; compared with the high fat diet group, * P ⁇ 0.05, ** P ⁇ 0.01.
  • FIG. 15 Effect of hFGF21 fusion protein FP4I on liver histopathology of high-fat diet-fed mice; Note: A: low-fat diet group; B: high-fat diet group; C: high-fat diet combined with hFGF21 fusion protein group.
  • FIG. 16 Effect of hFGF21 fusion protein FP4I on the pathological morphology of adipose tissue in mice fed with high fat diet; Note: A: low fat diet group; B: high fat diet group; C: high fat diet combined with hFGF21 fusion protein group.
  • the fusion proteins of the invention are typically prepared by biosynthetic methods. According to the nucleotide sequence of the present invention, one skilled in the art can conveniently prepare the nucleic acid of the present invention by various known methods. These methods are, for example but not limited to, PCR, DNA synthesis, etc. For specific methods, see J. Sambrook, Molecular Cloning Experiment Guide. As an embodiment of the present invention, the nucleic acid sequence of the present invention can be constructed by a method of segmentally synthesizing a nucleotide sequence and performing overlap extension PCR.
  • the invention also provides an expression vector comprising a sequence encoding a fusion protein of the invention and an expression control sequence operably linked thereto.
  • operably linked or “operably linked” is meant a condition in which portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, then it is operably linked to the coding sequence.
  • the expression vector may be a commercially available vector such as, but not limited to, pcDNA3, pIRES, pDR, pUC18 or the like which can be used for expression of a eukaryotic cell system.
  • pcDNA3, pIRES, pDR, pUC18 or the like which can be used for expression of a eukaryotic cell system.
  • One skilled in the art can select a suitable expression vector based on the host cell.
  • the recombinant enzyme expression vector of the present invention is prepared by cleavage and splicing of the enzyme and inserting the coding sequence of the fusion protein of the present invention into a suitable restriction site.
  • the invention also provides a host cell expressing a fusion protein of the invention comprising a coding sequence for a fusion protein of the invention.
  • the host cell is preferably a eukaryotic cell such as, but not limited to, CHO, COS cells, 293 cells, RSF cells and the like.
  • the cell is a CHO cell which can express the fusion protein of the present invention well, and a fusion protein having good binding activity and good stability can be obtained.
  • the present invention also provides a method for producing a fusion protein of the present invention using recombinant DNA, the steps of which include:
  • Introduction of the coding sequence into a host cell can employ a variety of known techniques in the art such as, but not limited to, calcium phosphate precipitation, protoplast fusion, lipofection, electroporation, microinjection, reverse transcription, phage Transduction method, alkali metal ion method.
  • the fusion protein obtained as described above can be purified to a substantially uniform property, such as a single band on SDS-PAGE electrophoresis.
  • a commercially available ultrafiltration membrane can be used to separate the protein, for example, from Millipore, Pellicon, etc., and the expression supernatant is first concentrated.
  • the concentrate may be further purified by gel chromatography or by ion exchange chromatography. For example, anion exchange chromatography (DEAE, etc.) or cation exchange chromatography.
  • the gel matrix may be a matrix commonly used for protein purification such as agarose, dextran, polyamide, and the like.
  • the Q- or SP- group is a preferred ion exchange group.
  • the purified product may be further purified by hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). All of the above purification steps can utilize different combinations to ultimately achieve a substantially uniform protein purity.
  • an affinity chromatography column containing a specific antibody, receptor or ligand of the fusion protein can be used for expression
  • the fusion protein was purified.
  • the fusion polypeptide bound to the affinity column can be eluted using conventional methods such as high salt buffer, pH change, and the like.
  • the amino terminus or carboxy terminus of the fusion protein may also contain one or more polypeptide fragments as a protein tag.
  • Any suitable label can be used in the present invention.
  • the label may be FLAG, HA, HA1, c-Myc, 6-His or 8-His, and the like. These tags can be used to purify the fusion protein.
  • Example 1 Construction of an expression plasmid encoding the hFGF21Fc fusion protein
  • the gene sequence encoding the ⁇ 1 microglobulin leader peptide and mature hFGF21 or its analog, flexible peptide linker, CTP rigid unit and human IgG Fc variant are artificially optimized CHO cell-preferred codons, full-length sequence Obtained by chemical synthesis methods.
  • the purified fusion protein gene of hFGF21 or its analog was digested with SpeI and EcoRI, and inserted into the corresponding restriction site of plasmid PXY1A1 modified by PCDNA3.1 to obtain a fusion gene expression plasmid.
  • This plasmid contains a cytomegalovirus early promoter which is an enhancer required for mammalian cells to express foreign genes at high levels.
  • the plasmid also contains a selectable marker to have kanamycin resistance in bacteria and G418 resistance in mammalian cells.
  • the PXY1A1 expression vector contains the mouse dihydrofolate reductase (DHFR) gene, thereby co-amplifying the fusion gene and the DHFR gene in the presence of methotrexate (MTX). (See U.S. Patent 4,399,216).
  • the present invention constructs a series of fusion proteins of hFGF21 comprising wild-type hFGF21 and its analogs, different lengths of flexible peptide linkers (Linker) and several different subtypes of IgG Fc variants (vFc).
  • the components are composed, and the position and length of the CTP rigid unit are also different.
  • the nucleotide sequence and translated amino acid sequence of FP4I are shown in FIG. 1 .
  • wild-type hFGF21 has the amino acid sequence shown in SEQ ID NO: 1 (denoted as hFGF21) and its isoform L174P hFGF21 (SEQ ID NO: 1 at position 174, Leu is replaced by Pro), and hFGF21 analog is preferred. comprises a truncated wild-type polypeptide hFGF21 hFGF21 (HPIP -), i.e., wild-type N- terminus of the mature protein deleted hFGF21 HPIP (having SEQ ID NO: 1 amino acid sequence of position 33-209).
  • the hFGF21 analog also preferably includes a variant in which an amino acid is substituted/substituted, such as variant Q55C, which is obtained by replacing Gln at position 55 of SEQ ID NO: 1 with Cys, and several other variants include A109T, L126R K150R, P199G and G202A are also substituted by the amino acid of the corresponding site of SEQ ID NO: 1.
  • the recombinant expression vector plasmid is transfected into a mammalian host cell line to express the hFGF21 fusion protein.
  • a preferred host cell line is a DHFR enzyme deficient CHO-cell (see U.S. Patent No. 4,818,679).
  • the host cell is selected from the CHO-derived cell line DXB11.
  • a preferred method of transfection is electroporation, and other methods, including calcium phosphate co-precipitation, lipofection, can also be used.
  • the transfected fusion protein gene was co-amplified with the DHFR gene in growth medium containing increasing concentrations of MTX.
  • Subclones with positive dilution DHFR expression were gradually pressurized, and transfectants capable of growing in up to 6 ⁇ M MTX medium were screened, the secretion rate was determined, and a cell line highly expressing the foreign protein was selected.
  • a cell line having a secretion rate of more than about 30 (preferably about 50) ⁇ g/10 6 (i.e., millions) of cells per 24 hours is subjected to adaptive suspension culture using serum-free medium, and then purified by conditioned medium. protein.
  • the conditioned medium containing the fusion protein was titrated to pH 7-8 with 1 N NaOH and then filtered through a 0.45 micron nitrocellulose filter. The filtrate was loaded onto a phosphate buffered saline (PBS) equilibrated Protein A column. After the fusion protein is bound to the Protein A column, the effluent components are discarded. The column was washed with PBS until the OD value at 280 nm was less than 0.01. The bound fusion protein was then eluted with 0.1 M citrate buffer pH 3.75. The eluate was neutralized with 0.4 volume of 1 M K 2 HPO 4 and the fractions containing the purified protein were combined and dialyzed against PBS.
  • PBS phosphate buffered saline
  • Example 4 Effect of single injection of hFGF21 fusion protein on glucose utilization rate in obese mice induced by high fat diet
  • mice 32 male C57BL/6J mice of SPF grade and 7 weeks old (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were selected. Breeding environment: temperature 22-25 ° C, relative humidity 45-65%, lighting time 12h / d.
  • Mice were randomly divided into 4 groups according to body weight: control group, hFGF21 fusion protein FP4I 2.5 mg/kg group (low dose group), 5 mg/kg group (middle dose group) and 10 mg/kg group (high dose group). The mice in the administration group were subcutaneously injected with the corresponding drug solution, and the control mice were subcutaneously injected with PBS buffer.
  • mice in each group were fasted for 16 hours to carry out the glucose tolerance test.
  • the fasting blood glucose level of the mice was measured, and 2 g/kg glucose solution was intraperitoneally injected.
  • the blood glucose levels after 15 min, 30 min, 60 min, 90 min, and 120 min of glucose injection were measured, and the area under the curve (iAUC) was calculated by the trapezoidal method.
  • the mice in each group continued the glucose tolerance test in the same manner as above. Data were expressed as mean ⁇ standard error (means ⁇ SEM) and data were analyzed using SPSS 18.0 statistical software. Normal distribution, one-way ANOVA was used for the mean difference between groups, LSD test was used for variance homogeneity, Dunnet T3 test for variance variance; non-parametric test for non-normal distribution, P ⁇ 0.05 for significant statistics difference.
  • hFGF21 fusion protein FP4I significantly improved the glucose utilization level of obese mice at 16 h after administration (P ⁇ 0.05).
  • high doses still significantly improved glucose utilization levels in obese mice 48 h after administration (P ⁇ 0.05).
  • the experimental results demonstrate that the hFGF21 fusion protein provided by the present invention has long-lasting effect on improving glucose utilization.
  • mice induced by high-fat diet were randomly divided into 10 groups (6 in each group): FP4A, FP4B, FP4C, FP4D, FP4E, FP4F, FP4G, FP4H, and FP4J, and the control group.
  • the mice in the drug-administered group were injected subcutaneously.
  • the corresponding solution was 10 mg/kg, and the control mice were injected subcutaneously with PBS buffer.
  • Glucose tolerance experiments were performed at 16 h and 48 h after injection, respectively.
  • hFGF21 fusion proteins FP4A, FP4B, FP4C, FP4D, FP4E, FP4F, FP4G, FP4H and FP4J significantly improved glucose utilization in obese mice at 16 h after administration (P ⁇ 0.05).
  • Figure 4a hFGF21 fusion proteins FP4A, FP4B, FP4C, FP4D, FP4E, FP4F, FP4G and FP4H significantly improved glucose utilization in obese mice 48 h after administration (P ⁇ 0.05), while FP4J group and control group There is no significant difference in blood glucose (Fig. 4b).
  • the hFGF21 fusion protein provided by the present invention has strong anti-proteolytic ability, is not easily degraded in the body, thereby maintaining a long physiological activity, and CTP-containing with respect to CTP-free FP4J.
  • the hFGF21 fusion protein FP4I showed a longer functional half-life in vivo, suggesting that the addition of CTP can further extend the in vivo functional half-life of hFGF21.
  • the mouse anti-hFGF21 antibody (R&D, product number MAB25373-100) was coated with HRP-labeled goat anti-human IgG-Fc mAb (Jackson, Inc., Cat. No. 109-035-098).
  • the data is then input into the analysis software PKSOLVER to obtain the pharmacokinetic parameters T 1/2 and AUC (0-t) of the drug to be tested in the blood.
  • Example 6 Experimental study of hFGF21 fusion protein in preventing obesity, nonalcoholic steatosis, insulin resistance and hypercholesterolemia in mice fed with high fat diet
  • the high fat diet combined with the FP4I group was injected subcutaneously with FP4I every 4 days at a dose of 3.6 mg/kg, and the low fat diet group and the high fat diet group were subcutaneously injected with PBS buffer for 116 days.
  • each group of mice was fasted for 16 hours, weighed and tested for fasting blood glucose, and the whole eye was taken by eyeball, centrifuged at 2000 ⁇ g for 15 min, and serum was separated. The liver tissue was separated, and after washing with physiological saline, the filter paper was blotted and weighed.
  • liver tissue Take 2 parts of liver tissue in the same part, 1 part is fixed in 10% formalin solution for HE staining, and the other part of liquid nitrogen is frozen and stored at -80 °C for liver index analysis.
  • One side of the epididymal adipose tissue was taken and fixed in a 10% formalin solution for HE staining.
  • mice were weighed once every 4 days, and the muscle, fat and body fluid contents of the mice were analyzed by time-domain NMR on the 104th day after the start of the experiment.
  • the Ouba XL-200 automatic biochemical analyzer measures the concentrations of ALT, AST, HDL-c, LDL-c, TG and TC in serum, and the specific operation is carried out according to the instrument manual.
  • mice in each group were fasted for 6 h (10:00 am-4:00 pm), and the blood glucose levels of each group were measured and weighed.
  • the insulin solution was administered by intraperitoneal injection at a lean mass of 0.75 IU/kg.
  • the blood glucose levels of the mice at 15 min, 30 min, 60 min, 90 min and 120 min after injection were measured, and the insulin tolerance curve was drawn.
  • the AUC area was calculated by the trapezoidal method.
  • the fasting serum insulin content of mice was measured by ELISA, and the insulin resistance index was calculated.
  • Insulin resistance index fasting blood glucose value (mmol / L) ⁇ fasting insulin content (mIU / L) / 22.5.
  • liver triglyceride content 2.4, liver triglyceride content
  • mice were accurately weighed 50 mg of liver tissue, and the TG content in liver tissue was measured by Folch method. Results are expressed as TG content per mg of liver tissue.
  • liver and adipose tissue of the same part were preserved in 10% formalin solution, and histopathological observation was performed by HE staining.
  • the body weight, body mass increase and body fat content of the high-fat diet group increased significantly, showing obvious obesity symptoms.
  • the hFGF21 fusion protein can significantly reduce the body mass increase and body fat content under high-fat diet feeding conditions. The results are shown in Figures 5-6.
  • mice fed high-fat diet showed significant symptoms of insulin resistance.
  • Serum insulin test results further indicate that mice have hyperinsulinemia and insulin resistance.
  • the hFGF21 fusion protein can significantly improve the insulin resistance of mice under high-fat diet feeding conditions and alleviate the increase of fasting blood glucose. The results are shown in Figure 10-13.
  • the liver tissue of the high-fat diet group showed obvious fatty degeneration, and the lipid droplet vacuoles were clearly visible and fused into a sheet-like structure, and the liver cell morphology was severely damaged.
  • the fat cell cross-sectional area is significantly increased.
  • the hFGF21 fusion protein significantly attenuated liver tissue steatosis and reduced the fat cell cross-sectional area.
  • hFGF21 fusion protein can effectively control the body mass of obese mice induced by high fat diet, improve insulin resistance, relieve liver steatosis and hypercholesterolemia.

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Abstract

具有改善的药学性质的人成纤维细胞生长因子21(hFGF21)或其类似物的融合蛋白，以及所述融合蛋白在制备治疗糖尿病、肥胖、非酒精性脂肪肝病、异常脂血症和/或代谢综合征等病症药物中的用途。

Description

人成纤维细胞生长因子21融合蛋白及其制备方法与用途 技术领域
本发明涉及人成纤维细胞生长因子21(hFGF21)融合蛋白，还涉及所述融合蛋白在制备治疗糖尿病、肥胖、异常脂血症和/或代谢综合征药物中的用途。
背景技术
成纤维细胞生长因子(Fibroblast growth factors,FGFs)家族是一类具有多种生理功能的多肽生长因子，已发现的哺乳动物FGF家族共有22个成员，分为7个亚家族。成纤维细胞生长因子21(FGF21)属于FGF19/21/23亚家族，主要在肝脏中表达，此外在与糖脂代谢相关的组织细胞，如脂肪组织、胰岛β细胞、肌肉组织中也有一定的表达。与其它FGF成员不同，该亚家族以内分泌的方式发挥作用，参与调控能量和胆酸内稳态、葡萄糖和脂质代谢，以及磷酸盐和维生素D的内稳态(Moore DD等,Science,2007,316:1436-1438；Beenken等,Nature Reviews Drug Discover,2009,8:235)。FGF21成熟蛋白由181个氨基酸组成，与FGF家族中绝大多数成员不同，FGF21无法与肝素特异性结合而促进细胞生长及分化。FGF21C-末端与其生物活性密切相关，FGF21的C-末端与辅助因子β-Klotho直接结合，然后激活FGFR受体及下游相关信号分子，继而发挥其生物效应(Yie J等,FEBS Lett,2009,583(1):19-24；和Micanovic R等,J Cell Physiol,2009,219(2):227-234)。
Kharitonenkov等首先阐明了FGF21对机体的代谢调节功能，FGF21通过调控葡萄糖转运子1(glucose transporter 1,GLUT1)表达并促进葡萄糖在3T3-L1细胞和人原代脂肪细胞中的摄取。后续实验显示，在基因控制或饮食诱导产生的肥胖小鼠中，FGF21显著降低小鼠体重和血糖水平，同时还减少肝脏和血清中的甘油三酯含量，对小鼠胰岛素敏感性也有重要影响(Kharitonenkov A等,Journal of clinical Investigation,2005,115(6):1627-1635；Coskun T等,Endocrinology,2008,149(12):6018-6027；Xu J等,Diabetes,2009,58(1):250-259)。饮食诱导的肥胖小鼠和ob/ob小鼠给予外源FGF21能够逆转其肝脏脂肪变性，增强肝脏胰岛素敏感性(包括减少肝脏葡萄糖产生和增加肝糖元含量)，从而改善全身葡萄糖不耐 受性及胰岛素抵抗(Xu J等,Diabetes,2009,58(1):250-259；Berglund ED等,Endocrinology,2009,150(9):4084-4093；Xu J等,Am J Physiol Endocrinol Metab,2009,297(5)E1105-1104)。另外，FGF21在糖尿病猴子中具有相同的代谢调节作用，同时还能减少低密度脂蛋白胆固醇以及增加高密度脂蛋白胆固醇浓度(Kharitonenkov A等,Endocrinology,2007,148(2):774-781)。综上所述，FGF21对肥胖的啮齿类、非人类灵长类动物都具有十分显著的有益代谢调节作用。不依赖胰岛素调节血糖浓度的FGF21，作为降糖药物大剂量用药时也不会导致低血糖。另外，FGF21是FGF家族中目前发现的唯一没有促有丝分裂作用的细胞因子，从而大大降低临床用药副作用风险。
大量临床前和临床研究显示，FGF21是治疗肥胖、2型糖尿病以及高脂血症等病症的潜在优良靶点。然而，由于其自身理化特性缺陷，使得天然FGF21很难被开发成临床治疗用生物制剂，主要原因包括：1.FGF21蛋白稳定性差，易受蛋白酶水解或酶促降解；2.FGF21构象非常不稳定，易发生聚集，低稳定性同时也增加了FGF21在大规模生产中的难度；3.天然FGF21半衰期非常短，人FGF21在小鼠体内半衰期0.5-1小时，在食蟹猴体内半衰期2-3小时(Kharitonenkov A等,Journal of clinical Investigation,2005,115:1627-1635)。多种蛋白长效化技术被报道用于延长重组FGF21的体内半衰期。例如，FGF21与PEG分子链接，增加分子量，降低肾小球滤过率，延长体内滞留时间(参见专利WO2005/091944,WO2006/050247,WO2008/121563和WO2012/066075)；FGF21与脂肪酸长链融合(能够结合血清白蛋白)(参见WO2010/084169和WO2012/010553)；或制备能与FGFR或FGFR/β-klotho复合物特异性结合的激动剂抗体模拟FGF21作用机制，来激活FGF/FGFR信号通路(参见WO2011/071783,WO2011/130417,WO2012/158704和WO2012/170438)；又或通过与Fc片段融合也可改善FGF21半衰期(参见WO2004/110472,WO2005/113606,WO2009/149171,WO2010/042747,WO2010/129503,WO2010/129600,WO2013/049247,WO2013/188181和WO2016/114633)。
在蛋白药物长效化技术领域，Fc融合技术运用最为广泛，因为它具有更小的临床副作用(例如，不易诱发过敏反应或因半衰期延长而加剧药物的毒性作用)。开发FGF21/Fc长效融合蛋白药物关键在于以下几点：其一，能否保持FGF21 的生物学活性。FGF21的C-末端含有β-Klotho的结合位点，因而Fc融合于FGF21C-末端会使其活性大幅降低，而融合在N-末端能最大程度地保留它与β-Klotho的结合亲和力。故现有技术中Fc片段大多融合于FGF21N-末端(Fc-FGF21)；其二，融合Fc后能否显著改善FGF21的药动学特性，有效延长其半衰期以满足临床上每周两次甚至每周一次给药的用药需求；因FGF21的C-末端还含有蛋白酶水解位点，极易发生降解，其水解代谢物的体外活性下降近200倍(Micanovic R等,J Cell Physiol,2009,219(2):227-234；Yie J等,FEBS Lett,2009,583(1):19-24)。据Fc-FGF21药代动力学研究结果显示，其半衰期改善并不显著，这是因为融合蛋白中FGF21的C-末端(尤其是在Pro171和Ser172之间)暴露，不能被Fc保护，因而易受蛋白酶攻击而发生降解(Hecht,R等,PLoS One,2012,7:e49345)。其三，如何降低接头序列或引入突变位点诱发免疫反应发生的风险；其四，融合Fc和/或引入突变位点是否可以改善FGF21的稳定性及其在高浓缩状态下的易聚合性。所以，理想的FGF21Fc融合蛋白，一方面FGF21C-末端的抗蛋白酶水解能力要增强，并且获得显著延长的半衰期；再一方面，FGF21C-末端与β-Klotho的结合亲和力不会因Fc的位阻效应而明显下降，导致其活性大幅下降；同时，融合配体及连接肽的引入还不增加其免疫源性，且还能改善其稳定性。
人绒毛膜促性腺激素(hCG)β链的羧基末端肽(以下称其为CTP)也具有延长某些蛋白体内半衰期的作用，因此一些专利文献公开的融合蛋白中选择CTP作为融合配体用于延长目的蛋白的半衰期。另外，CTP也可以作为接头，主要用于连接同一个蛋白的不同亚基。例如，中国专利CN103539860A、CN103539861A、CN103539868A和CN103539869A公开的融合蛋白中，CTP作为接头，位于促卵泡激素的beta亚基和alpha亚基之间；专利WO2005058953A2公开的融合蛋白中，CTP作为接头，用于连接糖蛋白激素的beta亚基和alpha亚基。
本发明人没有根据现有技术将CTP作为接头或者作为延长半衰期部分，而是将它与柔性肽接头(例如(GGGGS)n)连接组成新的接头序列，设置于FGF21和延长半衰期部分之间(如，免疫球蛋白Fc片段，但不包括现有技术所提示的CTP)之间，组成新的FGF21融合蛋白，从而进一步延长了半衰期，并且保持了良好的生物学活性和功能。
发明内容
本发明目的是解决FGF21半衰期短、稳定性差等问题，提供了一种药学性质改善的高糖基化的人FGF21融合蛋白，例如，增强的蛋白水解抗性、延长的体内半衰期并减少聚集。
本发明一方面，提供了一种高糖基化人成纤维细胞生长因子21(hFGF21)融合蛋白(以下简称hFGF21融合蛋白)，所述融合蛋白自N端至C端依次含有野生型人成纤维细胞生长因子21或其类似物(表示为hFGF21)、柔性肽接头(表示为L)、至少1个人绒毛膜促性腺激素β亚基的羧基末端肽刚性单元(以下表示为(CTP)n，n为1,2,3,4,或5)和融合配体(如，免疫球蛋白及其Fc片段、白蛋白或转铁蛋白)。
在一些实施例中，本发明公开的融合蛋白包含hFGF21野生型多肽，其中，野生型hFGF21多肽包含去除了1-28位氨基酸前导肽的SEQ ID NO：1所示序列；或包含去除了1-28位氨基酸前导肽且具有G141S或L174P取代的SEQ ID NO：1所示的同等型序列。
在另一些实施例中，本发明公开的融合蛋白包含hFGF21类似物，例如，相对于其野生型序列具有一个或多个氨基酸缺失、插入、添加或取代，以及缺失N-末端或C-末端的一个或多个氨基酸的截短形式；优选地，所述hFGF21类似物与野生型hFGF21的氨基酸序列至少70％同源；较优选地，所述hFGF21类似物与野生型hFGF21氨基酸序列至少80％同源；更优选地，所述hFGF21类似物与野生型hFGF21的氨基酸序列至少90％同源。最优选地，所述hFGF21类似物与野生型hFGF21的氨基酸序列至少95％同源。
其中，所述柔性肽接头优选非免疫原性的，并且在hFGF21和融合配体之间产生足够的距离，使相互之间的位阻效应降至最低。较佳地，使用含有2个或更多个氨基酸构成的柔性肽接头，且选自下列几种氨基酸：Gly(G)、Ser(S)、Ala(A)和Thr(T)。
优选地，所述柔性肽接头包含G和S残基。连接肽的长度对融合蛋白的活性非常重要。对本发明而言，优选地，所述柔性肽接头氨基酸组成的结构通式为(GS)a(GGS)b(GGGS)c(GGGGS)d，其中a，b，c和d是大于或等于0的整数，且a+b+c+d≥1。
本发明的一些实施例中，所述肽接头选自如下序列：
(i)L1：GGGGS；
(ii)L2：GSGGGSGGGGSGGGGS；
(iii)L3：GSGGGGSGGGGSGGGGSGGGGSGGGGS；
(iv)L4：GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
(v)L5：GGGSGGGSGGGSGGGSGGGS；
(vi)L6：GGSGGSGGSGGS。
其中，所述CTP刚性单元选自由人绒毛膜促性腺激素β亚基羧基末端第113至145位氨基酸所组成的全长或截短的序列，具体地，所述CTP刚性单元包含SEQ ID NO：2或其截短的序列。
优选地，所述CTP刚性单元包含至少2个糖基化位点；例如，本发明的一优选实施例中，所述CTP刚性单元包含2个糖基化位点，示例性地，所述CTP刚性单元包含SEQ ID NO：2N端的10个氨基酸，即SSSS*KAPPPS*；或所述CTP刚性单元包含SEQ ID NO：2C端的14个氨基酸，即S*RLPGPS*DTPILPQ；又如，另一实施例中，所述CTP刚性单元包含3个糖基化位点，示例性地，所述CTP刚性单元包含SEQ ID NO：2N端的16个氨基酸，即SSSS*KAPPPS*LPSPS*R；再如，另些实施例中，所述CTP刚性单元包含4个糖基化位点，示例性地，所述CTP刚性单元含28、29、30、31、32或33个氨基酸并开始于人绒毛膜促性腺激素β亚基的第113、114、115、116、117或118位，终止于第145位。具体地，所述CTP刚性单元包含SEQ ID NO：2N端的28个氨基酸，即SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ。在本文中，*代表糖基化位点。每种可能性都代表本发明的独立实施方式。
在另一些实施例中，本发明提供的CTP刚性单元与天然CTP氨基酸序列至少70％同源；在另一些实施例中，本发明提供的CTP刚性单元与天然CTP氨基酸序列至少80％同源；在另一些实施例中，本发明提供的CTP刚性单元与天然CTP氨基酸序列至少90％同源；在另一些实施例中，本发明提供的CTP刚性单元与天然CTP氨基酸序列至少95％同源。
示例性地，本发明所述CTP刚性单元可优选地包含如下序列：
(i)CTP1：SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(ii)CTP2：PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(iii)CTP3：SSSSKAPPPS；
(iv)CTP4：SRLPGPSDTPILPQ；
(v)CTP5：SSSSKAPPPSLPSPSR。
本发明一些实施例中，所述融合蛋白包含1个上述CTP刚性单元。
本发明所述融合蛋白还可包含1个以上的上述CTP刚性单元，优选地，包含2，3，4或5个上述CTP刚性单元。如本发明的一实施例中，所述融合蛋白包含3个CTP3刚性单元：SSSSKAPPPSSSSSKAPPPSSSSSKAPPPS(CTP3-CTP3-CTP3，或表示为(CTP3)3)；如本发明的另一实施例中，所述融合蛋白包含2个CTP5刚性单元：SSSSKAPPPSLPSPSRSSSSKAPPPSLPSPSR(CTP5-CTP5，或表示为(CTP5)2)。
其中，融合配体优选免疫球蛋白Fc片段；较优选地，Fc片段优选自人免疫球蛋白IgG、IgM、IgA及其变体的Fc片段；更优选自人IgG1、IgG2、IgG3或IgG4及其变体的Fc片段，其中，所述人IgG Fc变体(表示为vFc)包含位于野生型人IgG Fc中的至少一种氨基酸修饰，且Fc变体无裂解性，并显示出极小的Fc-介导的不良副作用(ADCC和CDC效应)和/或与FcRn受体的结合亲和力增强。
进一步地，人IgG Fc变体可选自下组：
(i)vFcγ1：含有Leu234Val、Leu235Ala和Pro331Ser突变的人IgG1绞链区、CH2和CH3区域(如SEQ ID NO：3所示氨基酸序列)；
(ii)vFcγ2-1：含有Pro331Ser突变的人IgG2绞链区、CH2和CH3区域(如SEQ ID NO：4所示氨基酸序列)；
(iii)vFcγ2-2：含有Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域(如SEQ ID NO：5所示氨基酸序列)；
(iv)vFcγ2-3：含有Pro331Ser、Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域(如SEQ ID NO：6所示氨基酸序列)。
(v)vFcγ4：含有Ser228Pro和Leu235Ala突变的人IgG4绞链区、CH2和CH3区域(如SEQ ID NO：7所示氨基酸序列)。
本发明所提供的IgG Fc变体包含但不限于(i)～(v)中所述5种变体，还可以是 IgG同种亚型间两类功能变体突变位点的组合或叠加，如上述(iv)中所述变体即是由(ii)和(iii)中的突变位点相叠加所获得的新的IgG2Fc的组合变体。
本发明所述融合蛋白中的Fc变体(vFc)，它含有人IgG如人IgG1、IgG2和IgG4的绞链区、CH2和CH3区域。这种CH2区域在228、234、235和331位(由EU计数系统确定)含有氨基酸突变。据信这些氨基酸突变能降低Fc的效应子功能。人IgG2不结合FcγR，但显示出极弱的补体活性。具有Pro331Ser突变的Fcγ2变体应比天然Fcγ2的补体活性更低，而且依旧是FcγR非结合子。IgG4Fc在激活补体级联中有缺陷，且它与FcγR的结合亲和力比IgG1低约一个数量级。与天然Fcγ4相比，具有Ser228Pro和Leu235Ala突变的Fcγ4变体应表现出最小的效应子功能。具有Leu234Val、Leu235Ala和Pro331Ser突变的Fcγ1也表现出比天然Fcγ1降低的效应子功能。这些Fc变体都比天然人IgG Fc更适于制备hFGF21及其类似物融合蛋白。而250和428位(由EU编号体系确定的位置)含有氨基酸突变，使得Fc区与新生儿受体FcRn的结合亲和力增加，从而进一步延长半衰期(Paul R等,J Biol Chem,2004,279:6213–6216)；上述两类功能的变体相互组合或叠加，获得新的组合变体，使其效应子功能降低的同时且延长了其半衰期。本发明所述Fc变体包含却不局限于上述几个位点的突变，也可引入其它位点的替换使得Fc具有降低的效应子功能和/或与FcRn受体的结合力增强，同时还不会致使Fc变体功能/活性降低或引起不良的构象变化，常见的突变位点可以参见Shields RL等,J Biol Chem,2001,276(9):6591-604。
本发明的一优选实施例中，所述融合蛋白的氨基酸序列如SEQ ID NO：8所示。
本发明所述的融合蛋白是糖基化的；优选地，所述融合蛋白是通过在哺乳动物细胞中表达而实现糖基化的；更优选地，所述融合蛋白是通过在中国仓鼠卵巢细胞中表达而实现糖基化的。
根据本发明的另一个方面，提供一种编码上述融合蛋白的DNA。本发明的一优选实施例中，所述融合蛋白的DNA序列如SEQ ID NO：9所示。
根据本发明的再一个方面，提供一种载体。该载体包含上述DNA。
根据本发明的再一个方面，提供一种宿主细胞。该宿主细胞包含上述载体，或者转染了上述载体。
在本发明的具体实施方式中，宿主细胞是CHO的衍生细胞株DXB-11。
根据本发明的再一个方面，提供一种药物组合物。该药物组合物包含药学上可接受的载体、赋形剂或稀释剂，以及有效量的上述融合蛋白。
根据本发明的再一个方面，提供所述融合蛋白在制备治疗与FGF21相关病症药物中的用途，例如，肥胖、1型或2型糖尿病、胰腺炎、血脂异常、非醇型脂肪肝病(NAFLD)、非醇型脂肪性肝炎(NASH)、胰岛素耐受、高胰岛素血症、葡萄糖不耐受、高血糖、代谢综合征、急性心肌梗塞、高血压、心血管病、动脉粥样硬化、外周动脉病、中风、心脏衰竭、冠心病、肾病、糖尿病并发症、神经病、与胰岛素受体的严重失活或突变相关的病症。
根据本发明的另一方面提供了一种从哺乳动物细胞系(如CHO衍生的细胞系)制备或生产所述融合蛋白的方法，包含以下步骤：
(a)将编码上述融合蛋白的DNA引入哺乳动物细胞；
(b)筛选步骤(a)中在其生长培养基中每24小时期间内，表达超过50μg/106个细胞的高产量细胞株；
(c)培养步骤(b)筛选获得的细胞株；
(d)收获步骤(c)得到的发酵液，纯化融合蛋白。
优选地，所述步骤(a)中的哺乳动物细胞为CHO细胞；更优选为CHO衍生细胞系DXB-11。
与现有产品相比，本发明所述hFGF21融合蛋白，具有如下的突出优点：
1、具有延长的体内功能半衰期。在高脂饮食诱导的肥胖小鼠给予hFGF21融合蛋白，给药16h，中、高剂量组均能显著改善肥胖小鼠葡萄糖利用水平，给药后48h，高剂量组仍可显著改善肥胖小鼠葡萄糖利用水平，说明hFGF21融合蛋白对于提高葡萄糖利用率具有长效性。同时，这也证明hFGF21融合蛋白具有显著增强的抗蛋白酶水解能力，在体内不易被降解而使活性降低或丧失。
2、获得了显著延长的体内循环半衰期。本发明所构建的融合蛋白，因Fc融合于FGF21的C-末端，Fc的保护作用使其对蛋白水解酶的易感性降低，所以较现有技术报道的N-末端Fc融合蛋白(Fc-FGF21)半衰期获得了大幅延长。另一方面，CTP刚性单元含有糖基，带负电、高度唾液酸化的CTP刚性单元能够抵 抗肾脏的清除作用，进一步延长融合蛋白的半衰期。本发明优选实施例中，证实含有CTP的hFGF21融合蛋白比不含CTP的hFGF21融合蛋白具有更长的体内循环半衰期和更高的生物利用度。优选实施例中显示，单次给药药代动力学数据显示，3mg/kg的hFGF21融合蛋白FP4I在SD大鼠体内循环半衰期T1/2为29.81±1.56h，而不含CTP的hFGF21融合蛋白FP4J的T1/2仅为22.43±1.45h，相对于天然hFGF21半衰期(在大鼠体内循环半衰期为1～1.5h)大幅度延长，大大降低给药频率。
3、现有技术中，FGF21的C-末端融合Fc(FGF21-Fc)往往导致其活性大幅降低，而本发明所构建的C-末端Fc融合蛋白，利用一种新型的连接肽，该连接肽由柔性肽和CTP刚性肽所组成，CTP刚性单元含有多个O-糖基侧链，能形成相对稳定的立体构象，可以有效地将hFGF21与Fc隔离开，从而最大限度地降低Fc带来的位阻效应，使FGF21保持了较佳的生物学活性。例如，hFGF21融合蛋白可以有效控制高脂饲料诱导的肥胖小鼠体质量，改善胰岛素抵抗和肝脏脂肪变性，表现出明显的抑制肥胖作用。
此外，CTP刚性单元糖基侧链的保护作用可以降低连接肽对蛋白酶的敏感性，使融合蛋白不易在连接区被降解。并且，CTP来源于天然人hCG，无免疫原性，相对于非天然编码的氨基酸序列更适宜用作连接肽。
4、对融合配体Fc进行突变，只保留Fc的长循环半衰期特性，降低或消除ADCC和CDC效应(例如，P331S)，从而增加了用药安全性。另外，与新生儿受体(FcRn)结合亲和力增强的Fc变体(例如，T250Q/M428L)可以进一步延长融合蛋白的半衰期。
5、本发明所构建的hFGF21融合蛋白在制备和储存过程中表现出更强的热稳定性，且具有较高的表达量。同时，在高浓缩制剂中，hFGF21融合蛋白也不易发生聚集。
定义
本发明所用的术语“人FGF21及hFGF21”是指野生型人FGF21多肽及其类似物。
野生型FGF21多肽
野生型hFGF21蛋白的序列可获自UNIPROT数据库，登录号为Q9NSA1。前体蛋白由209个氨基酸组成，包括信号肽(氨基酸1-28)和成熟蛋白(氨基酸29-209)。
尤其从US2001012628A1可得知在成熟蛋白中具有Pro替代Leu(在本发明SEQ ID NO：1的第174位)的野生型hFGF21同等型(isoform)或等位形式(allelicform)。另一种野生型hFGF21同等型的Gly被Ser取代(在本发明SEQ ID NO：1的第141位)。
从WO2003/011213可得知具有较短信号肽(本发明中SEQ ID NO：1第23位Leu丢失)的另一种同等型(参见WO2003/011213公布中的SEQIDNO：2，具有27个氨基酸残基的信号肽)。
本发明中，野生型hFGF21包含SEQ ID NO：1及L174P或G141S取代同等型去除前导肽后所示成熟蛋白部分序列(氨基酸29-209)；另外，还包括这些序列之前添加了上述27或28个氨基酸信号肽的前体蛋白全长序列。
hFGF21类似物
本发明所涉及的hFGF21类似物，是指由或可以由野生型人FGF21、特别是由或可以由SEQ ID NO：1推导或衍生的多肽，即通过其氨基酸序列修饰。这样的修饰(modification)、修改(amendment)或变化(change)可包括一个或多个氨基酸的取代、缺失和/或添加。在本文中有时使用术语“变体(mutein)”或“突变体”替代术语“类似物”。例如，可以在氨基酸序列中的C-末端、N-末端或内部添加和/或缺失氨基酸(在序列内部添加的氨基酸可以称为插入)。优选在C-末端和/或N-末端、更优选在N-末端添加和/或缺失氨基酸。
具有C-末端或N-末端缺失的氨基酸序列也可以称为截短的序列，所述截短的hFGF21多肽能够提供与未截短形式的成熟hFGF21多肽相似的，在一些情况下更优的活性，这是本领域已知的，它包括N-末端截短或C-末端截短的形式。
a、N-末端截短
在本发明的一些实施方案中，N-末端截短的hFGF21类似物包含选自缺失成熟hFGF21多肽的N-末端的1、2、3、4、5、6、7或8个氨基酸残基的多肽。具有少于9个氨基酸残基的N-末端截短的hFGF21多肽保留了成熟hFGF21多肽 降低个体血糖的能力。因此，在特定的实施方案中，本发明涵盖了具有N-末端截短1、2、3、4、5、6、7或8个氨基酸残基的截短形式的成熟hFGF21多肽或hFGF21蛋白变体。
b、C-末端截短
在本发明的一些实施方案中，C-末端截短的hFGF21类似物包含选自成熟hFGF21多肽的C-末端的1、2、3、4、5、6、7、8、9、10、11或12个氨基酸残基的多肽。在体外ELK-荧光素酶测定法(Yie J.等人，FEBS Letts,2009,583:19-24)中，具有少于13个氨基酸残基的C-末端截短的截短的FGF21多肽表现出的功效是野生型hFGF21功效的至少50％，提示这些hFGF21突变体保留了成熟hFGF21多肽降低个体血糖的能力。因此，在特定的实施方案中，本发明涵盖了具有C-末端截短1、2、3、4、5、6、7、8、9、10、11或12个氨基酸残基的截短形式的成熟hFGF21多肽或hFGF21蛋白变体。
hFGF21类似物的一个实例是野生型成熟hFGF21的截短形式，其中成熟蛋白的4个N-端氨基酸残基(HPIP)被去除，其公开于例如WO2006/065582中。这种截短的形式据信能在与野生型hFGF21相同的水平上刺激小鼠3T3-L1脂肪细胞的葡萄糖摄取。在本发明中，截短形式的hFGF21具有SEQ ID NO：1第33-209所示氨基酸序列，表示为hFGF21(HPIP-)。
hFGF21类似物的另一个实例是具有N-端Met的SEQ ID NO：1多肽(也称为“Met-hFGF21”)。N-端Met是当hFGF21在大肠杆菌(E.coli)中表达时添加的，参见例如WO2006/050247中的表6。
hFGF21类似物的另一个实例是SEQ ID NO：1所示hFGF21野生型前体蛋白中的一个和多个氨基酸被取代/替换，所述取代/替换包含但不限于：Q55C、A109T、L126R、G148C、K150R、P158S、S195A、P199G、G202A。
含有其它修饰的hFGF21类似物的氨基酸序列实例公开于例如WO2003/061712、WO2005/091944、WO2006/028595、WO2006/028714、WO2006/065582和WO2008/121563中。
hCG-β羧基末端肽(CTP)
CTP是一段来自人绒毛膜促性腺激素(hCG)的β-亚基羧基末端的短肽。四 种与生殖相关的多肽类激素促卵泡激素(FSH)、黄体生成素(LH)、促甲状腺素(TSH)和绒毛膜促性腺激素(hCG)含有相同的α-亚基和各自特异的β-亚基。与其它三种激素相比，hCG体内半衰期明显延长，这主要来源于其β-亚基上特有的羧基末端肽(CTP)(Fares FA等,Proc Natl Acad Sci USA,1992,89:4304-4308)。天然的CTP含有37个氨基酸残基，具有4个O-糖基化位点，终端是唾液酸残基。带负电、高度唾液酸化的CTP能够抵抗肾脏对其的清除作用，从而延长蛋白在体内的半衰期。然而，本发明人创造性地将至少一个CTP多肽与适当长度的柔性连接肽连接，共同作为连接肽，用于连接hFGF21与融合配体(如，免疫球蛋白Fc片段)。
本发明人发现，hFGF21的N端和C端序列对其功能发挥极为关键，且hFGF21空间构象复杂、脆弱，使它自身稳定性差，易降解且易聚合，再与融合配体连接，其位阻效应会对其正确折叠造成干扰，使其活性显著下降甚至丧失，或更易产生聚合体。通过在hFGF21与Fc变体间增加CTP刚性单元，相当于增加了一段刚性连接肽。一方面，保证了N-端融合的hFGF21不会影响Fc变体与FcRn的结合位点，从而影响半衰期；另外Fc的Protein A结合位点对于制备工艺中纯化步骤很重要，连接CTP刚性单元保证N-端融合的hFGF21也不会“罩住”它与protein A的结合位点。再一方面，CTP刚性单元的添加也使得约25KD大小的Fc片段不会干扰N-端融合的hFGF21的正确折叠，造成其生物学活性/功能的下降或丧失。
我们还证实单纯延长柔性连接肽的长度并不能明显改善Fc段对hFGF21活性的影响，过长的连接肽还会造成多聚体的形成以及对蛋白酶敏感性增加而易被降解，而CTP刚性单元的加入使得融合蛋白更加稳定。这可能解释为具有多个糖基侧链的CTP刚性多肽，相对于(GGGGS)n这类柔性连接肽的无规则卷曲，它可以形成稳定的立体构象，这种“阻隔”作用促使hFGF21和Fc段独立折叠形成正确的三维构象而互不影响各自的生物活性。再一方面，CTP糖基侧链的保护作用可以降低连接肽对蛋白酶的敏感性，使融合蛋白不易在连接区被降解。此外，CTP来源于天然人hCG，不具有免疫原性，因而相对于非天然编码的氨基酸序列更适宜用作连接肽。
IgG Fc变体
非裂解性Fc变体
Fc元件来源于免疫球蛋白IgG的恒定区Fc片段，它在消灭病原体的免疫防御中起重要作用。Fc介导的IgG的效应子功能发挥通过两种机制：(1)与细胞表面Fc受体(FcγRs)结合，由吞噬作用或裂解作用或杀伤细胞通过抗体依赖性细胞毒性(ADCC)途径消化病原体，或(2)与第一补体成分C1的C1q结合，引发补体依赖性细胞毒性(CDC)途径，从而裂解病原体。在四种人IgG亚型中，IgG1和IgG3能有效结合FcγRs，IgG4与FcγRs的结合亲和力较低，而IgG2与FcγRs的结合低得难以测定，所以人IgG2几乎没有ADCC效应。此外，人IgG1和IgG3还能有效结合C1q而激活补体级联反应。人IgG2与C1q结合相对弱，而IgG4不与C1q结合(Jefferis R等,Immunol Rev,1998,163:59-76)，所以人IgG2CDC效应也较弱。显然，没有一种天然IgG亚型是非常适合构建hFGF21/Fc融合蛋白的。为了得到不具效应子功能的非裂解性Fc，最有效方法是对Fc片段上补体、受体结合域突变改造，调节Fc与相关受体的结合亲和力，降低或消除ADCC和CDC效应，只保留Fc的长循环半衰期特性，而不产生细胞毒性。更多的非裂解性Fc变体所包含突变位点可以参见Shields RL等,J Biol Chem,2001,276(9):6591-604或中国发明专利CN 201280031137.2。
与新生儿受体(FcRn)结合亲和力增强的Fc变体
IgG的血浆半衰期取决于它与FcRn的结合，一般它们在pH6.0时结合，在pH7.4(血浆pH)时解离。通过对两者结合位点的研究，改造IgG上与FcRn结合的位点，使之在pH6.0时结合能力增加。已经证明对于结合FcRn重要的人Fcγ结构域的一些残基的突变可增加血清半衰期。已报道T250、M252、S254、T256、V308、E380、M428和N434中的突变可增加或降低FcRn结合亲和力(Roopenian等,Nat.Rview Immunology7:715-725,2007)。韩国专利号KR10-1027427公开了具有增加的FcRn结合亲和力的曲妥珠单抗(赫赛汀，Genentech)变体，并且这些变体包含选自257C、257M、257L、257N、257Y、279Q、279Y、308F和308Y的一个或更多个氨基酸修饰。韩国专利公开号KR2010-0099179提供了贝伐单抗(阿瓦斯汀，Genentech)变体并且这些变体包含 N434S、M252Y/M428L、M252Y/N434S和M428L/N434S的氨基酸修饰，显示增加的体内半衰期。此外，Hinton等也发现T250Q和M428L 2个突变体分别使与FcRn的结合增加3和7倍。同时突变2个位点，则结合增加28倍。在恒河猴体内，M428L或T250QM/428L突变体显示血浆半衰期增加2倍(Paul R.Hinton等,J Immunol,2006,176:346-356)。更多的与新生儿受体(FcRn)结合亲和力增强的Fc变体所包含突变位点可以参见中国发明专利CN201280066663.2。此外，有研究对五种人源化抗体的Fc段进行T250Q/M428L突变不仅改善了Fc与FcRn的相互作用，且在随后的体内药代动力学试验中，发现以皮下注射方式给药，Fc突变抗体与野生型抗体相比药代动力学参数有所改善，如体内暴露量增加，清除率降低，皮下生物利用度提高(Datta-Mannan A等,MAbs.Taylor&Francis,2012,4(2):267-273.)。
“与胰岛素受体严重失活性突变相关的病症”描述了患有胰岛素受体(或其直接下游的可能蛋白)突变对象中的病况，所述突变导致严重的胰岛素耐受，但通常没有2型糖尿病中常见的肥胖。在许多方面，患有这些病况对象表现出1型糖尿病和2型糖尿病的综合症状。因而受累对象按严重程度递增基本分为若干类别，包括：A型糖尿病抗性、C型胰岛素抗性(AKA HAIR-AN综合征)、Rabson-Mendenhall综合征，Donohue氏综合征或矮怪病(Leprechaunism)。这些病症与非常高的内源性胰岛素水平相关，导致血糖水平升高。因而受累对象还存在多种与“胰岛素毒性”相关临床特征，包括雄激素过多、多囊卵巢综合征(PCOS)、多毛症和黑棘皮病(皮肤褶皱过度生长和色素沉积)。
“糖尿病并发症”是由慢性高血糖引起的身体其它部位的功能障碍如糖尿病肾病、糖尿病神经病变、糖尿病足(足部溃疡和循环低下)和眼部病变(视网膜病)。糖尿病还增加心脏病以及骨和关节病症风险。糖尿病的其它长期并发症包括皮肤问题、消化问题、性功能障碍和牙齿与牙龈问题。
“代谢综合征”(metabolic syndrome,MS)是多种代谢成分异常聚集的病理状态，包括：(1)腹部肥胖或超重；(2)动脉粥样硬化血脂异常，如高甘油三酯(TG)血症及高密度脂蛋白胆固醇(HDL-C)低下；(3)高血压；(4)胰岛素抗性和/或葡萄糖耐量异常。有些标准中还包括微量白蛋白尿、高尿酸血症及促炎症状态(C-反应蛋白CRP)增高及促血栓状态(纤维蛋白原增高和纤溶酶原抑制物-1，PAI-1) 增高。
“血脂异常”是脂蛋白代谢病症，包括脂蛋白过度生产或缺陷。血脂异常可以表现为血液中总胆固醇、低密度脂蛋白(LDL)胆固醇和甘油三酯浓度升高，和高密度脂蛋白(HDL)胆固醇浓度减少。
“非醇型脂肪肝病(NAFLD)”是与醇消耗无关的肝病，其特征是肝细胞脂肪变性。
“非醇型脂肪性肝炎(NASH)”是与醇消耗无关的肝病，其特征是肝细胞脂肪变性，伴随小叶内炎症和纤维化。
附图说明
图1、显示了根据本发明实施例在PCDNA3.1表达载体内SpeI/EcoRI(酶切位点以
Figure PCTCN2017079871-appb-000001
标示出)片段的hFGF21融合蛋白的核苷酸序列及推导的氨基酸序列，由α1微球蛋白前导肽(1-19，以
Figure PCTCN2017079871-appb-000002
标示)、hFGF21(20-200，P取代L)、柔性肽接头(201-227，以
Figure PCTCN2017079871-appb-000003
标示)、CTP1(228-255，以
Figure PCTCN2017079871-appb-000004
标示)和vFc(256-478)构成。
图2-a、hFGF21融合蛋白FP4I还原与非还原SDS-PAGE电泳结果，从左至右条带依次为：NR(非还原性)；M(蛋白Marker)；R(还原性)。
图2-b、hFGF21融合蛋白FP4I SEC-HPLC检测结果。
图3-a、hFGF21融合蛋白FP4I单次注射16h后糖耐量实验曲线(means±SEM，n＝8)
图3-b、hFGF21融合蛋白FP4I单次注射16h后糖耐量实验iAUC(means±SEM，n＝8)；统计学差异标记注释：与对照组相比，*P＜0.05。
图3-c、hFGF21融合蛋白FP4I单次注射48h后糖耐量实验曲线(means±SEM，n＝8)。
图3-d、hFGF21融合蛋白FP4I单次注射48h后糖耐量实验iAUC(means±SEM，n＝8)；统计学差异标记注释：与对照组相比，*P＜0.05。
图4-a、几种hFGF21融合蛋白单次注射16h后糖耐量实验iAUC(means±SEM，n＝8)；统计学差异标记注释：与对照组相比，*P＜0.05。
图4-b、几种hFGF21融合蛋白单次注射48h后糖耐量实验iAUC(means±SEM，n＝8)；统计学差异标记注释：与对照组相比，*P＜0.05。
图5、hFGF21融合蛋白FP4I对高脂饲料喂养条件下小鼠体重增幅的影响(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01；与高脂饲料组相比，*P＜0.05，**P＜0.01。
图6、hFGF21融合蛋白FP4I对高脂饲料喂养条件下小鼠身体组成的影响(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01；与高脂饲料组相比，*P＜0.05，**P＜0.01。
图7、hFGF21融合蛋白FP4I对高脂饲料喂养条件下小鼠肝功能的影响(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01；与高脂饲料组相比，*P＜0.05，**P＜0.01。
图8、hFGF21融合蛋白FP4I对高脂饲料喂养小鼠肝脏质量的影响(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01；与高脂饲料组相比，*P＜0.05，**P＜0.01。
图9、hFGF21融合蛋白FP4I对高脂饲料喂养小鼠肝脏三酰甘油含量的影响(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01；与高脂饲料组相比，*P＜0.05，**P＜0.01。
图10-a、hFGF21融合蛋白FP4I对高脂饲料喂养小鼠胰岛素耐量影响实验的血糖曲线(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01；与高脂饲料组相比，*P＜0.05，**P＜0.01。
图10-b、hFGF21融合蛋白FP4I对高脂饲料喂养小鼠胰岛素耐量影响实验AUC(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01；与高脂饲料组相比，*P＜0.05，**P＜0.01。
图11、hFGF21融合蛋白FP4I对高脂饲料喂养小鼠空腹血糖水平的影响(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01；与高脂饲料组相比，*P＜0.05，**P＜0.01。
图12、hFGF21融合蛋白FP4I对高脂饲料喂养小鼠空腹胰岛素水平的影响(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01；与高脂饲料组相比，*P＜0.05，**P＜0.01。
图13、hFGF21融合蛋白FP4I对高脂饲料喂养小鼠胰岛素抵抗指数的影响(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01； 与高脂饲料组相比，*P＜0.05，**P＜0.01。
图14、hFGF21融合蛋白FP4I对高脂饲料喂养条件下小鼠血清脂质的影响(means±SEM，n＝8)；统计学差异标记注释：与低脂饲料组相比，#P＜0.05，##P＜0.01；与高脂饲料组相比，*P＜0.05，**P＜0.01。
图15、hFGF21融合蛋白FP4I对高脂饲料喂养小鼠肝脏组织病理形态学影响；注：A：低脂饲料组；B：高脂饲料组；C：高脂饲料联合hFGF21融合蛋白组。
图16、hFGF21融合蛋白FP4I对高脂饲料喂养小鼠脂肪组织病理形态学影响；注：A：低脂饲料组；B：高脂饲料组；C：高脂饲料联合hFGF21融合蛋白组。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
本发明融合蛋白通常由生物合成的方法制备。根据本发明所述的核苷酸序列，本技术领域人员可方便地用各种已知方法制得本发明的编码核酸。这些方法例如但不限于：PCR，DNA人工合成等，具体的方法可参见J.萨姆布鲁克，《分子克隆实验指南》。作为本发明的一种实施方式，可通过分段合成核苷酸序列再进行重叠延伸PCR的方法来构建本发明的编码核酸序列。
本发明还提供了一种表达载体，包含编码本发明的融合蛋白的序列以及与之操作性相连的表达调控序列。所述的“操作性相连”或“可操作地连于”指这样一种状况，即线性DNA序列的某些部分能够调节或控制同一线性DNA序列其它部分的活性。例如，如果启动子控制序列的转录，那么它就是可操作地连于编码序列。
表达载体可采用市售的例如但不限于：pcDNA3、pIRES、pDR，pUC18等可用于真核细胞系统表达的载体。本领域技术人员可以根据宿主细胞来选择合适的表达载体。
根据已知空载表达载体的酶切图谱，本领域技术人员可按照常规方法通过限 制性酶剪切与拼接，将本发明的融合蛋白的编码序列插入合适的限制性位点，制得本发明的重组表达载体。
本发明还提供了表达本发明融合蛋白的宿主细胞，其中含有本发明的融合蛋白的编码序列。所述的宿主细胞优选的是真核细胞，例如但不限于CHO，COS细胞，293细胞，RSF细胞等。作为本发明的优选方式，所述的细胞是CHO细胞，其可良好地表达本发明的融合蛋白，可获得结合活性良好，稳定性良好的融合蛋白。
本发明还提供一种用重组DNA制备本发明融合蛋白的方法，其步骤包括：
1)提供编码融合蛋白的核酸序列；
2)将1)的核酸序列插入到合适的表达载体，获得重组表达载体；
3)将2)的重组表达载体导入合适的宿主细胞；
4)在适合表达的条件下培养转化宿主细胞；
5)收集上清液，并纯化融合蛋白产物。
将所述编码序列导入宿主细胞可采用本领域的多种已知技术，例如但不限于：磷酸钙沉淀，原生质体融合，脂质体转染，电穿孔，微注射，反转录法，噬菌体转导法，碱金属离子法。
有关宿主细胞的培养和表达可参见Olander RM Dev Biol Stand,1996,86:338。可通过离心去除悬浮液中的细胞和残渣，收集清液。可通过琼脂糖凝胶电泳技术进行鉴定。
可将上述制备获得的融合蛋白纯化为基本均一的性质，例如在SDS-PAGE电泳上呈单一条带。例如，当重组蛋白为分泌表达时，可以采用商品化的超滤膜来分离所述蛋白，例如Millipore、Pellicon等公司产品，首先将表达上清浓缩。浓缩液可采用凝胶层析的方法进一步加以纯化，或采用离子交换层析的方法纯化。例如阴离子交换层析(DEAE等)或阳离子交换层析。凝胶基质可为琼脂糖、葡聚糖、聚酰胺等常用于蛋白纯化的基质。Q-或SP-基团是较为理想的离子交换基团。最后，还可用羟基磷灰石吸附层析，金属螯合层析，疏水相互作用层析和反相高效液相色谱(RP-HPLC)等方法对上述纯化产物进一步精制纯化。上述所有纯化步骤可利用不同的组合，最终使蛋白纯度达到基本均一。
可利用含有所述融合蛋白的特异性抗体、受体或配体的亲和层析柱对表达的 融合蛋白进行纯化。根据所使用的亲和柱的特性，可利用常规的方法，如高盐缓冲液、改变pH等方法洗脱结合在亲和柱上的融合性多肽。可选择地，所述的融合蛋白的氨基端或羧基端还可含有一个或多个多肽片段，作为蛋白标签。任何合适的标签都可以用于本发明。例如，所述的标签可以是FLAG，HA，HA1，c-Myc，6-His或8-His等。这些标签可用于对融合蛋白进行纯化。
实施例1、构建编码hFGF21Fc融合蛋白的表达质粒
编码α1微球蛋白(α1microglobulin)前导肽和成熟hFGF21或其类似物、柔性肽接头、CTP刚性单元和人IgG Fc变体的基因序列都是人工优化过的CHO细胞偏爱密码子，全长序列经化学合成方法获得。为了便于将目的片段插入表达载体的特定位点，在所合成的片段5’和3’端各有一个限制性酶切位点，分别为SpeI和EcoRI。验证后的hFGF21或其类似物融合蛋白基因用SpeI和EcoRI酶切，然后插入到经PCDNA3.1改造后的质粒PXY1A1相应酶切位点间，得到了融合基因表达质粒。该质粒含巨细胞病毒早期启动子，它是哺乳动物细胞高水平表达外源基因所需的增强子。该质粒还含有选择性标记物，从而在细菌中可以具有卡那霉素抗性，而在哺乳动物细胞中可以具有G418抗性。另外，当宿主细胞是DHFR基因表达缺陷型时，PXY1A1表达载体含有小鼠的二氢叶酸还原酶(DHFR)基因，从而在存在氨甲蝶呤(MTX)时能共扩增融合基因和DHFR基因(参见美国专利US 4,399,216)。
如表1所示，本发明构建了一系列hFGF21的融合蛋白，它们包含野生型hFGF21及其类似物、不同长度的柔性肽接头(Linker)和几种不同亚型的IgG Fc变体(vFc)元件组成，而且CTP刚性单元位置和长度也不同。为了验证含有至少1个，并且不同长度的CTP刚性单元均具有较高的生物学活性，我们构建了融合蛋白FP4A、FP4B、FP4C、FP4D、FP4E、FP4F、FP4G、FP4H和FP4I；同时还构建了不含CTP刚性单元的FP4J。其中，FP4I的核苷酸序列及翻译的氨基酸序列如图1所示。
本实施例中野生型hFGF21具有如SEQ ID NO：1所示的氨基酸序列(表示为hFGF21)和其同等型L174PhFGF21(SEQ ID NO：1第174位Leu被Pro取代)，而hFGF21类似物优选地包括截短的野生型hFGF21多肽hFGF21(HPIP-)，即野 生型hFGF21成熟蛋白的N-末端缺失了HPIP(具有SEQ ID NO：1第33-209位所示氨基酸序列)。hFGF21类似物还优选地包括一个氨基酸被取代/替换的变体，例如变体Q55C，是由SEQ ID NO：1第55位的Gln被Cys所替换而得，另外几种变体包括A109T、L126R、K150R、P199G和G202A也是由SEQ ID NO：1相应位点氨基酸被替换而得。
表1、所构建hFGF21融合蛋白的组成
Figure PCTCN2017079871-appb-000005
实施例2、融合蛋白在转染细胞系中的表达
将重组表达载体质粒转染入哺乳动物宿主细胞系，以表达hFGF21融合蛋白。为了稳定高水平的表达，优选的宿主细胞系是DHFR酶缺陷型CHO-细胞(参见美国专利US 4,818,679)，本实施例中宿主细胞选取CHO衍生细胞株DXB11。一种优选的转染方法是电穿孔，也可以使用其它方法，包括磷酸钙共沉降、脂转染。在电穿孔中，用设置为300V电场和1500μFd电容的Gene Pulser电穿孔仪(Bio-Rad Laboratories,Hercules,CA)，在比色杯内的5×107个细胞中加入50μg高纯度的表达质粒。在转染两天后，将培养基改成含0.6mg/mL G418的生长培养基。用抗人IgG Fc的ELISA分析方法，筛选对选择用药具有抗性的转染子。也可用抗hFGF21的ELISA进行融合蛋白表达量的定量。通过极限稀释96孔培养板，亚克隆产生高水平融合蛋白的孔。
为了实现融合蛋白较高水平的表达，宜用受MTX药物抑制的DHFR基因进行共扩增。在含有递增浓度MTX的生长培养基中，用DHFR基因共扩增转染 的融合蛋白基因。极限稀释DHFR表达阳性的亚克隆，逐步加压并筛选出能在高达6μM MTX培养基中生长的转染子，测定其分泌率，筛选出高表达外源蛋白的细胞系。将分泌率超过约30(较佳地约50)μg/106(即百万)个细胞/24小时的细胞系使用无血清培养基的进行适应性悬浮培养，然后再用条件培养基纯化融合蛋白。
实施例3、融合蛋白的纯化与定性
用1N NaOH将含有融合蛋白的条件培养基滴定到pH 7～8，然后用0.45微米的硝酸纤维素过滤器过滤。将滤液加样到磷酸盐缓冲液盐水(PBS)平衡的Protein A柱上。待融合蛋白结合于Protein A柱后，弃去流出的组分。用PBS洗涤该柱，直到280nm处的OD值低于0.01。然后用0.1M pH为3.75的柠檬酸缓冲液洗脱结合的融合蛋白。洗脱液用0.4体积的1M K2HPO4中和，合并含有纯化蛋白的组分，并用PBS透析。然后用0.22微米的硝酸纤维素过滤器过滤，并存储在4℃。在非还原和还原条件下，由SDS-PAGE对蛋白产物进行鉴定、分析。纯化的FP4I蛋白SDS-PAGE电泳结果如图2-a所示，在非还原条件下，仅有一条清晰的带(约130KD)，无聚合体。在还原条件下，只呈现一条带(大约65KD)，无降解产物。另外，用SEC-HPLC检测融合蛋白FP4I样品的纯度，结果如图2-b所示，主峰面积百分比达到98.87％，无聚合体。以上结果表明，本发明所构建的融合蛋白具有良好的稳定性，不易降解和聚集。
实施例4、hFGF21融合蛋白单次注射对高脂饮食诱导的肥胖小鼠葡萄糖利用率的影响
选取SPF级别、7周龄雄性C57BL/6J小鼠32只(购自上海斯莱克实验动物有限公司)。饲养环境：温度22-25℃，相对湿度45-65％，照明时间12h/d。适应性饲养1周后，给予高脂饲料饲养(D12492饲料，美国Research diets公司产品)12周。小鼠按体重随机分为4组：对照组、hFGF21融合蛋白FP4I 2.5mg/kg组(低剂量组)、5mg/kg组(中剂量组)和10mg/kg组(高剂量组)。给药组小鼠皮下注射相应药物溶液，对照组小鼠皮下注射PBS缓冲液。注射后各组小鼠禁食16h，开展糖耐量实验。检测小鼠空腹血糖值，腹腔注射2g/kg葡萄糖溶液， 检测注射葡萄糖15min、30min、60min、90min和120min后的血糖值，梯形法计算曲线下基线上增加面积(iAUC)。给药后48h，各组小鼠继续开展糖耐量实验，方法同上。数据以均数±标准误(means±SEM)形式表示，采用SPSS18.0统计软件分析数据。正态分布，多组间均数差异采用单因素方差分析，方差齐性采用LSD检验，方差不齐采用Dunnet T3检验；非正态性分布采用非参数检验，P<0.05表示具有显著性统计学差异。
从图3a和图3b可知，以对照组作为参照，给药后16h，hFGF21融合蛋白FP4I中剂量和高剂量能显著改善肥胖小鼠葡萄糖利用水平(P<0.05)。从图3c和图3d可知，给药后48h，高剂量仍可显著改善肥胖小鼠葡萄糖利用水平(P<0.05)。实验结果说明本发明提供的hFGF21融合蛋白对于提高葡萄糖利用率具有长效性。
同时，按上述方法测定其他几种hFGF21融合蛋白的体内药效。高脂饮食诱导的肥胖小鼠按体重随机分为10组(每组6只)：FP4A、FP4B、FP4C、FP4D、FP4E、FP4F、FP4G、FP4H和FP4J和对照组，给药组小鼠皮下注射10mg/kg相应溶液，对照组小鼠皮下注射PBS缓冲液。分别于注射后16h和48h开展糖耐量实验。结果显示，给药后16h，与对照组相比，hFGF21融合蛋白FP4A、FP4B、FP4C、FP4D、FP4E、FP4F、FP4G、FP4H和FP4J均能显著改善肥胖小鼠葡萄糖利用水平(P<0.05)(图4a)；给药后48h，hFGF21融合蛋白FP4A、FP4B、FP4C、FP4D、FP4E、FP4F、FP4G和FP4H仍能显著改善肥胖小鼠葡萄糖利用水平(P<0.05)，而FP4J组与对照组相比，血糖已无显著性差异(图4b)。上述结果同时还表明，本发明所提供的hFGF21融合蛋白具有较强的抗蛋白酶水解能力，在体内不易被降解，从而维持了较长的生理学活性，并且相对于不含CTP的FP4J，含CTP的hFGF21融合蛋白FP4I表现出更长的体内功能半衰期，这说明添加CTP能更进一步延长hFGF21的体内功能半衰期。
实施例5、hFGF21融合蛋白大鼠体内单次给药药代动力学实验
6只雄性SPF级SD大鼠(购自上海斯莱克实验动物有限公司)，体重180±10g。饲养环境：温度22-25℃，相对湿度45-65％，照明时间12h/d。适应性饲养一周后，尾静脉注射给予3mg/kg的FP4I和FP4J，分别于给药前，给药后 1h、4h、7h、24h、48h、72h、96h、120h、149h、168h、192h、216h和240h眼眶取血0.3ml，全血静置后以2000×g离心15min分离得血清。用双抗夹心ELISA测定时以鼠抗hFGF21抗体(R&D公司，货号MAB25373-100)包被，以HRP标记羊抗人IgG-Fc单抗(Jackson公司，货号109-035-098)进行检测。后将数据输入分析软件PKSOLVER，得出待测药物在血液中药代动力学参数T1/2和AUC(0-t)。
结果显示，3mg/kg的FP4I在SD大鼠体内的循环半衰期T1/2为29.81±1.56h，AUC(0-t)为1716711±201507ng/ml·h。而天然FGF-21大鼠体内循环半衰期为1～1.5h，可知FP4I体内半衰期大幅度延长。3mg/kg的FP4J在SD大鼠体内的循环半衰期T1/2为22.43±1.45h，AUC(0-t)为1210604±191426ng/ml·h。可知FP4I比FP4J具有更长的体内循环半衰期和更高的生物利用度。
实施例6、hFGF21融合蛋白预防高脂饲料喂养条件下小鼠肥胖、非酒精性脂肪变性、胰岛素耐受和高胆固醇血症形成的实验研究
一、模型建立与分组给药
7周龄C57BL/6J雄性小鼠24只(购自上海斯莱克实验动物有限公司)。饲养环境：温度22-25℃，相对湿度45-65％，照明时间12h/d。适应性饲养1周后，按体重随机分为3组：低脂饲料组(low fat diet，LFD)，高脂饲料组(high fat diet，HFD)，高脂饲料联合hFGF21融合蛋白FP4I 3.6mg/kg组(HFD+FP4I 3.6mg/kg)。低脂饲料为D12450B饲料，高脂饲料为D12492饲料，均为美国Research diets公司产品。高脂饲料联合FP4I组按3.6mg/kg剂量每4天皮下注射FP4I一次，低脂饲料组和高脂饲料组皮下注射PBS缓冲液，连续给药116天。末次给药周期后，各组小鼠禁食16小时，称重并检测空腹血糖值，摘眼球取全血，2000×g离心15min，分离得血清。分离肝脏组织，生理盐水洗净后滤纸吸干，称重。取相同部位2份肝脏组织，1份固定于10％福尔马林溶液用于HE染色，其余一份液氮急冻后保存于-80℃，用于肝脏指标含量分析。取一侧附睾周脂肪组织，固定于10％福尔马林溶液用于HE染色。
二、指标检测
2.1、体质量观察
小鼠每4天称体质量一次，实验开始后第104天，时域核磁共振法分析小鼠身体中肌肉、脂肪和体液含量。计算实验前后小鼠体质量变化，体质量增幅＝实验结束时小鼠体质量-实验开始时小鼠体质量。
2.2、血清生化检测
欧霸XL-200全自动生化分析仪检测血清中ALT、AST、HDL-c、LDL-c、TG和TC浓度，具体操作按照仪器说明书进行。
2.3、胰岛素耐量实验、空腹胰岛素水平及胰岛素耐受指数
实验开展118天后，各组小鼠禁食6h(10:00am-4:00pm)，检测各组小鼠血糖值，称重。按瘦质量0.75IU/kg腹腔注射给予胰岛素溶液，检测注射后15min、30min、60min、90min和120min各组小鼠血糖值，绘制胰岛素耐量曲线，梯形法计算AUC面积。ELISA法检测小鼠空腹血清胰岛素含量，并计算胰岛素耐受指数。胰岛素耐受指数＝空腹血糖值(mmol/L)×空腹胰岛素含量(mIU/L)/22.5。
2.4、肝脏三酰甘油含量
每只小鼠精确称取50mg肝脏组织，Folch法检测肝脏组织中TG含量。结果以每mg肝脏组织TG含量表示。
2.5、病理组织检测
取同一部位保存于10％福尔马林溶液中的小鼠肝脏及脂肪组织，HE染色进行病理形态学观察。
三、统计与分析
数据以均数±标准误(means±SEM)形式表示，采用SPSS18.0统计软件分析数据。正态分布，多组间均数差异采用单因素方差分析，方差齐性采用LSD检验，方差不齐采用Dunnet T3检验；非正态性分布采用非参数检验，P<0.05表示具有显著性统计学差异。
四、结果
4.1、hFGF21融合蛋白对高脂饲料喂养小鼠体质量及脂肪含量的影响
与低脂饲料组相比，高脂饲料组小鼠体质量，体质量增幅及体内脂肪含量均显著上升，表现出明显的肥胖症状。hFGF21融合蛋白能显著降低高脂饲料喂养条件下体质量增幅及体内脂肪含量。结果见图5～6。
4.2、hFGF21融合蛋白对高脂饲料喂养小鼠肝功能、肝脏脂肪变性的影响
如图7所示，与低脂饲料组相比，高脂饲料组小鼠血清ALT水平显著上升，表现出明显的肝功能损伤和脂质代谢紊乱。与高脂饲料组相比，hFGF21融合蛋白显著降低小鼠血清ALT水平。如图8和图9所示，高脂饲料喂养小鼠肝质量和肝脏三酰甘油含量显著升高，而hFGF21融合蛋白可显著降低高脂饲料喂养小鼠肝质量和肝脏三酰甘油含量。
4.3、hFGF21融合蛋白对高脂饲料喂养小鼠胰岛素耐受的影响。
胰岛素耐量实验显示，高脂饲料喂养小鼠出现明显的胰岛素耐受症状。血清胰岛素检测结果进一步表明小鼠存在高胰岛素血症及胰岛素抵抗。hFGF21融合蛋白可显著改善高脂饲料喂养条件下小鼠胰岛素抵抗症状，缓解空腹血糖升高。结果见图10-13。
4.4、hFGF21融合蛋白对高脂饲料喂养小鼠高胆固醇血症的影响
如图14所示，与低脂饲料组相比，高脂饲料组小鼠血清TC和LDL-c含量均显著上升，表现出脂质代谢紊乱。与高脂饲料组相比，hFGF21融合蛋白显著降低小鼠血清TC和LDL-c水平。
4.5、病理形态学检测
如图15和16所示，与低脂饲料组组相比，高脂饲料组组小鼠肝脏组织发生明显的脂肪变性，脂滴空泡清晰可见且融合成片状结构，肝细胞形态严重破坏，脂肪细胞横截面积显著增加。与高脂饲料组相比，hFGF21融合蛋白显著缓解肝脏组织脂肪变性，减少脂肪细胞横截面积。
综合以上研究结果，证实hFGF21融合蛋白可以有效地控制高脂饲料诱导的肥胖小鼠的体质量、改善胰岛素抵抗、缓解肝脏脂肪变性和高胆固醇血症。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (25)

  1. 人成纤维细胞生长因子21融合蛋白，所述融合蛋白从N端至C端依次包含野生型hFGF21或其类似物、柔性肽接头、至少1个人绒毛膜促性腺激素β亚基羧基末端肽刚性单元和融合配体；其中，融合配体选自免疫球蛋白及其Fc片段、人血清白蛋白和转铁蛋白，优选地，所述融合配体为免疫球蛋白Fc片段。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白是糖基化的；优选地，所述融合蛋白是通过在哺乳动物细胞中表达而实现糖基化的；更优选地，所述融合蛋白是通过在中国仓鼠卵巢细胞中表达而实现糖基化的。
  3. 如权利要求1所述的融合蛋白，其特征在于，所述野生型hFGF21包含去除了1-28位氨基酸前导肽的SEQ ID NO：1所示序列；或包含去除了1-28位氨基酸前导肽且具有G141S或L174P取代的SEQ ID NO：1所示同等型序列。
  4. 如权利要求1所述的融合蛋白，其特征在于，所述hFGF21类似物相对于野生型hFGF21的氨基酸序列具有一个或多个氨基酸缺失、插入、添加或取代，以及缺失N-末端或C-末端的一个或多个氨基酸；优选地，所述hFGF21类似物与野生型hFGF21的氨基酸序列至少70％同源；较优选地，所述hFGF21类似物与野生型hFGF21氨基酸序列至少80％同源；更优选地，所述hFGF21类似物与野生型hFGF21的氨基酸序列至少90％同源；最优选地，所述hFGF21类似物与野生型hFGF21的氨基酸序列至少95％同源。
  5. 如权利要求4所述的融合蛋白，其特征在于，所述hFGF21类似物缺失N-末端的1、2、3、4、5、6、7或8个氨基酸残基。
  6. 如权利要求5所述的融合蛋白，其特征在于，所述hFGF21类似物缺失N-末端4个氨基酸HPIP。
  7. 如权利要求4所述的融合蛋白，其特征在于，所述hFGF21类似物缺失C-末端1、2、3、4、5、6、7、8、9、10、11或12个氨基酸残基。
  8. 如权利要求4所述的融合蛋白，其特征在于，所述hFGF21类似物包含选自Q55C、A109T、L126R、G148C、K150R、P158S、S195A、P199G和G202A的一个或多个氨基酸的取代。
  9. 如权利要求1所述的融合蛋白，其特征在于，所述柔性肽接头含有2个或更多个选自G、S、A和T的氨基酸。
  10. 如权利要求9所述的融合蛋白，其特征在于，所述柔性肽接头氨基酸组成的 结构通式为(GS)a(GGS)b(GGGS)c(GGGGS)d，其中a，b，c和d是大于或等于0的整数，且a+b+c+d≥1。
  11. 如权利要求10所述的融合蛋白，其特征在于，所述柔性肽接头的氨基酸选自如下序列：
    (i)GGGGS；
    (ii)GSGGGSGGGGSGGGGS；
    (iii)GSGGGGSGGGGSGGGGSGGGGSGGGGS；
    (iv)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
    (v)GGGSGGGSGGGSGGGSGGGS；
    (vi)GGSGGSGGSGGS。
  12. 如权利要求1所述的融合蛋白，其特征在于，所述人绒毛膜促性腺激素β亚基的羧基末端肽刚性单元包含SEQ ID NO：2或其截短的序列；其中，所述截短的序列包含至少2个糖基化位点。
  13. 如权利要求12所述的融合蛋白，其特征在于，所述人绒毛膜促性腺激素β亚基的羧基末端肽刚性单元包含以下氨基酸序列：
    (i)SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
    (ii)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
    (iii)SSSSKAPPPS；
    (iv)SRLPGPSDTPILPQ；
    (v)SSSSKAPPPSLPSPSR。
  14. 如权利要求1所述的融合蛋白，其特征在于，所述人绒毛膜促性腺激素β亚基的羧基末端肽刚性单元与权利要求13所述融合蛋白中的CTP氨基酸序列至少具有70％，80％，90％或95％的同一性。
  15. 如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白包含1、2、3、4或5个人绒毛膜促性腺激素β亚基的羧基末端肽刚性单元。
  16. 如权利要求1所述融合蛋白，其特征在于，所述人免疫球蛋白Fc片段为具有降低的ADCC效应和/或CDC效应和/或与FcRn受体的结合亲和力增强的变体。
  17. 如权利要求16所述融合蛋白，其特征在于，所述Fc片段选自人IgG Fc变 体；更优选地，所述人IgG Fc变体选自：
    (i)含有Leu234Val、Leu235Ala和Pro331Ser突变的人IgG1绞链区、CH2和CH3区域；
    (ii)含有Pro331Ser突变的人IgG2绞链区、CH2和CH3区域；
    (iii)含有Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域；
    (iv)含有Pro331Ser、Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域；
    (v)含有Ser228Pro和Leu235Ala突变的人IgG4绞链区、CH2和CH3区域。
  18. 如权利要1所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO：8。
  19. 编码如权利要求1-18中任一项所述融合蛋白的DNA分子。
  20. 如权利要求19所述的DNA分子，其特征在于，所述DNA分子包含如SEQ ID NO：9所示的序列。
  21. 一种载体，其特征在于，包含如权利要求19或20所述的DNA分子。
  22. 一种宿主细胞，其特征在于，包含如权利要求21所述的载体，或者转染了权利要求19或20所述DNA分子的载体。
  23. 一种药物组合物，其特征在于，包括药学上可接受的载体、赋形剂或稀释剂，以及有效剂量的如权利要求1-18中任一项所述的融合蛋白。
  24. 一种制备如权利要求1-18中任一项所述融合蛋白的方法，所述方法包括：
    (a)将权利要求19或20所述编码融合蛋白的DNA序列引入哺乳动物细胞；
    (b)筛选步骤(a)中在其生长培养基中每24小时期间内，表达超过50μg/106(百万)个细胞的高产细胞株；
    (c)培养步骤(b)筛选到的细胞株，表达融合蛋白；
    (d)收获步骤(c)中得到的发酵液，纯化融合蛋白；优选地，所述步骤(a)中的哺乳动物细胞为CHO细胞；更优选地，所述哺乳动物细胞为CHO衍生细胞系DXB-11。
  25. 如权利要求1-18中任一项所述的融合蛋白在制备用于治疗肥胖、1型或2型糖尿病、胰腺炎、血脂异常、非醇型脂肪肝病(NAFLD)、非醇型脂肪性肝炎 (NASH)、胰岛素耐受、高胰岛素血症、葡萄糖不耐受、高血糖、代谢综合征、急性心肌梗塞、高血压、心血管病、动脉粥样硬化、外周动脉病、中风、心脏衰竭、冠心病、肾病、糖尿病并发症、神经病、与胰岛素受体的严重失活或突变相关的病症药物中的用途。
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