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CN113603794B - Synergistic bifunctional proteins for regulating blood glucose and lipids - Google Patents

Synergistic bifunctional proteins for regulating blood glucose and lipids Download PDF

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CN113603794B
CN113603794B CN202111020303.6A CN202111020303A CN113603794B CN 113603794 B CN113603794 B CN 113603794B CN 202111020303 A CN202111020303 A CN 202111020303A CN 113603794 B CN113603794 B CN 113603794B
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董炤
周驰
冯雄
张吉余
贾世香
李强
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Anyuan Pharmaceutical Technology Shanghai Co ltd
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Abstract

The present invention relates to a synergistic bifunctional protein regulating blood glucose and lipid, comprising a human GLP-1 analog and human FGF21. The invention provides a preparation method of the synergistic type bifunctional protein, and also provides application of the synergistic type bifunctional protein in preparing medicines for treating type 2 diabetes, obesity, dyslipidemia, fatty liver disease and/or metabolic syndrome. The synergistic dual-functional protein provided by the invention can synergistically regulate blood sugar and lipid levels in vivo, and can meet multiple requirements of type 2 diabetics on reducing blood sugar, relieving liver steatosis, reducing weight and improving circulatory lipid metabolism disorder.

Description

Synergistic bifunctional proteins for regulating blood glucose and lipids
Technical Field
The invention relates to GLP-1-FGF21 synergistic bifunctional protein and a pharmaceutical composition thereof, and also relates to application of the synergistic bifunctional protein in preparation of drugs for treating type 2 diabetes, obesity, hyperlipidemia, fatty liver disease and/or metabolic syndrome.
Background
Glucagon-like peptide-1 (GLP-1) is a 36 amino acid incretin secreted by mammalian intestinal L cells, stimulating insulin secretion from islet beta cells in a glucose-dependent manner by binding to and activating the GLP-1 receptor (GLP-1R), inhibiting release of glucagon by islet alpha cells, exerting a biological effect of controlling blood glucose, and simultaneously exhibiting a biological effect of inhibiting gastrointestinal motility and controlling appetite (Knudsen LB, J Med Chem 2004, 4128-4134). Natural human GLP-1 is readily inactivated in vivo by dipeptidyl peptidase IV (DDP-IV) to make its half-life short. Exendin-4 is extracted from saliva of south African Exendin, has 39 amino acids, has 53% homology with the amino acid sequence of human GLP-1 and similar biological activity, but the amino terminal second position of Exendin-4 replaces Ala in human GLP-1 by Gly, so that the Exendin-4 is resistant to enzymatic degradation of DPP-IV to a certain extent, and thus the in vivo circulation half-life is prolonged. Exendin-4 has a special Trap-cage structure at the carboxyl end, so that the binding affinity of the Exendin-4 and a GLP-1 receptor is obviously higher than that of human GLP-1 (Neidigh JW et al, biochemistry,2001, 40:13188-13200), and under the equimolar concentration, exendin-4 shows stronger effect of promoting islet beta cells to release insulin. Two slightly different blood glucose metabolism regulators are marketed, and most typically, liraglutide from Norand Extra peptide from Alaskan are administered 2-3 times a day to effectively control blood glucose levels in type 2 diabetics. However, the high injection frequency results in a significant increase in the cost of patient treatment and poor clinical compliance. To extend the in vivo half-life and bioavailability of GLP-1 and Exendin-4 analogues, fc fragments or HSA fusion techniques have been applied to the development of such long acting drugs. There are currently marketed products, dulaglutide (Dulaglutide) from Gift corporation and Abiglutide (Albiglutide) from the company Gelanin Smith. The most prominent clinical manifestation is dolalutin, a GLP-1higg4 Fc fusion protein (dulaglutine), with an average half-life of up to 90 hours (chinese patent CN 1802386B), with clinical indication type 2 diabetes mellitus, recommended administration method of 1 subcutaneous injection per week. Clinical studies have shown that dolaluin is effective in controlling postprandial blood glucose and glycosylated hemoglobin in diabetics and reducing weight in obese patients by suppressing appetite, but has various degrees of gastrointestinal adverse reactions. Epidemiological studies have shown that most type 2 diabetics are associated with non-alcoholic fatty liver disease and symptoms of lipid metabolism disorders (Radaelli MG et al J Endocrinol Invest,2017, s 40618). The current clinical research results do not support GLP-1 or Exendin-4 analogues to have the effect of treating fatty liver and hyperlipidemia (Petit JM, diabetes Metab,2017,43,2S28-2S 33) independent of the weight loss effect, so that the products cannot completely meet all the treatment requirements of type 2 diabetics.
The fibroblast growth factor (Fibroblast growth factors, FGFs) family has 22 members, 7 subfamilies, of which the FGF19 subfamilies exert physiological activity in an endocrine fashion, and are involved in regulating energy and bile acid homeostasis, glucose and lipid metabolism, phosphate and vitamin D homeostasis (Moore DD et al, science,2007,316:1436-1438 and Beenken et al, nature Reviews Drug Discover,2009, 8:235). FGF21 is one of the members of the FGF19 subfamily, having 182 amino acids, and in vivo, the FGF21 carboxy-terminus first binds to the cofactor β -Klotho transmembrane protein and the amino-terminus in turn binds to FGFR to form a stable FGF21/β -Klotho/FGFR complex, activating downstream related signaling molecules (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 is physiologically active and shows insulin-independent glucose utilization promoting functions (Kharitonenkov A et al, J Clin Invest,2005,115 (6): 1627-1635), insulinotropic sensitization (Duthchak PA et al, cell,2012,148,387-393), inhibition of liver lipid neogenesis, promotion of liver fatty acid beta oxidation, lowering serum triglyceride levels (Xu J et al, diabetes,2009,58,250-259); by inhibiting liver SREBP-2 synthesis, serum total cholesterol and low density lipoprotein levels are reduced, thereby alleviating hypercholesterolemia (Lin Z et al, circulation,2015,131,1861-1871).
Taken together, FGF21 exhibits beneficial metabolic regulation effects on metabolic diseases such as obesity, type 2 diabetes, nonalcoholic fatty liver disease, and hyperlipidemia. Meanwhile, FGF21 is the only cytokine currently found in the FGF family that has no mitogenic effect, greatly reducing its potential carcinogenicity in clinical use (Wu X et al, proc Natl Acad Sci USA,2010, 170:14158-14163). However, natural FGF21 is difficult to develop into a therapeutic biological agent due to its own physical and chemical property deficiency, and the main reasons include: 1. FGF21 protein has poor stability and is easy to hydrolyze by protease; 2. the FGF21 is unstable in conformation and easy to aggregate, so that the difficulty in large-scale production of FGF21 is increased; 3. natural FGF21 has a short half-life, human FGF21 has a half-life in mice of 0.5-1 hour and in cynomolgus monkeys of 2-3 hours (khatritnenkov a et al, J Clin Invest,2005, 115:1627-1635). Various protein longevity techniques have been applied to extend the in vivo half-life of recombinant FGF 21. For example, FGF21 links to PEG molecules, increases molecular weight, decreases glomerular filtration rate, prolongs in vivo residence time (see patent WO2005/091944, WO2006/050247, WO2008/121563, and WO 2012/066075); FGF21 is fused with long chain fatty acids (capable of binding serum albumin) (see WO2010/084169 and WO 2012/010553); or preparing an agonist antibody capable of specifically binding to FGFR or FGFR/beta-klotho complex to mimic the mechanism of action of FGF21 to activate FGF/FGFR signaling pathway (see WO2011/071783, WO2011/130417, WO2012/158704 and WO 2012/170438); or by fusion to the Fc fragment, FGF21 half-life can also be improved (see WO2004/110472, WO2005/113606, WO2009/149171, WO2010/042747, WO2010/129503, WO2010/129600, WO2013/049247, WO2013/188181 and WO 2016/114633). Currently, there is no long-acting FGF21 protein on the market, but three long-acting FGF21 proteins, LY2405319 from Gift corporation, PF-05231023 from Jupiter corporation and BMS986036 from Bai Zhu Shi Gui Bao, are in clinical trial. Of these, LY2405319 and PF-05231023 only show weight loss, lowering serum TG levels, and do not show positive therapeutic effects on glycemic control in type 2 diabetics (Gaich G et al, cell Metab,2013,18:333-340 and Dong JQ et al, br J Clin Pharmacol,2015,80-1051-1063). BMS986036 shows good therapeutic effect in a clinical study on non-alcoholic fatty liver disease, but it did not develop a test study on glycemic control in type 2 diabetics. The above results show that the FGF21 long-acting protein alone can not meet the most critical glycemic control requirements in the treatment of type 2 diabetics, although it can exhibit various pharmacodynamic activities of losing weight, treating non-alcoholic fatty liver disease and hyperlipidemia.
Recently, it has been reported that the combination of GLP-1 and FGF21 has a synergistic effect in blood glucose control. For example, CN102802657a discloses that the use of GLP-1 and FGF21 compositions can synergistically reduce db/db mouse blood glucose levels. But the drug combination not only increases the administration frequency of patients and reduces the compliance of patients to treatment, but also greatly increases the treatment cost. In addition, a bifunctional protein prepared by fusing GLP-1 and FGF21 has also been reported, and in order to solve the problem of easy degradation of FGF21 in vivo, researchers often introduce corresponding mutations into natural FGF21 molecules, but this also inevitably increases the potential immunogenicity of the bifunctional protein (WO 2017/074123 and CN 104024273B). In addition, the observed synergistic FGF21 effects of GLP-1 are multi-apparent in controlling blood glucose, whereas their therapeutic effects in other metabolic diseases such as obesity, non-alcoholic fatty liver and lipid metabolism disorders are lacking in comparison to the commercial long-acting GLP-1 analog products. The lack of investigation above may involve the following reasons: (1) Natural GLP-1 or FGF21 are unstable in vivo, either molecule cannot remain structurally intact and stable in vivo, and it is not possible to produce a synergistic effect functionally; (2) In the process of fusing GLP-1 and FGF21 into single proteins, the respective three-dimensional conformation needs to be maintained to the greatest extent without mutual interference so as to realize functional synergy, and the molecular design level needs to be carefully treated; (3) The function of GLP-1 and FGF21 both depend on binding to their respective receptors, and in which case they are in dynamic equilibrium, a large number of in vitro and in vivo experiments are required to verify that none of the currently published patent or other non-patent documents are relevant.
In summary, if a GLP-1-FGF21 synergistic dual-function protein medicine with enhanced stability can be developed in the field, the effects of prolonged half-life and low immunogenicity can be realized, and multiple requirements of reducing blood sugar, relieving liver steatosis, reducing weight and improving circulatory lipid metabolism disorder, which are needed to be solved by many type 2 diabetics, can be met.
Disclosure of Invention
The invention aims to provide a bifunctional protein containing a human GLP-1 analogue and human FGF21 and having synergistic effect in terms of blood sugar and lipid regulation, a preparation method and application thereof, and aims to overcome the defects of unstable structure, short in-vivo half-life and the like of natural GLP-1 and FGF21, keep the physiological effects of GLP-1 on powerful blood sugar reduction and FGF21 in the treatment of insulin sensitization, weight reduction, fatty liver and hypercholesteremia, and relieve gastrointestinal adverse reactions caused by GLP-1 to a certain extent.
In one aspect of the present invention, there is provided a synergistic bifunctional protein capable of synergistically regulating blood sugar and lipid, comprising, in order from the N-terminus to the C-terminus, a human glucagon-like peptide-1 analog (hereinafter abbreviated as GLP-1 analog), a connecting peptide 1 (hereinafter abbreviated as L1), a human fibroblast growth factor 21 (hereinafter abbreviated as FGF 21), a connecting peptide 2 (hereinafter abbreviated as L2) and a human immunoglobulin Fc fragment (hereinafter abbreviated as Fc fragment); wherein the connecting peptide 1 consists of only flexible peptide; the connecting peptide 2 consists of a flexible peptide and a rigid peptide, the rigid peptide in turn consisting of at least 1 rigid unit, and the rigid unit comprising the carboxy-terminal peptide of the β subunit of human chorionic gonadotrophin or a truncated sequence thereof.
Wherein the GLP-1 analogue refers to analogues, fusion peptides and derivatives thereof which are obtained by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of human GLP-1 (shown as SEQ ID NO: 1) and can keep the GLP-1 activity. For example, the GLP-1 analogs include, but are not limited to, the sequences set forth in SEQ ID NO:2,3,4 or 5. In a preferred embodiment of the invention, the GLP-1 analog is as set forth in SEQ ID NO:2, in another embodiment of the present invention, the GLP-1 analogue is as set forth in SEQ ID NO: shown at 5.
Wherein the "linker peptide 1 (L1)" is a short peptide acting as a linker between the GLP-1 analog and FGF 21. The linker peptide 1 is preferably non-immunogenic and creates a sufficient distance between the GLP-1 analogue and FGF21 to minimize steric effects with respect to each other so as not to affect or seriously affect the correct folding and steric conformation of the GLP-1 analogue and FGF 21. The skilled artisan can design the linker peptide according to methods conventional in the art. Preferably, a flexible peptide comprising 2 or more amino acids is used and is selected from the following amino acids: gly (G), ser (S), ala (A) and Thr (T); more preferably, the connecting peptide 1 comprises G and S residues. The length of the linker peptide is very important for the activity of the bifunctional protein, preferably consisting of 5-30 amino acids. In a preferred embodiment of the present invention, the amino acid sequence of the connecting peptide 1 is GGGGGGGSGGGGSGGGGS.
Wherein said "FGF21" is a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; or a sequence comprising the amino acid leader peptide at positions 1-28 removed and having a substitution of G141S or L174P: 6. In a preferred embodiment of the invention, FGF21 comprises the amino acid sequence of SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.
Wherein the "connecting peptide 2 (L2)" is a short peptide acting as a linker between FGF21 and Fc fragment. The connecting peptide consists of a flexible peptide and a rigid peptide, wherein the flexible peptide contains 2 or more amino acid residues selected from Gly (G), ser (S), ala (A) and Thr (T). Preferably, the flexible peptide comprises G and S residues. For the present invention, preferably, the flexible peptide amino acid composition has a structural formula of (GS) a (GGS) b (GGGS) c (GGGGS) d, wherein a, b, c and d are integers greater than or equal to 0, and a+b+c+d is not less than 1.
In some embodiments of the invention, the L2 comprises a flexible peptide selected from the group consisting of:
(i)GGGGS；
(ii)GSGGGSGGGGSGGGGS；
(iii)GSGGGGSGGGGSGGGGSGGGGSGGGGS；
(iv)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
(v)GGGSGGGSGGGSGGGSGGGS；
(vi)GGSGGSGGSGGS。
wherein the rigid peptide comprising connecting peptide 2 (L2) consists of one or more rigid units selected from the group consisting of full-length or truncated sequences consisting of amino acids 113 to 145 of the carboxy terminus of the β subunit of human chorionic gonadotrophin (hereinafter CTP rigid units); specifically, the CTP rigid unit comprises SEQ ID NO:7 or a truncated sequence thereof.
Preferably, said CTP rigid unit comprises at least 2 glycosylation sites; for example, in a preferred embodiment of the present invention, said CTP rigid unit comprises 2 glycosylation sites, illustratively said CTP rigid unit comprises SEQ ID NO: 10 amino acids at 7N-terminal, SSSS KAPPPS; or the CTP rigid unit comprises SEQ ID NO: 14 amino acids at 7C-terminal, s×rlpgps×dtpilpq; as another example, in another embodiment, the CTP rigid unit comprises 3 glycosylation sites, illustratively, the CTP rigid unit comprises SEQ ID NO: 16 amino acids at the 7N-terminal, SSSS KAPPPS LPSPS R; as another example, the CTP rigid unit comprises 4 glycosylation sites, illustratively, the CTP rigid unit comprises 28, 29, 30, 31, 32 or 33 amino acids and starts at position 113, 114, 115, 116, 117 or 118 of the human chorionic gonadotrophin β subunit and ends at position 145. Specifically, the CTP rigid unit comprises SEQ ID NO: the 28 amino acids at the 7N-terminal, SSSS KAPPPS LPSPS RLPGPS DTPILPQ. Herein, represents a glycosylation site. Each possibility represents a separate embodiment of the invention.
Illustratively, the CTP rigid unit encompassed by L2 of the present invention may preferably comprise the following sequence:
(i)CTP 1 ：SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(ii)CTP 2 ：PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(iii)CTP 3 ：SSSSKAPPPS；
(iv)CTP 4 ：SRLPGPSDTPILPQ；
(v)CTP 5 ：SSSSKAPPPSLPSPSR。
In other embodiments, 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.
In some embodiments of the present invention, L2 comprises 2,3,4 or 5 CTP rigid units as described above. In one embodiment of the present invention, L2 of said synergistic bifunctional protein comprises 2 CTPs 3 Rigid unit: SSSSKAPPPSSSSSKAPPPS (CTP) 3 -CTP 3 Or expressed as (CTP) 3 ) 2 )。
Wherein the "Fc fragment" is selected from the group consisting of an Fc fragment of human immunoglobulin IgG, igM, igA and variants thereof; more preferably from human IgG1, igG2, igG3 or IgG4 and variants thereof, 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 FcRn receptor; most preferably, the human IgG Fc variant is selected from the group consisting of:
(i) vFcγ1: human IgG1 hinge region, CH2, and CH3 regions (amino acid sequence shown as SEQ ID NO: 8) containing Leu234Val, leu235Ala, and Pro331Ser mutations;
(ii) vFc gamma 2-1: human IgG2 hinge region, CH2, and CH3 region (amino acid sequence as shown in SEQ ID NO: 9) containing Pro331Ser mutation;
(iii) vFc gamma 2-2: human IgG2 hinge region, CH2, and CH3 region (amino acid sequence shown as SEQ ID NO: 10) containing mutations of Thr250Gln and Met428 Leu;
(iv) vFc gamma 2-3: human IgG2 hinge region, CH2, and CH3 region (as shown in SEQ ID NO: 11) containing Pro331Ser, thr250Gln, and Met428Leu mutations.
(v) vfcγ4: human IgG4 hinge region, CH2, and CH3 region (as shown in SEQ ID NO: 12) containing Ser228Pro and Leu235Ala mutations.
The Fc variants provided by the present invention include, but are not limited to, the 5 variants described in (i) to (v), but may also be combinations or stacks of mutation sites of two types of functional variants between IgG isotypes, as described in (iv) above, i.e., the novel combination variants of IgG2 Fc obtained by stacking mutation sites in (ii) and (iii).
Fc variants (vfcs) in the synergistic bifunctional proteins of the present invention comprise hinge, CH2 and CH3 regions of human IgG such as human IgG1, igG2 and IgG 4. Such CH2 regions contain amino acid mutations at positions 228, 234, 235 and 331 (as determined by the EU counting system). These amino acid mutations are believed to reduce the effector function of Fc. Human IgG2 does not bind fcγr but shows very weak complement activity. The fcγ2 variant with the Pro331Ser mutation should have lower complement activity than native fcγ2 and still be an fcγr non-binder. IgG4 Fc is defective in activating the complement cascade and its binding affinity to fcγr is about an order of magnitude lower than IgG 1. Compared to native fcγ4, fcγ4 variants with Ser228Pro and Leu235Ala mutations should exhibit minimal effector function. Fcγ1 with Leu234Val, leu235Ala and Pro331Ser mutations also showed reduced effector function compared to native fcγ1. These Fc variants are all more suitable than native human IgG Fc for the preparation of FGF21 and its analog bifunctional proteins. And positions 250 and 428 (positions determined by the EU numbering system) contain amino acid mutations that increase the binding affinity of the Fc region to the neonatal receptor FcRn, thereby further extending half-life (Paul R et al, J Biol Chem,2004, 279:6213-6216); the variants of the two functions are combined or overlapped to obtain a novel combined variant, so that the effector function is reduced and the half life of the combined variant is prolonged. The Fc variants of the present invention contain mutations which are not limited to the several sites described above, but substitutions at other sites may be introduced to provide Fc with reduced effector function and/or enhanced binding to the FcRn receptor, without also causing reduced function/activity or adverse conformational changes in the Fc variants, as is common for mutation sites, see Shields RL et al, J Biol Chem,2001,276 (9): 6591-604.
In a preferred embodiment of the present invention, the amino acid sequence of the synergistic bifunctional protein is as shown in SEQ ID NO: shown at 13. In another preferred embodiment of the present invention, the amino acid sequence of the synergistic bifunctional protein is as shown in SEQ ID NO: 15.
The synergistic bifunctional protein is glycosylated; preferably, the bifunctional protein is glycosylated by expression in mammalian cells; more preferably, the bifunctional protein is glycosylated by expression in chinese hamster ovary cells.
According to another aspect of the present invention, there is provided a DNA encoding the above-mentioned synergistic bifunctional protein. In a preferred embodiment of the present invention, the DNA sequence of the synergistic bifunctional protein is as set forth in SEQ ID NO: 14.
According to yet another aspect of the present invention, a carrier is provided. The vector comprises the DNA described above.
According to yet another aspect of the invention, a host cell is provided. The host cell comprises the vector or is transfected with the vector.
In a specific embodiment of the invention, the host cell is the derived cell line DXB-11 of CHO.
According to yet another aspect of the present invention, a pharmaceutical composition is provided. The pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or diluent and an effective amount of the synergistic dual-function protein.
According to a further aspect of the present invention there is provided a method of preparing or producing said synergistic bifunctional protein from a mammalian cell line (such as a CHO-derived cell line) comprising the steps of:
(a) Introducing into mammalian cells a DNA encoding the synergistic bifunctional protein described above;
(b) The expression in the selection step (a) is more than 20. Mu.g/10 every 24 hours in its growth medium 6 High-yield cell lines of individual cells;
(c) Culturing the cell strain obtained by screening in step (b);
(d) Harvesting the fermentation broth obtained in the step (c), and purifying the synergistic bifunctional protein.
Preferably, the mammalian cells in step (a) are CHO cells; more preferably the CHO derived cell line DXB-11.
According to a further aspect of the present invention there is provided the use of said synergistic bifunctional protein in the manufacture of a medicament for the treatment of FGF 21-related diseases and GLP-1-related diseases, as well as other metabolic, endocrine and cardiovascular diseases, including obesity, type 1 and type 2 diabetes mellitus, pancreatitis, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular diseases, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, gastroparesis, disorders associated with severe inactivating mutations of the insulin receptor; preferably, the disease includes obesity, type 1 and type 2 diabetes, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, metabolic syndrome.
Compared with the existing products, the synergistic bifunctional protein of the present invention has a plurality of outstanding advantages, and the preferred bifunctional protein FP4I-2 of the present invention is specifically illustrated below:
1. prolong half-life in vivo and maintain blood glucose reducing activity in vivo for a longer time. The glucose tolerance results carried out on the C57BL/6 mice show that 144 hours after single administration of FP4I-2 still show good glucose utilization promoting capacity, which is obviously superior to the GLP-1 analogues liraglutide, exenatide and natural FGF21 on the market. Compared with the GLP-1-Fc fusion protein dolapride, the FP4I-2 has more excellent blood sugar control effect in a model mouse with type 2 diabetes.
2. Better biosafety and tolerability. The first administration of dolapride induces serious gastrointestinal adverse effects. In db/db mice, the mice given FP4I-2 for the first time had significantly increased food intake for 24 hours relative to mice given dolantin, indicating that the synergistic bifunctional protein FP4I-2 was effective in alleviating the symptoms of anorexia induced by gastrointestinal adverse reactions.
3. The synergistic type bifunctional protein shows good fatty liver treatment effect. Compared with dolaluin, the FP4I-2 can obviously reduce the liver quality of db/db mice and improve liver functions, and the functional improvement in the aspect does not depend on GLP-1 related ingestion inhibition, so that the physiological activity of FGF21 is fully reflected. The in vivo functional half-life of FP4I-2 is significantly prolonged relative to native FGF21, and the frequency of administration of FP4I-2 in animal models is 2 times per week, which would increase clinical feasibility.
4. The C-terminal sequence of FGF21 is critical to the activity, the N-terminal fusion of FGF21 is mostly adopted by the double proteins reported in the prior art, the free C-terminal is more beneficial to maintaining the activity of the double proteins, but the C-terminal of FGF21 also contains a protease hydrolysis site, the double proteins are extremely easy to degrade, the exposed C-terminal is more susceptible to be attacked by protease to degrade, and in order to solve the problem, the prior art avoids using natural FGF21, and introduces corresponding mutation to improve the stability of the double proteins, but the potential immunogenicity of the double proteins is also necessarily increased. The bifunctional protein constructed by the invention adopts natural FGF21, and is connected with Fc fragment at the C terminal. The bifunctional protein provided by the invention not only has obviously prolonged in vivo circulation half-life, but also has synergistic effects in terms of blood sugar and lipid regulation, which shows that the constructed bifunctional protein better maintains the functions of two active molecules and has extremely strong stability, and the bifunctional protein is beneficial to a novel connecting peptide adopted between FGF21 and Fc variants, the connecting peptide consists of a flexible peptide and a CTP rigid peptide, the CTP rigid unit contains a plurality of O-glycosyl side chains, a relatively stable three-dimensional conformation can be formed, FGF21 and Fc can be effectively isolated, thereby maximally reducing the steric effect brought by Fc, and ensuring that FGF21 maintains better biological activity. In addition, the protecting action of the glycosyl side chain of the CTP rigid unit can cover the enzymolysis site of FGF21, reduce the sensitivity of the FGF21 to protease and achieve the purpose of stabilizing protein.
5. Mutating Fc only retains the long circulation half-life characteristics of Fc, reducing or eliminating ADCC and CDC effects (e.g., P331S), thereby increasing drug safety. In addition, fc variants (e.g., T250Q/M428L) with increased binding affinity to neonatal receptor (FcRn) may further extend the half-life of the synergistic bifunctional protein.
Detailed Description
Human GLP-1 analogues
The term "human GLP-1 analogue" as used herein refers to analogues, fusion peptides and derivatives thereof which are obtained by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of human GLP-1 (shown as SEQ ID NO: 1) and which are capable of maintaining the activity of human GLP-1. For example, the human GLP-1 analogs include, but are not limited to, the sequence set forth in SEQ ID NO:2,3,4 or 5.
Human FGF21
The term "human FGF21" as used herein refers to wild-type human FGF21 polypeptide.
The sequence of the wild-type FGF21 protein is available from UNIPROT database under accession No. 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).
In particular, from US2001012628A1 it is known that wild type FGF21 isoforms (isoforms) or allelic forms (allelicforms) having Pro substitution Leu in the mature protein (as shown in positions 47-227 of SEQ ID NO:13 in the present invention); the other wild-type FGF21 isoform has Ser substituted Gly (Gly at position 141 of SEQ ID NO:6 of the present invention is substituted or replaced with Ser).
Another isoform with a shorter signal peptide (Leu missing at position 23 of SEQ ID NO:6 in the present invention) is known from WO 2003/01213 (see SEQ ID NO:2 in the publication WO 2003/01213, signal peptide with 27 amino acid residues).
In the present invention, wild-type FGF21 comprises SEQ ID NO:6 and L174P or G141S substitution isoforms with the leader peptide removed, the mature protein portion sequence (amino acids 29-209) shown; in addition, the full-length sequence of the precursor protein to which the 27 or 28 amino acid signal peptide is added before the sequences is also included.
hCG-beta Carboxy Terminal Peptide (CTP)
CTP is a short peptide derived from the carboxy terminus of the β -subunit of human chorionic gonadotrophin (hCG). Four reproduction-related polypeptide hormones Follicle Stimulating Hormone (FSH), luteinizing Hormone (LH), thyrotropin (TSH) and chorionic gonadotropin (hCG) contain the same α -subunit and each specific β -subunit. hCG has a significantly longer in vivo half-life than the other three hormones, mainly derived from the unique carboxy-terminal peptide (CTP) on its β -subunit (Fares FA et al Proc Natl Acad Sci USA,1992, 89:4304-4308). The natural CTP contains 37 amino acid residues with 4O-glycosylation sites, terminating with sialic acid residues. The negatively charged, highly sialylated CTPs are able to resist their clearance by the kidneys, thereby extending the half-life of the protein in vivo. However, the present inventors creatively linked a rigid peptide consisting of at least one CTP rigid unit with a flexible peptide of appropriate length, together as a linking peptide 2, for linking FGF21 with an Fc fragment.
The inventor finds that the N-terminal and C-terminal sequences of FGF21 play a very critical role in the functions of the FGF21, and the FGF21 has complex and fragile spatial conformation, so that the FGF21 has poor self stability, is easy to degrade and polymerize, is connected with a fusion ligand, and has steric effect to interfere with the correct folding of the FGF, so that the activity of the FGF21 is obviously reduced or even lost, or a polymer is more easily generated. By adding CTP rigid units between FGF21 and Fc variants, this corresponds to the addition of a stretch of rigid linking peptides. On the one hand, the N-terminal fused FGF21 is ensured not to influence the binding site of the Fc variant and FcRn, thereby influencing half-life; in addition, the Protein A binding site of Fc is important for the purification step in the preparation process, and the rigid unit connected with CTP ensures that FGF21 fused at the N-terminal does not "cover" the binding site of the FGF21 with Protein A. In yet another aspect, the addition of CTP rigid units is such that an Fc fragment of about 25KD size does not interfere with the correct folding of N-terminally fused FGF21, resulting in a decrease or loss of its biological activity/function. This may be explained by CTP rigid polypeptides having multiple glycosyl side chains, which may form stable steric conformations relative to random crimping of such flexible linking peptides as (GGGGS) n, which "blocking" effect promotes independent folding of FGF21 and Fc segments into the correct three-dimensional conformation without affecting the respective biological activities. In yet another aspect, the protecting effect of the CTP glycosyl side chain may reduce the sensitivity of the linker peptide to proteases, making the synergistic bifunctional protein less susceptible to degradation at the linker region. Furthermore, CTP is derived from natural human hCG and is not immunogenic and thus is more suitable as a connecting peptide than a non-naturally encoded amino acid sequence.
IgG Fc variants
Non-lytic Fc variants
The Fc element is derived from the constant region Fc fragment of immunoglobulin IgG, which plays an important role in the immune defenses against pathogens. Fc-mediated effector function of IgG plays a role by two mechanisms: (1) Binding to cell surface Fc receptors (fcγrs), digesting the pathogen by phagocytosis or lysis or killing of cells via Antibody Dependent Cellular Cytotoxicity (ADCC) pathways, or (2) binding to C1q of the first complement component C1, triggering Complement Dependent Cytotoxicity (CDC) pathways, thereby lysing the pathogen. Of the four human IgG subtypes, igG1 and IgG3 bind efficiently to fcγrs, igG4 binds with low affinity to fcγrs, and IgG2 binds with low affinity to fcγrs, which is difficult to measure, so human IgG2 has little ADCC effect. In addition, human IgG1 and IgG3 can bind efficiently 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. Clearly, none of the native IgG subtypes is well suited for constructing GLP-1-FGF21 bifunctional proteins. In order to obtain non-cleaving Fc without effector function, the most effective approach is to mutate complement and receptor binding domains on the Fc fragment, modulate the binding affinity of Fc to related receptors, reduce or eliminate ADCC and CDC effects, and only preserve the long circulation half-life properties of Fc without generating cytotoxicity. Further non-lytic Fc variants can include mutation sites as described in Shields RL et al, J Biol Chem,2001,276 (9): 6591-604 or Chinese invention patent CN 201280031137.2.
Fc variants with enhanced binding affinity to neonatal receptor (FcRn)
The plasma half-life of IgG depends on its binding to FcRn, typically they bind at pH6.0 and dissociate at pH7.4 (plasma pH). By studying both binding sites, the site on IgG that binds FcRn was engineered to increase binding capacity at ph 6.0. Mutations at some residues of the human fcγ domain important for binding 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,2007, 7:715-725). Korean patent No. KR 10-1027427 discloses trastuzumab (herceptin, genentech) variants with increased FcRn binding affinity, and these variants comprise one or more amino acid modifications selected from 257C, 257M, 257L, 257N, 257Y, 279Q, 279Y, 308F and 308Y. Korean patent publication No. KR 2010-0099179 provides bevacizumab (avastin, genentech) variants and these variants comprise amino acid modifications of N434S, M252Y/M428L, M Y/N434S and M428L/N434S, showing an increased in vivo half-life. In addition, hinton et al also found that the T250Q and M428L 2 mutants increased binding to FcRn by 3 and 7 fold, respectively. At the same time 2 sites were mutated, binding was increased 28-fold. In rhesus monkeys, either the M428L or T250QM/428L mutants showed a 2-fold increase in plasma half-life (Paul R.Hinton et al, J Immunol,2006, 176:346-356). More Fc variants with enhanced binding affinity to neonatal receptor (FcRn) contain mutation sites as described in chinese patent CN201280066663.2. In addition, T250Q/M428L mutations in the Fc fragments of five humanized antibodies were studied to not only improve Fc interaction with FcRn, but also in subsequent in vivo pharmacokinetic experiments, it was found that administration by subcutaneous injection improved the pharmacokinetic parameters of the Fc mutant antibodies compared to wild-type antibodies, such as increased in vivo exposure, decreased clearance, and increased subcutaneous bioavailability (Datta-Mannan A et al, MAbs. Taylor & Francis,2012,4 (2): 267-273).
The term "FGF 21-related disorder", "GLP-1-related disorder" includes obesity, type 1 and type 2 diabetes, pancreatitis, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, acute myocardial infarction, hypertension, cardiovascular disease, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, gastroparesis, disorders associated with severe inactivating mutations of insulin receptors.
A "disorder associated with a severe inactivating mutation of the insulin receptor" describes a condition in a subject suffering from a mutation of the insulin receptor (or a possible protein immediately downstream thereof) that results in severe insulin resistance, but generally without the obesity common in type 2 diabetes. In many aspects, subjects suffering from these conditions exhibit a combination of type 1 diabetes and type 2 diabetes. The affected objects are thus substantially classified into several categories by increasing severity, including: type a diabetes resistance, type C insulin resistance (AKA HAIR-AN syndrome), rabson-Mendenhall syndrome, donohue's syndrome or dwarfism (lepichahauism). These conditions are associated with very high endogenous insulin levels, resulting in elevated blood glucose levels. Thus, the affected subjects also present a variety of clinical features associated with "insulin toxicity," including hyperandrogenism, polycystic ovary syndrome (PCOS), hirsutism, and acanthosis nigricans (overgrowth of skin folds and pigmentation).
"diabetic complications" are dysfunctions of other parts of the body caused by chronic hyperglycemia such as diabetic nephropathy, diabetic neuropathy, diabetic foot (foot ulcers and hypoperfusions) 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 dental and gingival problems.
"Metabolic syndrome" (metabolic syndrome, MS) is a pathological condition in which a variety of metabolic components are abnormally aggregated, including: (1) abdominal obesity or overweight; (2) Atherosclerosis dyslipidemias such as hypertriglyceridemia (TG) and high density lipoprotein cholesterol (HDL-C) lowering; (3) hypertension; (4) abnormality in insulin resistance and/or glucose tolerance. Some criteria also include microalbuminuria, hyperuricemia, and elevated pro-inflammatory states (C-reactive protein) and elevated pro-thrombotic states (elevated fibrinogen and elevated plasminogen inhibitor-1).
"dyslipidemia" is a disorder of lipoprotein metabolism, including overproduction or deficiency of lipoproteins. Dyslipidemia may manifest as elevated concentrations of total cholesterol, low Density Lipoprotein (LDL) cholesterol and triglycerides, and reduced concentrations of High Density Lipoprotein (HDL) cholesterol in the blood.
"non-alcoholic fatty liver disease (NAFLD)" is a liver disease that is not related to alcohol consumption and is characterized by liver cell steatosis.
"non-alcoholic steatohepatitis (NASH)" is a liver disease that is not related to alcohol consumption and is characterized by hepatocyte steatosis, accompanied by intra-lobular inflammation and fibrosis.
"atherosclerosis" is a vascular disease characterized by irregularly distributed lipid deposits on the intima of large and medium-sized arteries, resulting in narrowing of the arterial lumen and eventual progression to fibrosis and calcification.
Drawings
FIG. 1, shows the nucleotide sequence and deduced amino acid sequence of the bifunctional protein FP4I-2 of the SpeI/EcoRI fragment in a PCDNA3.1 expression vector according to an embodiment of the invention, consisting of the alpha 1 microglobulin leader peptide (1-19), GLP-1 analog (20-47), L1 (48-65), FGF21 mature protein (66-246), L2 (247-301) and IgG2 Fc (302-524).
FIG. 2a, reducing SDS-PAGE electrophoresis of GLP-1-FGF21 bifunctional protein FP 4I-2.
FIG. 2b, SEC-HPLC profile of GLP-1-FGF21 bifunctional protein FP 4I-2.
FIG. 3a, GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2, sugar tolerance experimental curves (means.+ -. SEM, n=8) after 16h of single injection.
FIG. 3b, GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 were glucose-tolerant after 16h after a single injection The test iaauc (means±sem, n=8); statistical difference signature annotation: in comparison with the control group, * P＜0.05， ** P＜0.01。
FIG. 4a, GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2, sugar tolerance experimental curves (means.+ -. SEM, n=8) after 96h of single injection.
FIG. 4b, GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 sugar tolerance assay iAUC (means+ -SEM, n=8) after 96h of single injection; statistical difference signature annotation: in comparison with the control group, * P＜0.05， ** P＜0.01。
FIG. 5a, GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2, sugar tolerance experimental curves (means.+ -. SEM, n=8) after 144h of single injection. FIG. 5b, GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 sugar tolerance assay iAUC (means+ -SEM, n=8) after 144h of single injection; statistical difference signature annotation: in comparison with the control group, * P＜0.05， ** P＜0.01。
FIG. 6a, sugar tolerance experimental curves (means.+ -. SEM, n=8) of Exendin4-FGF21 bifunctional protein FP4I-3 after 16h single injection.
FIG. 6b, sugar tolerance test iAUC (means+ -SEM, n=8) after 16h of single injection of Exendin4-FGF21 bifunctional protein FP 4I-3; statistical difference signature annotation: in comparison with the control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## P＜0.01。
FIG. 7a, sugar tolerance experimental curves (means.+ -. SEM, n=8) of Exendin4-FGF21 bifunctional protein FP4I-3 after a single injection for 96 h.
FIG. 7b, sugar tolerance test iAUC (means+ -SEM, n=8) after a single injection of Exendin4-FGF21 bifunctional protein FP4I-3 for 96 h; statistical difference signature annotation: in comparison with the control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## P＜0.01。
FIG. 8a, sugar tolerance experimental curves (means.+ -. SEM, n=8) of Exendin4-FGF21 bifunctional protein FP4I-3 after 144h of single injection.
FIG. 8b, sugar tolerance test iAUC (means+ -SEM) after 144h of Exendin4-FGF21 bifunctional protein FP4I-3N=8); statistical difference signature annotation: in comparison with the control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## P＜0.01。
FIG. 9, effect of GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 on food intake 24h after first administration of db/db mice (means.+ -. SEM, n=6); statistical difference signature annotation: in comparison with the control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## P＜0.01。
FIG. 10 effect of multiple doses of GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 on the glycosylated hemoglobin values of db/db mice (means+ -SEM, n=6); statistical difference signature annotation: in comparison with the control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05，##P＜0.01。
FIG. 11, effect of multiple doses of GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 on cumulative food intake of db/db mice (means+ -SEM, n=6); statistical difference signature annotation: in comparison with the control group, * P＜0.05， ** P is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## P＜0.01。
FIG. 12 influence of GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 on body weight of obese mice induced by high fat diet (means+ -SEM, n=7); statistical difference signature annotation: in comparison with the obese control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## p is less than 0.01; in contrast to the FP4I-1 group, & P＜0.05， && P＜0.01。
FIG. 13, influence of GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 on liver quality of high fat diet-induced obese mice (means+ -SEM, n=7); statistical difference signature annotation: in comparison with the obese control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## P＜0.01。
FIG. 14, GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 liver of obese mice induced by high fat dietEffect of dirty triglyceride content (means±sem, n=7); statistical difference signature annotation: in comparison with the obese control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## P＜0.01。
FIG. 15 effect of GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 on serum triglyceride levels of high fat diet-induced obese mice (means+ -SEM, n=7); statistical difference signature annotation: in comparison with the obese control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## p is less than 0.01; in contrast to the FP4I-1 group, & P＜0.05， && P＜0.01。
FIG. 16 effect of GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 on serum total cholesterol content of obese mice induced by high fat diet (means+ -SEM, n=7); statistical difference signature annotation: in comparison with the obese control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## P＜0.01。
FIG. 17 effect of GLP-1-FGF21 bifunctional proteins FP4I-1 and FP4I-2 on serum low density lipoprotein levels of obese mice induced by high fat diet (means+ -SEM, n=7); statistical difference signature annotation: in comparison with the obese control group, * P＜0.05， ** p is less than 0.01; in contrast to the group of dolastatin, # P＜0.05， ## P＜0.01。
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
The synergistic bifunctional proteins of the present invention are generally prepared by biosynthetic methods. The coding nucleic acids according to the invention can be prepared by various known methods, as is convenient to the person skilled in the art, on the basis of the nucleotide sequences according to the invention. Such as, but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in J.Sam Brookfield, guidelines for molecular cloning experiments. As one embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing nucleotide sequences in segments followed by overlap extension PCR.
The invention also provides an expression vector comprising a sequence encoding the enhanced bifunctional protein of the invention and an expression control sequence operably linked thereto. The term "operably linked" or "operably linked" refers to a condition in which certain 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 transcription of a sequence, it is operably linked to a coding sequence.
The expression vector may be commercially available such as, but not limited to: pcDNA3, pIRES, pDR and pUC18, etc. can be used for eukaryotic cell system expression vectors. The selection of an appropriate expression vector can be made by one skilled in the art depending on the host cell.
According to the restriction enzyme map of the known no-load expression vector, a person skilled in the art can insert the coding sequence of the synergistic bifunctional protein of the invention into a proper restriction site by restriction enzyme cleavage and splicing according to a conventional method to prepare the recombinant expression vector of the invention.
The invention also provides a host cell for expressing the synergistic bifunctional protein of the invention, which contains the coding sequence of the synergistic bifunctional protein of the invention. The host cell is preferably a eukaryotic cell such as, but not limited to, CHO, COS, 293 and RSF cells, and the like. As a preferred mode of the invention, the cell is a CHO cell, which can well express the synergistic type bifunctional protein of the invention, and can obtain the synergistic type bifunctional protein with good binding activity and good stability.
The invention also provides a method for preparing the synergistic dual-function protein by using the recombinant DNA, which comprises the following steps:
1) Providing a nucleic acid sequence encoding a synergistic bifunctional protein;
2) Inserting the nucleic acid sequence of 1) into a proper expression vector to obtain a recombinant expression vector;
3) Introducing the recombinant expression vector of 2) into a suitable host cell;
4) Culturing the transformed host cell under conditions suitable for expression;
5) The supernatant was collected and the synergistic bifunctional protein product was purified.
The coding sequence may be introduced into the host cell using a variety of techniques known in the art, such as, but not limited to: calcium phosphate precipitation, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription, phage transduction, alkali metal ion.
For culture and expression of host cells see Olander RM Dev Biol Stand,1996,86:338. Cells and residues in the suspension can be removed by centrifugation and the supernatant collected. Identification can be performed by agarose gel electrophoresis techniques.
The synergistic bifunctional proteins prepared as described above may be purified to have substantially homogeneous properties, e.g., as a single band on SDS-PAGE electrophoresis. For example, when the recombinant protein is expressed as a secretion, a commercially available ultrafiltration membrane may be used to isolate the protein, for example, from a company product such as Millipore, pellicon, and the expression supernatant is first concentrated. The concentrate can be further purified by gel chromatography or ion exchange chromatography. Such as anion exchange chromatography (DEAE, etc.) or cation exchange chromatography. The gel matrix may be agarose, dextran, polyamide, etc. commonly used for protein purification. The Q-or SP-groups are the preferred ion exchange groups. Finally, the purified product can be further refined and purified by methods such as hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography, reverse phase high performance liquid chromatography (RP-HPLC) and the like. All of the purification steps described above can be used in different combinations to achieve a substantially uniform protein purity.
The expressed bifunctional proteins may be purified using an affinity chromatography column containing antibodies, receptors or ligands specific for the bifunctional protein. Depending on the nature of the affinity column used, the fusion polypeptide bound to the affinity column may be eluted using conventional methods, such as high salt buffers, pH changes, and the like. Optionally, the amino-or carboxy-terminus of the bifunctional protein may further comprise one or more polypeptide fragments as protein tags. Any suitable label may be used with the present invention. For example, the tag may be FLAG, HA, HA1, c-Myc,6-His or 8-His, etc. These tags can be used to purify the bifunctional proteins.
EXAMPLE 1 construction of synergistic bifunctional protein expression plasmid
The gene sequences encoding the alpha 1 microglobulin leader peptide, GLP-1 analogues, L1, FGF21 mature protein, L2 (comprising flexible peptide units and rigid peptide units) and human IgG Fc variants are all artificially optimized CHO cell preference codons, and the full-length sequence is obtained by a chemical synthesis method. To facilitate insertion of the desired fragment into a specific site of the expression vector, a restriction enzyme site was located at each of the 5 'and 3' ends of the synthesized fragment, speI and EcoRI, respectively. The verified synergistic type bifunctional protein gene is digested by SpeI and EcoRI, and then inserted into corresponding digestion sites of a plasmid PXY1A1 modified by PCDNA3.1, and a fusion gene expression plasmid (the nucleotide sequence and the translated amino acid sequence of the synergistic type bifunctional protein FP4I-2 are exemplarily shown in FIG. 1) is obtained. The plasmid contains cytomegalovirus early promoter, which is an enhancer required for mammalian cells to express exogenous genes at high levels. The plasmid also contains a selectable marker, which may be kanamycin resistant in bacteria and G418 resistant in mammalian cells. In addition, when the host cell is deficient in DHFR gene expression, the PXY1A1 expression vector contains a mouse dihydrofolate reductase (DHFR) gene, thereby enabling co-amplification of the fusion gene and DHFR gene in the presence of Methotrexate (MTX) (see U.S. Pat. No. 4,399,216).
We constructed a variety of synergistic bifunctional proteins comprising GLP-1 and FGF21, the present invention exemplifies three of FP4I-1, FP4I-2 and FP4I-3 whose amino acid compositions are shown in Table 1 (L1 and L2 are underlined, respectively, and mutant amino acids in the Fc variants are boxed).
TABLE 1 amino acid composition of each synergistic bifunctional protein
Example 2 expression of synergistic bifunctional proteins in transfected cell lines
The recombinant expression vector plasmid is transfected into a mammalian host cell line to express the synergistic bifunctional protein. For stable high level expression, the preferred host cell line is DHFR enzyme deficient CHO-cells (see US 4,818,679), which in this example is selected from the CHO derived cell line DXB11. One preferred transfection method is electroporation, other methods may also be used, including calcium phosphate co-sedimentation, lipofection. In electroporation, 5X 10 in a cuvette was performed using a Gene Pulser electroporator (Bio-Rad Laboratories, hercules, calif.) set to 300V electric field and 1500 μFd capacitance 7 50. Mu.g of high purity expression plasmid was added to each cell. Two days after transfection, the medium was changed to a growth medium containing 0.6mg/mL G418. Transfectants resistant to selection drugs were screened using ELISA assay against human IgG Fc. The amount of expression of the synergistic bifunctional protein can also be quantified by ELISA against human FGF21 or human GLP-1. Subcloning produced wells of high levels of synergistic bifunctional proteins by limiting dilution of 96-well plates.
In order to achieve higher levels of expression of the synergistic bifunctional protein, it is desirable to co-amplify the DHFR gene inhibited by MTX drugs. The transfected, enhanced bifunctional protein gene was co-amplified with the DHFR gene in growth medium containing increasing concentrations of MTX. Limiting dilution of the subclones positive for DHFR expression, gradually pressurizing and screening out transfectants which can grow in the MTX culture medium with the concentration of up to 6 mu M, measuring the secretion rate of the transfectants, and screening out the cell line with high expression of the exogenous protein. The secretion rate is greater than about 10 (preferably about 20) μg/10 6 (i.e., millions) of cells/24The cell line was subjected to adaptive suspension culture using serum-free medium, and then the synergistic bifunctional protein was purified using conditioned medium.
EXAMPLE 3 purification and characterization of synergistic bifunctional proteins
The FP4I-2 purification and characterization method is exemplarily described in this example. The cell culture supernatant is subjected to clarification treatment such as high-speed low-temperature centrifugation, 0.22 mu m sterilization filtration and the like, and is purified by three steps of protein A affinity, anion exchange and hydrophobic chromatography, and the specific method is as follows: in the first step, protein A affinity chromatography is adopted, the balance solution is PBS buffer solution, the eluent is citrate buffer solution with pH of 3.5, and the eluted target protein is neutralized by 1M Tris solution. Intermediate purification the high resolution anion exchange packing Q Sepharose HP (GE company) was selected to remove residual impurity proteins. The binding mode was used, eluting with 20mM Tris-HCl,0.2M NaCl,pH 7.5 solution and eluting with 20mM Tris-HCl,0.3M NaCl,pH 7.5 solution. The precise purification step selects Butyl Sepharose FF (GE company) to remove the polymer, utilizes the different hydrophobicity of FP4I-2 monomer and the polymer, the monomer with weak hydrophobicity directly flows through, the polymer with strong hydrophobicity is combined on the medium, the flow through mode of hydrophobic chromatography is selected, and the balancing solution is PBS buffer solution.
The results of qualitative analysis of the target protein product are shown in FIG. 2a and FIG. 2b. The theoretical molecular weight of the FP4I-2 single chain is about 53KD, and SDS-PAGE electrophoresis under reducing condition shows that the actual size of the FP4I-2 single chain molecule is about 70KD because of glycosylation sites. The target protein was obtained by the same method as that for FP4I-1 and-3.
Example 4 Effect of synergistic bifunctional protein Single injection on glucose utilization in C57BL/6 mice
SPF-grade, 8 week old male C57BL/6J mice (purchased from Fukang Biotechnology Co., ltd., beijing) were selected. Feeding environment: the temperature is 22-25 ℃, the relative humidity is 45-65%, and the illumination time is 12h/d. After 1 week of adaptive feeding, the mice were randomly divided into control group, dolapride 120nmol/kg group, FP4I-2 120nmol/kg group and FP4I-1 120nmol/kg group (n=7) according to body weight. The mice in the administration group are subcutaneously injected with the corresponding drug solution, and the mice in the control group are subcutaneously injected with PBS buffer. Each group of mice was fasted for 16h after injection and a glucose tolerance experiment was performed. The fasting blood glucose of the mice was measured, 2g/kg glucose solution was intraperitoneally injected, and blood glucose levels after 15min, 30min, 60min, 90min and 120min of glucose injection were measured, and the area of increase (iaauc) on the baseline under the curve was calculated by the trapezoidal method. The mice of each group continued to develop the glucose tolerance test 96h and 144h after the administration, as above. Data are presented as mean ± standard error (means ± SEM) and analyzed using SPSS18.0 statistical software. Normal distribution, wherein the mean difference among multiple groups adopts single-factor variance analysis, the variance uniformity adopts LSD (least squares) test, and the variance uniformity adopts Dunnet T3 test; non-normal distribution using non-parametric test, P <0.05 indicates significant statistical differences.
As can be seen from fig. 3a and 3b, fp4I-1 and FP4I-2 significantly improved the glucose utilization level (P < 0.01) in mice at 16h post-dosing, with the control group given PBS buffer as a reference. From FIGS. 4a and 4b, it can be seen that 96h after administration, FP4I-1 and FP4I-2 also significantly improved the glucose utilization level in mice (P < 0.01). From FIGS. 5a and 5b, it can be seen that 144h after administration, FP4I-1 and FP4I-2 still showed improved glucose utilization levels in mice (P < 0.05). The experimental result shows that under the condition of sudden rise of the glucose level in the body, the GLP-1-FGF21 bifunctional protein provided by the invention can quickly regulate the glucose in the body to restore to the normal physiological level by promoting the release and secretion of insulin, has long-acting property, and can be applied to the treatment of diabetes and complications thereof caused by absolute or relative deficiency of insulin. The mice did not develop symptoms of hypoglycaemic shock or death after administration of FP4I-1 and FP4I-2 with simultaneous fasting for 16h, indicating that the bifunctional protein did not lead to a hypoglycaemic response similar to that caused by insulin.
Meanwhile, the glucose utilization activity of the Exendin4-FGF21 bifunctional protein FP4I-3 was determined as described above. C57BL/6 mice were split into control, du-Lalutide and FP4I-3 groups by body weight. Each of the administration groups was subcutaneously administered 120nmol/kg of the drug solution, and the control group was administered PBS buffer. Sugar tolerance experiments were performed at 16h, 96h, 144h post injection. As shown in fig. 6a and 6b, FP4I-3 significantly improved the glucose utilization level in mice (P < 0.01) but significantly weaker than dolapride (P < 0.01) compared to the control group at 16h post-dose. From fig. 7a and 7b, it can be seen that 96h after dosing, fp4i-3 significantly improved the glucose utilization level in mice (P < 0.01), but significantly lower than dolapride group (P < 0.01). As shown in FIGS. 8a and 8b, 144h after administration, FP4I-3 improved the glucose utilization capacity of mice to be lost (P > 0.05).
From the blood glucose lowering performance of the FP4I-3 animal, the Exendin-4 does not form a synergistic effect with FGF21 unexpectedly, the regulation effect on blood glucose is obviously weaker than that of a long-acting GLP-1 analogue, namely, the dolapride, and the functional half-life is obviously shortened compared with that of the dolapride. The preferred GLP-1-FGF21 type bifunctional proteins FP4I-2 and FP4I-1 have high stability in vivo, are not easy to be degraded and inactivated, and have longer in vivo pharmacodynamic activity maintenance time compared with the Exendin4-FGF21 type bifunctional protein FP 4I-3. The above results indicate that the combination of the three functional parts of the GLP-1 analogue, FGF21 and Fc fragment in the bifunctional protein is not random, arbitrary, wherein the selection of the GLP-1 analogue, the structure of the linker peptide, and the sequence of fusion, and even the difference in glycosylation, affect to a different extent whether the conformation of the bifunctional protein is correct or stable, thus ultimately determining whether the active molecules are functionally coordinated with each other, and whether the half-life can be effectively prolonged.
Example 5 Experimental study of the Effect of synergistic bifunctional protein on blood glucose control in db/db mice
Male db/db mice at 8 weeks of age purchased from Shanghai Laek laboratory animal Limited, feeding Environment: the temperature is 22-25 ℃, the relative humidity is 45-65%, and the illumination time is 12h/d. After 1 week of feeding per cage, adaptive singles were randomly divided into 4 groups according to body weight, blood glucose level and feeding level: control group, dolapride group, FP4I-1 group, FP4I-2 group (n=7). The control group was subcutaneously injected with PBS buffer, and the other groups were subcutaneously injected with 120nmol/kg of the corresponding drug solution, 2 times per week, 8 times in total. Daily intake of each mouse was recorded. After the last dosing cycle, each group of mice was fasted for 16 hours, and the whole blood was collected from the eyeballs and 5 μl of the whole blood was used for glycosylated hemoglobin detection. Data are expressed as mean ± standard deviation (x ± s), and data are analyzed using SPSS 18.0 statistical software. Normal distribution, wherein the mean difference among multiple groups adopts single-factor variance analysis, the variance uniformity adopts LSD (least squares) test, and the variance uniformity adopts Dunnet T3 test; non-normal distribution using non-parametric test, P <0.05 indicates significant statistical differences.
As shown in FIG. 9, mice fed significantly (P < 0.01) within 24 hours to the FP4I-1 and FP4I-2 mice after the first administration compared to the Duraglutide group. The results show that the GLP-1-FGF21 synergistic bifunctional protein can obviously relieve the severe gastrointestinal adverse reaction symptoms induced after the first administration of the long-acting GLP-1 receptor agonist drugs. As shown in FIG. 10, the group FP4I-1, the group FP4I-2 and the dolapride can significantly reduce the value of db/db mouse glycosylated hemoglobin (P < 0.01), and the values of the glycosylated hemoglobin of the group FP4I-1 and the group FP4I-2 are significantly lower than those of the dolapride group (P < 0.05). The db/db mouse is a spontaneous hyperglycemia mouse accompanied by serious insulin resistance, and the GLP-1-FGF21 synergistic bifunctional protein provided by the invention has better characteristics than dolapride in the aspect of long-term blood glucose control of the db/db mouse. According to the data of example 4, GLP-1-FGF 21-enhanced bifunctional proteins were not significantly superior to dolapride in promoting insulin release and secretion. Natural FGF-21 shows good insulin sensitization in the candy clamp experiments (Xu J et al, diabetes,2009, 58:250-259), and no direct evidence exists at present that the dolapride has insulin sensitization in vivo. Thus, in summary, the GLP-1-FGF21 synergistic bifunctional proteins exhibit the advantage in glycemic control as a result of the synergistic effect of GLP-1 in promoting insulin secretion and release and FGF21 in enhancing insulin sensitivity. As shown in FIG. 11, in the experimental period, the cumulative food intake of the FP4I-1 and FP4I-2 mice is significantly higher than that of the Yu Dula Lu peptide mice (P < 0.05), and the results show that the blood sugar control capacity of the FP4I-1 and FP4I-2 mice on type 2 diabetes is stronger than that of the Du Lu peptide under the condition of excluding food intake intervention factors.
Example 6 Experimental study of the Effect of synergistic bifunctional protein on the treatment of obesity-induced obesity in high fat diet mice and fatty liver and lipid metabolism disorder
C57BL/6 mice at 8 weeks of age purchased from Shanghai Laek laboratory animal Co., ltd., feeding environment: the temperature is 22-25 ℃, the relative humidity is 45-65%, and the illumination time is 12h/d. After 1 week of adaptive feeding, 7 mice were given low fat feed (D12450B, research diets) and the remaining mice were given high fat feed (D12451, research diets). After 40 weeks, obese mice were acclimatized for 1 week, single mice per cage, and then the obese mice were randomly divided into: obesity control group, dularlutide group, obesity diet control group, FP4I-1 group and FP4I-2 group (n=7). Wherein the daily feeding of the obese diet control group, the FP4I-1 group and the FP4I-2 group mice is consistent with the daily feeding of the Duraglutide group mice. The obese control group and the obese diet control group were subcutaneously injected with PBS buffer, and each of the administration groups was subcutaneously injected with 120nmol/kg of the corresponding drug solution 1 time every 6 days for a total of 2 times. The body weight of each mouse before and after the start of the experiment was recorded. After the last dosing period, each group of mice was fasted for 16 hours, whole blood was collected from the orbit, centrifuged at 2000 Xg for 15min, and serum was isolated. And detecting the serum lipid content by a full-automatic biochemical analyzer. Liver tissue was isolated, washed with physiological saline, blotted dry with filter paper, and weighed. About 50mg of liver tissue was taken from the same site, and the triglyceride content in the liver tissue was measured by Folch method. Results are expressed as triglyceride content per mg liver tissue. Data are presented as mean ± standard error (means ± SEM) and analyzed using SPSS18.0 statistical software. Normal distribution, wherein the mean difference among multiple groups adopts single-factor variance analysis, the variance uniformity adopts LSD (least squares) test, and the variance uniformity adopts Dunnet T3 test; non-normal distribution using non-parametric test, P <0.05 indicates significant statistical differences.
As shown in fig. 12 to 17, in the obese mice induced by the high fat diet, the weight, liver mass, liver triglyceride content, serum total cholesterol and serum low density lipoprotein content of the mice were significantly reduced (P < 0.01) after the dulcin treatment. The dolapride can cause serious gastrointestinal adverse reaction and has a certain function of suppressing appetite of the nerve center, so that the food intake of animals is greatly reduced. In this example, the above index was also significantly reduced after daily administration of the same diet as dolapride group mice in the obese diet control group, with no significant statistical difference (P > 0.05) from the dolapride group. The results show that the effects of dolapride in reducing weight, relieving liver steatosis and regulating lipid metabolism disorder are basically dependent on the inhibition of ingestion without other independent treatment mechanisms. After the fat mice in the FP4I-1 group and the FP4I-2 group are given the same food intake as the mice in the dolapride group, compared with the dolapride group, the weight and serum triglyceride level of the fat mice in the FP4I-2 group are obviously reduced (P is less than 0.01), which shows that the FP4I-2 has the functions of reducing the synthesis of fat in the body and promoting the utilization of fat metabolism, and can be applied to the treatment of obesity and metabolic syndrome caused by obesity.
Compared with the dolastatin group, the liver quality and the liver triglyceride content of the FP4I-2 group mice are obviously reduced (P is less than 0.01 or P is less than 0.05), which indicates that the FP4I-2 can effectively reduce excessive deposition of fat in liver tissues and restore liver functions, and the FP4I-2 can be used for treating various liver diseases such as liver steatosis-induced nonalcoholic fatty liver, nonalcoholic steatohepatitis, liver fibrosis, liver cirrhosis and the like.
Compared with the dolastatin group, the serum total cholesterol and serum low density lipoprotein content of the FP4I-2 group mice are obviously reduced (P is less than 0.01 or P is less than 0.05), which indicates that the FP4I-2 can be applied to the treatment of various cardiovascular and cerebrovascular diseases such as hypercholesterolemia and induced hypertension, coronary heart disease, chronic heart failure, cerebral infarction, atherosclerosis and the like. Compared with the dolapride group, the FP4I-1 group mice have a certain tendency to decrease in body weight, liver mass, liver triglyceride content, serum triglyceride, serum total cholesterol and serum low density lipoprotein, but have no statistical differences.
The research results show that the FP4I-1 and the FP4I-2 can treat obesity, fatty liver disease and lipid metabolism disorder through the physiological activity of FGF21, are not completely dependent on the ingestion regulation effect of GLP-1 analogues, have the most prominent treatment effect of the FP4I-2, are obviously superior to that of the dolapride, and can fill the gap in the clinical treatment of the dolapride. In conclusion, the therapeutic target of FP4I-2 is richer than that of dolapride, and meets the requirements of clinical diversified treatment.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
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Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Ser Ile Glu Lys Thr Ile
100 105 110
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
115 120 125
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
130 135 140
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
145 150 155 160
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
165 170 175
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
180 185 190
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu
195 200 205
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 220
<210> 12
<211> 229
<212> PRT
<213> vFcγ4 amino acid composition ()
<400> 12
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225
<210> 13
<211> 505
<212> PRT
<213> FP4I-2 amino acid composition ()
<400> 13
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Pro
35 40 45
Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln
50 55 60
Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu
65 70 75 80
Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu
85 90 95
Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu
100 105 110
Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu
115 120 125
Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu
130 135 140
Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu
145 150 155 160
Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro
165 170 175
Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro
180 185 190
Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser
195 200 205
Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser
210 215 220
Tyr Ala Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser
245 250 255
Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro
260 265 270
Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Val Glu Cys Pro Pro Cys
275 280 285
Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
290 295 300
Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
305 310 315 320
Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
325 330 335
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
340 345 350
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His
355 360 365
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
370 375 380
Gly Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln
385 390 395 400
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
405 410 415
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
420 425 430
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
435 440 445
Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
450 455 460
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
465 470 475 480
Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln
485 490 495
Lys Ser Leu Ser Leu Ser Pro Gly Lys
500 505
<210> 14
<211> 1380
<212> DNA
<213> FP4I-2 nucleotide composition ()
<400> 14
caccctattc ccgatagctc ccccctcctg cagttcggag gccaggtgag gcagcggtac 60
ctgtacaccg acgacgctca gcagaccgaa gctcacctgg agatcaggga ggatggaacc 120
gtcggcggag ctgctgacca gtcccccgag agcctgctgc agctgaaggc cctgaagccc 180
ggagtcatcc agatcctggg cgtgaagacc tcccggtttc tgtgtcagcg gcccgatgga 240
gccctgtacg gctccctgca ttttgacccc gaggcctgta gcttcaggga gctgctgctg 300
gaagacggct acaacgtgta ccagagcgaa gctcacggac tgcccctgca cctgcctggc 360
aacaaatccc ctcacaggga ccccgctccc aggggacctg ccaggttcct gcctctgccc 420
ggactgcctc ctgctcctcc cgaacctcct ggcatcctcg ctcctcagcc ccctgatgtc 480
ggcagcagcg accctctgtc catggtcggc cccagccaag gcaggagccc ttcctacgct 540
tccggatccg gtggcggtgg ctccggtgga ggcggaagcg gcggtggagg atcaggcggt 600
ggaggtagcg gcggaggcgg tagctccagc tctagtaaag ctccccctcc ttccctgccc 660
tcaccctcaa gactgcctgg accttccgac actcccatcc tgccacaggt ggagtgccct 720
ccatgtccag caccccctgt cgcaggtcca tctgtgttcc tgtttccacc caagcctaaa 780
gaccagctga tgatctcccg caccccagaa gtcacctgtg tggtcgtgga tgtgagccat 840
gaagaccccg aggtccagtt caattggtac gtggatggcg tcgaggtgca caacgctaag 900
acaaaaccta gagaagagca gttcaactct acctttcgcg tcgtgagtgt gctgacagtc 960
gtgcaccagg actggctgaa tggcaaggag tataagtgca aagtgagcaa caaaggactg 1020
cctgcctcaa tcgaaaagac tatttccaag accaaaggac agccaagaga gccccaggtg 1080
tacaccctgc ctccaagccg cgaagagatg actaaaaatc aggtctctct gacctgtctg 1140
gtgaaggggt tttatcctag tgatatcgcc gtggaatggg agtcaaacgg tcagccagag 1200
aacaattaca agaccacacc ccctatgctg gacagcgatg ggtctttctt tctgtatagc 1260
aaactgacag tggacaagtc tcggtggcag cagggtaacg tcttctcttg cagtgtgctg 1320
cacgaagcac tgcacaatca ttacacccag aagtcactgt cactgagccc aggaaaatga 1380
<210> 15
<211> 500
<212> PRT
<213> FP4I-1 amino acid composition ()
<400> 15
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly
20 25 30
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Pro Ile
35 40 45
Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg
50 55 60
Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile
65 70 75 80
Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser
85 90 95
Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly
100 105 110
Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr
115 120 125
Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu
130 135 140
Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro
145 150 155 160
Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg
165 170 175
Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro
180 185 190
Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser
195 200 205
Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr
210 215 220
Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
225 230 235 240
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Ser Ser Lys
245 250 255
Ala Pro Pro Pro Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Glu
260 265 270
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Ala
275 280 285
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
290 295 300
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
305 310 315 320
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
325 330 335
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
340 345 350
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
355 360 365
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
370 375 380
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
385 390 395 400
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
405 410 415
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
420 425 430
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
435 440 445
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
450 455 460
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
465 470 475 480
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
485 490 495
Ser Leu Gly Lys
500

Claims (4)

1. A synergistic dual-function protein is prepared by the following method,
(a) The coded amino acid sequence is shown as SEQ ID NO:13 or 15 into mammalian cells, said mammalian cells being the CHO derived cell line DXB-11;
(b) The expression in the selection step (a) is more than 20. Mu.g/10 every 24 hours in its growth medium 6 High-yield cell lines of individual cells;
(c) Culturing the cell strain screened in the step (b) to express the synergistic bifunctional protein;
(d) Harvesting the fermentation broth obtained in the step (c), and purifying the synergistic bifunctional protein.
2. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent and an effective amount of the synergistic dual functional protein as claimed in claim 1.
3. The use of the synergistic bifunctional protein of claim 1, for the preparation of a medicament for the treatment of obesity and metabolic syndrome caused by obesity, type 1 or type 2 diabetes, liver steatosis-induced nonalcoholic fatty liver, nonalcoholic steatohepatitis, liver fibrosis and liver cirrhosis of a variety of liver diseases, dyslipidemia, or hypercholesterolemia and its induced hypertension, coronary heart disease, chronic heart failure, cerebral infarction and atherosclerosis of a variety of cardiovascular and cerebrovascular diseases.
4. Use of a synergistic bifunctional protein of claim 1, in the manufacture of a medicament for treating non-alcoholic steatohepatitis or cardiovascular disease.
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