Patent Application
20210380654
The present invention relates to a human fibroblast growth factor 21 (hFGF21) fusion protein, and use of the fusion protein for the manufacture of a medicament for the treatment of diabetes, obesity, dyslipidemia and/or metabolic syndrome.
Fibroblast growth factors (FGFs) are a family of polypeptide growth factors exhibiting 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. It is mainly expressed in the liver, but also in cells of tissues related to glycolipid metabolism, such as adipose tissue, islet β cells and muscle tissue. Unlike other FGF members, this subfamily acts in an endocrine manner, and regulates energy and bile acid homeostasis, glucose and lipid metabolism, and phosphate and vitamin D homeostasis (Moore D D et. al., Science, 2007, 316:1436-1438; Beenken et. al., Nature Reviews Drug Discover, 2009, 8: 235). 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 co-factor β-Klotho, and then activates the FGFR receptor and downstream related signaling molecules to exert 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).
The metabolic regulation function of FGF21 on the body was first elucidated by Kharitonenkov et. al. FGF21 promoted the uptake of glucose in 3T3-L1 cells and human primary adipocytes by regulating the expression of glucose transporter 1 (GLUT1). Subsequent experiments showed that FGF21 significantly reduced body weight and blood glucose levels, as well as triglyceride levels in the liver and serum, and significantly affected insulin sensitivity in genetically-controlled or diet-induced obese mice (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). Administration of exogenous FGF21 to diet-induced obese mice and ob/ob mice can reverse hepatic steatosis and enhance hepatic insulin sensitivity (including reduction of hepatic glucose production and increase of hepatic glycogen content), thereby improving systemic glucose intolerance and insulin resistance (Xu J et. al., Diabetes, 2009, 58(1):250-259; Berglund E D et. al., Endocrinology, 2009, 150(9):4084-4093; Xu J et. al., Am J Physiol Endocrinol Metab, 2009, 297(5)E1105-1104). In addition to the same metabolic regulation effects as described above, FGF21 also reduces the concentration of low density lipoprotein cholesterol and increases the concentration of high density lipoprotein cholesterol in diabetic monkeys (Kharitonenkov A et. al., Endocrinology, 2007, 148(2):774-781). In summary, FGF21 has a significant beneficial metabolic regulation effect on obese rodent and non-human primates. FGF21, which regulates blood glucose levels independent of insulin, does not cause hypoglycemia when administered as a hypoglycemic agent in large doses. In addition, 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.
A large number of preclinical and clinical research have shown that FGF21 is a potential excellent target for the treatment of obesity, type 2 diabetes and hyperlipidemia. However, due to its own physical and chemical properties, native FGF21 is difficult to be developed into clinical therapeutic biological agents mainly because of the reasons as follows: 1. FGF21 protein has poor stability and is susceptible to protease hydrolysis or enzymatic degradation; 2. FGF21 has a very unstable conformation and is prone to aggregation. The low stability also increases the difficulty of FGF21 production in large-scale; 3. The native FGF21 has a very short half-life. Human FGF21 has an in vivo half-life of 0.5-1 hour in mice, and of 2-3 hours in cynomolgus monkeys (Kharitonenkov A et. al., Journal of clinical Investigation, 2005, 115:1627-1635). A variety long-acting technologies for protein have been reported to extend the in vivo half-life of recombinant FGF21, for example, by linking FGF21 to a PEG molecule to increase the molecule weight thereof to reduce the glomerular filtration rate and prolong the in vivo retention time (see patents WO2005/091944, WO2006/050247, WO2008/121563 and WO2012/066075); infusing FGF21 with a fatty acid long chain which is capable of binding to serum albumin (see WO2010/084169 and WO2012/010553); or preparing an agonist antibody that specifically binds to the FGFR or FGFR/β-klotho complex to activate the FGF/FGFR signaling pathway by mimicking the action mechanism of FGF21 (see WO2011/071783, WO2011/130417, WO2012/158704 and WO2012/170438); or infusing with a Fc fragment to improve the half-life of FGF21 (see WO2004/110472, WO2005/113606, WO2009/149171, WO2010/042747, WO2010/129503, WO2010/129600, WO2013/049247, WO2013/188181 and WO2016/114633).
In the field of long-acting technologies for protein, Fc fusion technology is the most widely used because it leads to fewer clinical side effects, for example, it is not easy to induce an allergic reaction or increase the toxicity of the drug due to the prolonged half-life. The key to the development of FGF21/Fc long-acting fusion protein drugs lies in the following points. First, whether the biological activity of FGF21 can be maintained. The C-terminus of FGF21 contains a binding site for β-Klotho, and thus fusion of Fc to the C-terminus of FGF21 results in a significant decrease in its activity, while fusion to the N-terminus retains its binding affinity to β-Klotho to the utmost extent. Therefore, in the prior art, the Fc fragment is mostly fused to the N-terminus of FGF21 (Fc-FGF21). Second, whether the fusion of Fc can significantly improve the pharmacokinetic properties of FGF21 and effectively prolong its half-life to meet the clinical requirement for administrating twice a week or even once a week. The FGF21 is highly susceptible to degradation because it contains a protease hydrolysis site at the C-terminus, and the in vitro activity of its hydrolyzed metabolites is reduced by nearly 200-fold (Micanovic R et. al., J Cell Physiol, 2009, 219(2):227-234; Yie Jet. al., FEBS Lett, 2009, 583(1):19-24). According to pharmacokinetic studies on Fc-FGF21, its half-life was not significantly improved because the C-terminus (especially between Pro171 and Ser172) of FGF21 in the fusion protein was exposed and was not protected by Fc, and thus it is susceptible to degradation by protease attack (Hecht, R et. al., PLoS One, 2012, 7:e49345). Third, how to reduce the risk of an immune response induced by the linker sequence or the introduced mutation site. Fourth, whether the fusion of Fc and/or the introduction of a mutation site can improve the stability of FGF21 and its polymerizability in a highly concentrated state. Therefore, for an ideal FGF21 Fc fusion protein, on the one hand, the anti-proteolytic ability of the C-terminus of FGF21 should be enhanced as well as the half-life should be significantly extended; on the other hand, the binding affinity of the C-terminus of FGF21 to β-Klotho should not be significantly reduced due to the steric effect of Fc, otherwise will result in a significant decrease in its activity; at the same time, the introduction of the fusion ligand and the linker peptide should not increase its immunogenicity, but improving its stability.
The carboxyl terminal peptide (hereinafter referred to as CTP) of the human chorionic gonadotropin (hCG) beta chain has the effect of prolonging the in vivo half-life of certain proteins. Thus CTP is used in fusion proteins as disclosed in some patent documents as a fusion ligand for prolonging the half-life of protein of interest. In addition, CTP can also be used as a linker mainly for linking different subunits of the same protein. For example, CTP is used as a linker between the beta and alpha subunits of follicle stimulating hormone, as disclosed in Chinese Patent Nos. CN103539860A, CN103539861A, CN103539868A, and CN103539869A. As another example, CTP is used as a linker between the beta and alpha subunits of glycoprotein hormone as disclosed in the patent WO2005058953A2.
The present inventors do not use CTP as a linker or as a half-life prolonging moiety as suggested by the prior art, but instead connect it to a flexible peptide linker (e.g., (GGGGS)n) to constitute a new linker sequence. The new linker is located between FGF21 and the half-life prolonging moiety (e.g., the immunoglobulin Fc fragment, which does not contain the CTP as suggested by the prior art) to constitute a new FGF21 fusion protein, further prolonging the half-life and maintaining the biological activity and function of FGF21.
The object of the present invention is to solve the problems of short half-life and 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.
In one aspect, the present invention provides a highly glycosylated human fibroblast growth factor 21 (hFGF21) fusion protein, which is hereinafter referred to as hFGF21 fusion protein. The fusion protein comprises, in order from the N-terminus to C-terminus, wild-type human fibroblast growth factor 21 or an analog thereof (denoted as hFGF21), a flexible peptide linker (denoted as L), at least one rigid unit comprising the carboxyl terminal peptide of human chorionic gonadotropin β subunit (hereinafter referred to as (CTP)n, wherein n is 1, 2, 3, 4, or 5) and a fusion ligand (such as an immunoglobulin and a Fc fragment thereof, an albumin or a transferrin).
In some embodiments, the fusion protein disclosed herein comprises a wild-type hFGF21 polypeptide, wherein the wild-type hFGF21 polypeptide comprises a sequence as shown in SEQ ID NO: 1 from which amino acids 1-28 (a leader peptide) are removed; or an isoform of the sequence as shown in SEQ ID NO: 1 from which amino acids 1-28 (a leader peptide) are removed and having G141S or L174P substitution.
In other embodiments, the fusion protein disclosed herein comprises an hFGF21 analog, for example, an hFGF21 analog having one or more amino acid deletions, insertions, additions or substitutions relative to its wild-type sequence, and a truncated form having one or more amino acid deletions at the N- or C-terminus. Preferably, the amino acid sequence of the hFGF21 analog is at least 70% homologous to that of wild-type hFGF21; more preferably, the amino acid sequence of the hFGF21 analog is at least 80% homologous to that of wild-type hFGF21; more preferably, the amino acid sequence of the hFGF21 analog is at least 90% homologous to that of wild-type hFGF21; and most preferably, the amino acid sequence of the hFGF21 analog is at least 95% homologous to that of wild-type hFGF21.
The flexible peptide linker is preferably non-immunogenic and can generate sufficient distance between hFGF21 and the fusion ligand to minimize the steric effects between each other. Preferably, a flexible peptide linker consisting of two or more amino acids selected from the group consisting of Gly (G), Ser (S), Ala (A) and Thr (T) is used.
Preferably, the flexible peptide linker comprises residues G and S. The length of the linker peptide plays a very important role in the activity of the fusion protein. For the purposes of the present invention, the flexible peptide linker preferably has the structural formula of (GS)a(GGS)b(GGGS)c(GGGGS)d, wherein each a, b, c, and d is an integer equal to or greater than 0, and a+b+c+d≥1.
In some embodiments of the present invention, the peptide linker is selected from the group consisting of:
The CTP rigid unit is selected from a full-length sequence consisting of carboxyl-terminal amino acids 113 to 145 of human chorionic gonadotropin β subunit, or a truncated sequence thereof. Specifically, the CTP rigid unit comprises the amino acid sequence as shown in SEQ ID NO: 2 or a truncated sequence thereof.
Preferably, the CTP rigid unit contains at least 2 glycosylation sites. For example, in a preferred embodiment of the present invention, the CTP rigid unit contains 2 glycosylation sites. Illustratively, the CTP rigid unit contains N-terminal 10 amino acids of SEQ ID NO: 2, i.e. SSSS*KAPPPS*; alternatively, the CTP rigid unit contains C-terminal 14 amino acids of SEQ ID NO: 2, i.e. S*RLPGPS*DTPILPQ. As another example, in another embodiment, the CTP rigid unit contains 3 glycosylation sites. Illustratively, the CTP rigid unit contains N-terminal 16 amino acids of SEQ ID NO: 2, i.e. SSSS*KAPPPS*LPSPS*R. As another example, in other embodiments, the CTP rigid unit contains 4 glycosylation sites. Illustratively, the CTP rigid unit contains 28, 29, 30, 31, 32, or 33 amino acids, starting from position 113, 114, 115, 116, 117, or 118 and ending at position 145 of the human chorionic gonadotropin beta subunit. Specifically, the CTP rigid unit contains N-terminal 28 amino acids of SEQ ID NO: 2, i.e. SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ. Herein, * represents a glycosylation site. Each possibility represents a separate embodiment of the present invention.
In other embodiments, the amino acid sequence of the CTP rigid unit provided by the present invention is at least 70% homologous to that of native CTP. In other embodiments, the amino acid sequence of the CTP rigid unit provided by the present invention is at least 80% homologous to that of native CTP. In other embodiments, the amino acid sequence of the CTP rigid unit provided by the present invention is at least 90% homologous to that of native CTP. In other embodiments, the amino acid sequence of the CTP rigid unit provided by the present invention is at least 95% homologous to that of native CTP.
Illustratively, the CTP rigid unit of the present invention may preferably comprise the following sequences:
In some embodiments of the present invention, the fusion protein comprises one CTP rigid unit as described above.
The fusion protein of the present invention may comprise more than one, preferably 2, 3, 4 or 5, CTP rigid units as described above. In an embodiment of the present invention, the fusion protein comprises three CTP3 rigid units: SSSSKAPPPSSSSSKAPPPSSSSSKAPPPS (CTP3-CTP3-CTP3, or expressed as (CTP3)3). In another embodiment of the present invention, the fusion 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 selected from the group consisting of Fc fragments of human immunoglobulin IgG, IgM, IgA and variants thereof, more preferably from the group consisting of Fc fragments of human IgG1, IgG2, IgG3 and IgG4 and variants thereof. The human IgG Fc variant (denoted as vFc) comprises at least one amino acid modification in the wild-type human IgG Fc. In addition, 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.
Further, the human IgG Fc variant may be selected from the group consisting of:
(i) vFcγ1: hinge, CH2 and CH3 regions of human IgG1 with mutations Leu234Val, Leu235Ala, and Pro331Ser (the amino acid sequence as shown in SEQ ID NO: 3);
(ii) vFcγ2-1: hinge, CH2 and CH3 regions of human IgG2 with mutation Pro331Ser (the amino acid sequence as shown in SEQ ID NO: 4);
(iii) vFcγ2-2: hinge, CH2 and CH3 regions of human IgG2 with mutations Thr250Gln and Met428Leu (the amino acid sequence as shown in SEQ ID NO: 5);
(iv) vFcγ2-3: hinge, CH2 and CH3 regions of human IgG2 with mutations Pro331Ser, Thr250Gln and Met428Leu (the amino acid sequence as shown in SEQ ID NO: 6).
The IgG Fc variants provided by the present invention include, but are not limited to, the five variants as described in (i) to (v), and may also be those obtained by combining or adding the mutation sites of two functional variants of the same IgG subtype. For example, the variant as described in (iv) is a new IgG2 Fc combination variant obtained by adding the mutation sites in (ii) and (iii).
The Fc variant (vFc) in the fusion protein of the present invention comprises hinge, CH2 and CH3 regions of human IgG such as human IgG1, IgG2 and IgG4. The CH2 region contains amino acid mutations at positions 228, 234, 235 and 331 (as defined by the EU numbering system). It is believed that these amino acid mutations reduce the effector functions of Fc. Human IgG2 Fc does not bind to FcγR but shows extremely weak complement activity. An Fcγ2 variant with mutation Pro331Ser should have less complement activity than native Fcγ2 while remain as a non-binder to FcγR. IgG4 Fc is deficient in activating the complement cascade, and its binding affinity to FcγR is about an order of magnitude lower than that of IgG1. An Fcγ4 variant with mutations Ser228Pro and Leu235Ala should exhibit minimal effector functions as compared to the native Fcγ4. An Fcγ1 variant with mutations Leu234Val, Leu235Ala and Pro331Ser also should exhibit decreased effector functions than the native Fcγ1. These Fc variants are more suitable for the preparation of fusion proteins of hFGF21 and an analog thereof than native human IgG Fcs. The amino acid mutations at positions 250 and 428, as defined by the EU numbering system, increase the binding affinity of the Fc region to the neonatal receptor FcRn, thereby further prolonging the half-life (Paul R et al., J Biol Chem, 2004, 279:6213-6216). The above two types of functional variants are combined or added on each other to generate new combination variants to reduce the effector functions while prolonging the half-life. The Fc variants of the present invention contain mutations at, but not limited to, the above-described sites, and substitutions may be introduced at other sites such that Fcs have reduced effector functions and/or enhanced binding affinity to FcRn. Meantime, these mutations should not lead to reduction of the function/activity or undesirable conformational changes of the Fc variants. Common mutation sites may be found in Shields R L et al., J Biol Chem, 2001, 276(9):6591-604.
In a preferred embodiment of the present invention, the fusion protein has the amino acid sequence as shown in SEQ ID NO: 8.
The fusion protein of the present invention is glycosylated. Preferably, the fusion protein is glycosylated by expressing the same in mammalian cells. More preferably, the fusion protein is glycosylated by expressing the same in Chinese hamster ovary cells.
According to another aspect of the present invention, a DNA encoding the fusion protein described above is provided. In a preferred embodiment of the present invention, the fusion protein has the DNA sequence as shown in SEQ ID NO: 9.
According to another aspect of the present invention, a vector is provided. This vector contains the DNA described above.
According to another aspect of the present invention, a host cell is provided. The host cell comprises or is transfected with the vector described above.
In a specific embodiment of the present invention, the host cell is CHO-derived cell line DXB-11.
According to still 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 fusion protein described above.
According to still another aspect of the present invention, there is provided use of the fusion protein for the preparation of a medicament for treating a condition associated with FGF21, such as 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 arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, neuropathy, and disorders associated with severe inactivation or mutation in the insulin receptor.
According to another aspect of the present invention, a method for preparing or producing the fusion protein from a mammalian cell line, such as a CHO-derived cell line, is provided, which comprises the steps of:
(a) introducing a DNA encoding the fusion protein described above into a mammalian cell;
(b) screening the high-yielding cell line in step (a) which expresses more than 50 μg/106 cells per 24 hours in its growth medium;
(c) growing the cell line obtained in step (b);
(d) harvesting the fermentation broth obtained in step (c) and purifying the fusion protein.
Preferably, the mammalian cell used in the step (a) is a CHO cell, more preferably, CHO-derived cell line DXB-11.
Compared with the current products, the hFGF21 fusion protein of the present invention has the following outstanding advantages:
1. Prolonged in vivo functional half-life. Glucose utilization levels in obese mice were significantly improved both in medium- and high-dose groups at 16 h after administration of hFGF21 fusion protein to obese mice induced with high-fat diet, and the glucose utilization levels in obese mice were still significantly improved in the high-dose group at 48 h after administration, indicating that the hFGF21 fusion protein has long-lasting effects on improving glucose utilization, at the same time, also demonstrating that the hFGF21 fusion protein has a significantly enhanced anti-proteolytic ability, allowing it to be less susceptible to degradation that results in activity reduction or loss.
2. Significantly prolonged in vivo circulating half-life. Since the protective effects of Fc, which is fused at the C-terminus of FGF21, reduces the susceptibility of FGF21 to proteolytic enzyme, the fusion protein constructed by the present invention has a significantly longer half-life than the fusion proteins in which Fc are fused at the N-terminus (Fc-FGF21) as reported in the prior art. On the other hand, the CTP rigid unit contains a glycosyl group. The negatively charged, highly sialylated CTP rigid unit is capable of resisting the clearance of the kidney, which further prolongs the half-life of the fusion protein. In a preferred embodiment of the present invention, it is demonstrated that 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 pharmacokinetic data obtained after a single-dose in a preferred embodiment showed that the in vivo circulating half-life T1/2 of 3 mg/kg hFGF21 fusion protein FP4I in SD rats was 29.81±1.56 h, whereas the T1/2 of CTP-free hFGF21 fusion protein FP4J was only 22.43±1.45 h, which is greatly prolonged compared with the half-life of native hFGF21 (the in vivo circulating half-life in rats is 1˜1.5 h), allowing the frequency of administration to be greatly reduced.
3. In the prior art, the C-terminal fusion of Fc to FGF21 (FGF21-Fc) often leads to a significant decrease in its activity. However, the C-terminal Fc fusion protein constructed by the present invention utilizes a novel linker peptide which is consisted of a flexible peptide and a CTP rigid peptide. The CTP rigid unit can form a relatively stable stereoscopic conformation by containing a plurality of O-glycosyl side chains, which effectively isolates hFGF21 from Fc, thereby minimizing the steric hindrance effect generated by Fc and allowing FGF21 to maintain a better biological activity. For example, the hFGF21 fusion protein can effectively control the body weight of obese mice induced by high-fat diet, improve insulin resistance and hepatic steatosis, and exhibit significant inhibition of obesity. In addition, the protective effect of the CTP rigid unit 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. Moreover, CTP is derived from native human hCG and thus is non-immunogenic and more suitable for being used as a linker peptide than a non-naturally encoded amino acid sequence.
4. Mutations such as P331S have been introduced in the fusion ligand Fc, retaining only the properties of Fc of long circulating half-life, and reducing or eliminating the ADCC and CDC effects, thereby increasing drug safety. In addition, Fc variants with enhanced binding affinity to neonatal receptor (FcRn) (such as T250Q/M428L) can further prolong the half-life of the fusion protein.
5. 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, the hFGF21 fusion protein is also less likely to aggregate in highly concentrated preparations.
The term “human FGF21” and “hFGF21” as used in the present invention refer to a wild-type human FGF21 polypeptide as well as an analog thereof.
The sequence of the wild-type FGF21 protein is available from the UNIPROT database with an Accession No. Q9NSA1. The precursor protein consists of 209 amino acids, including a signal peptide (amino acids 1-28) and a mature protein (amino acids 29-209).
An isoform or allelic form of wild-type FGF21 having a Pro instead of Leu in the mature protein at position 174 of SEQ ID NO: 1 of the present invention is known from US 2001012628 A1. Another isoform of wild-type FGF21 having a Ser instead of Gly at position 141 of SEQ ID NO: 1 of the present invention is known.
Another isoform having a shorter signal peptide in which Leu at position 23 of SEQ ID NO: 2 of the present invention is missing is known from WO 2003/011213 (see SEQ ID NO: 2 of WO2003/011213, having a signal peptide of 27 amino acid residues).
In the present invention, the wild-type hFGF21 comprises the sequence of the mature protein moiety (amino acids 29-209) obtained by removing the leader peptide from the sequence as shown in SEQ ID NO: 1 or an isoform thereof having substitution L174P or G141S. In addition, the full-length sequence of the precursor protein containing the signal peptide of 27 or 28 amino acids is also included.
hFGF21 Analogs
The hFGF21 analog as used in the present invention refers to a polypeptide which is or can be deduced or derived from wild-type human FGF21, in particular from SEQ ID NO: 1, by modification of the amino acid sequence thereof. Such modification, amendment or change may include substitution, deletion, and/or addition of one or more amino acids. The term “mutein” or “variant” is sometimes used herein instead of the term “analog”. For example, amino acids may be added and/or deleted at the C-terminus, at the N-terminus, or internally in the amino acid sequence (an amino acid added internally in a sequence may be referred to as an insertion). Preferably amino acids are added and/or deleted at the C and/or 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. The truncated hFGF21 polypeptide, including forms of N-terminal truncation or C-terminal truncation, is capable of providing an activity similar to, in some cases better than, the un-truncated form of the mature hFGF21 polypeptide, as is known in the art.
a. N-Terminal Truncations
In some embodiments of the present invention, the hFGF21 analog having a N-terminal truncation is selected from a polypeptide in which 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues at the N-terminus of the mature hFGF21 polypeptide are deleted. FGF21 polypeptides having N-terminal truncations of fewer than 9 amino acid residues retain the ability of the mature FGF21 polypeptide to reduce blood glucose in an individual. Accordingly, in particular embodiments, the present invention encompasses truncated forms of the mature FGF21 polypeptide or FGF21 protein variants having N-terminal truncations of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues.
b. C-Terminal Truncations
In some embodiments of the present invention, the hFGF21 analog having a C-terminal truncation is selected from a polypeptide in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues at the C-terminus of the mature hFGF21 polypeptide are deleted. FGF21 polypeptides having C-terminal truncations of fewer than 13 amino acid residues exhibited at least 50% of the efficacy of wild-type FGF21 in an in vitro ELK-luciferase assay (Yie J. et al. FEBS Letts 583:19-24 (2009)), indicating that these FGF21 mutants retain the ability of the mature FGF21 polypeptide to reduce blood glucose in an individual. Accordingly, in particular embodiments, the present invention encompasses truncated forms of the mature FGF21 polypeptide or FGF21 protein variants having C-terminal truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues.
One example of an FGF21 analogue is the truncated form of wild-type mature FGF21 in which four N-terminal amino acid residues (HPIP) of the mature protein are removed, which is disclosed in, e.g., WO 2006/065582. This truncated form is believed to stimulate glucose uptake in mouse 3T3-L1 adipocytes at the same level as the wild-type FGF21. In the present invention, the truncated form of hFGF21 has the amino acid sequence of amino acid 33-209 of SEQ ID NO: 1, designated as hFGF21 (HPIP−).
A further example of an FGF21 analogue is the polypeptide of SEQ ID NO:1 which has an N-terminal Met (also designated as “Met-hFGF21”). The N-terminal Met is added when hFGF21 is expressed in E. coli, see e.g. WO 2006/050247, Table 6.
Another example of an hFGF21 analog is a wild-type hFGF21 precursor protein as shown in SEQ ID NO: 1 in which one or more amino acids are substituted/replaced. The substitution/replacement includes, but is not limited to, Q55C, A109T, L126R, G148C, K150R, P158S, S195A, P199G and G202A.
Examples of amino acid sequences of hFGF21 analogs containing other modifications are disclosed in, for example, WO2003/061712, WO2005/091944, WO2006/028595, WO2006/028714, WO2006/065582 and WO2008/121563.
hCG-β Carboxyl Terminal Peptide (CTP)
CTP is a short peptide from the carboxyl terminus of the human chorionic gonadotropin (hCG) beta subunit. Four kinds of reproduction-related polypeptide hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), and human chorionic gonadotropin (hCG) contain the same alpha subunit and their respective specific beta subunits. Compared with the other three hormones, hCG has a significantly prolonged in vivo half-life, which is mainly due to the specific carboxyl terminal peptide (CTP) on the hCG β-subunit (Fares F A et al., Proc Natl Acad Sci USA, 1992, 89: 4304-4308). The native CTP contains 37 amino acid residues, including four O-glycosylation sites, and sialic acid residues at the terminus. The highly sialylated, negatively charged CTP can resist the clearance by the kidney, thereby prolonging the in vivo half-life of the protein. The present inventors creatively connect at least one CTP peptide with a flexible peptide linker having an appropriate length to form a new peptide linker, for linking hFGF21 to a fusion ligand e.g., an immunoglobulin Fc fragment.
The present inventors have found that the N-terminal and C-terminal sequences of hFGF21 play critical roles in its function. The hFGF21 has a complex and fragile spatial conformation which makes itself poorly stable, easily degradable and easy to polymerize. The steric hindrance effect of the fusion ligand linked to the hFGF21 will interfere with the correct folding of hFGF21, resulting in a significant decrease or even loss of activity, or higher probability of producing a polymer. The addition of a CTP rigid unit between hFGF21 and an Fc variant is equivalent to the addition of a rigid peptide linker. On one hand, the addition of the CTP peptide ensures that the N-terminally fused hFGF21 does not affect the binding site in Fc variant for FcRn, thus having no effect on the half-life. In addition, the protein A binding site in Fc is important for purification steps. The addition of a CTP rigid unit ensures that the N-terminally fused hFGF21 will not “cover” its binding site for protein A. On the other hand, the addition of a CTP rigid unit prevents the Fc fragment having a size of about 25 kD from interfering with the correct folding of the N-terminally fused hFGF21, thus leading to no loss or decline of the biological activity/function of the hFGF21.
We have also demonstrated that simply prolonging the length of the flexible linker does not significantly relief the effect of the Fc fragment on hFGF21 activity. An over-length linker peptide can result in the generation of polymers and increased sensitivity to proteases, making hFGF21 susceptible to degradation. However, the addition of a CTP rigid unit makes the fusion protein more stable. This might because that the rigid CTP peptide containing multiple glycosyl side chains can form a stable steric conformation compared to the random coil of a flexible linker such as (GGGGS)n. This “block” effect causes the hFGF21 and Fc fragment to fold independently into correct three-dimensional conformations without affecting the biological activities of each other. Moreover, the protective effect of the glycosyl side chains of CTP reduces the sensitivity of the peptide linker to proteases, such that the fusion protein is less susceptible to degradation in the linking region. Furthermore, 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, and plays an important role in eradicating pathogens in immune defense. The Fc-mediated effector functions of IgG function through two mechanisms as follows: (1) After binding to Fc receptors (FcγRs) on the cell surface, pathogens are broken down by phagocytosis or lysis or by killer cells through the antibody-dependent cell-mediated cytotoxicity (ADCC) pathway. (2) Alternatively, after binding to C1q of the first complement component C1, the complement-dependent cytotoxicity (CDC) pathway is triggered and thus pathogens are lysed. Among the four subtypes of human IgG, IgG1 and IgG3 are able to bind to FcγRs effectively, and IgG4 has lower binding affinity for FcγRs. The binding of IgG2 to FcγRs is too low to be measured, so human IgG2 has little ADCC effects. In addition, human IgG1 and IgG3 can also effectively bind to C1q to activate the complement cascade. Human IgG2 binds weakly to C1q and IgG4 does not bind to C1q (Jefferis R et. al., Immunol Rev, 1998, 163: 59-76), so the CDC effect of human IgG2 is also weak. Obviously, none of the native IgG subtypes is well suitable for constructing hFGF21/Fc fusion proteins. In order to obtain non-lytic Fc variants without effector functions, the most effective method is to mutate the complement- and receptor-binding regions of the Fc segment and adjust the binding affinity of Fc for related receptors to reduce or eliminate the ADCC and CDC effects and retain only the long in vivo half-life of Fc without the generation of cytotoxicity. More mutation sites contained in non-lytic Fc variants can be found in Shields R L et al., J Biol Chem, 2001, 276(9):6591-604 or China Patent No. CN 201280031137.2.
Fc Variants with Enhanced Affinity to the Neonatal Receptor FcRn
The plasma half-life of IgG depends on its binding to FcRn. Typically, IgG binds to FcRn at pH 6.0 and dissociates from FcRn at pH 7.4 (plasma pH). Through the study on the binding sites of the two, the sites on IgG that bind to FcRn are modified to increase the binding affinity at pH 6.0. It has been proven that mutations of some residues in the human Fey domain, which are important for the binding to FcRn, can increase the serum half-life. Mutations in residues T250, M252, S254, T256, V308, E380, M428 and N434 have been reported to increase or decrease the FcRn-binding affinity (Roopenian et al., Nat. Review Immunology 7:715-725, 2007). Trastuzumab (Herceptin, Genentech) variants, disclosed in Korean Patent No. KR 10-1027427, show increased FcRn-binding affinity, and these variants contain one or more amino acid modifications selected from the group consisting of 257C, 257M, 257L, 257N, 257Y, 279Q, 279Y, 308F and 308Y. Bevacizumab (Avastin, Genentech) variants, provided in Korean Patent No. KR 2010-0099179, show prolonged in vivo half-life and these variants contain amino acid modifications N434S, M252Y/M428L, M252Y/N434S and M428L/N434S. In addition, Hinton et al. also found that two variants T250Q and M428L increased the binding affinity for FcRn by 3 and 7 times, respectively. When the two sites were mutated simultaneously, the binding affinity was increased by 28 times. In rhesus macaque, the M428L or T250Q/M428L variant shows a 2-fold increase in plasma half-life (Paul R. Hinton et al., J Immunol, 2006, 176:346-356). More mutation sites contained in Fc variants with increased binding affinity for FcRn can be found in China Patent No. CN201280066663.2. In addition, studies show that the T250Q/M428L mutations in the Fc regions of five humanized antibodies improve the interaction between the Fc domain and FcRn. Moreover, in subsequent in vivo pharmacokinetic tests, compared to wild-type antibodies, the Fc mutated antibodies show improved pharmacokinetic parameters, such as increased in vivo exposure, reduced clearance, and increased subcutaneous bioavailability, when administered via subcutaneous injection (Datta-Mannan A et al., MAbs. Taylor & Francis, 2012, 4(2):267-273).
The term “disorders associated with severe inactivating mutations in the insulin receptor” describes conditions in subjects with mutations in the insulin receptor (or possible proteins directly downstream from it) which cause severe insulin resistance but are often occurred without the obesity commonly found in type 2 diabetes. In many ways, subjects afflicted with these conditions manifest hybrid symptoms of type 1 diabetes and Type 2 diabetes. Subjects thereby afflicted fall into several categories of roughly increasing severity, including: Type A Insulin Resistance, Type C Insulin Resistance (AKA HAIR-AN Syndrome), Rabson-Mendenhall Syndrome and Donohue's Syndrome or Leprechaunism. These disorders are associated with very high endogenous insulin levels, resulting in hyperglycemia. Subjects thereby afflicted also present with various clinical features associated with “insulin toxicity,” including hyperandrogenism, polycystic ovarian syndrome (PCOS), hirsuitism, and acanthosis nigricans (excessive growth and pigmentation in the folds of the skin).
“Diabetic complications” are dysfunctions in other parts of the body caused by chronic hyperglycemia such as diabetic nephropathy, diabetic neuropathy, diabetic feet (foot ulcers and poor circulation), and ocular lesions (retinopathies). Diabetes also increases the risk for heart disease and bone and joint disorders. Other long-term complications of diabetes include skin problems, digestive problems, sexual dysfunction and problems with teeth and gums.
“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 and dyslipidemia, such as hypertriglyceridemia (TG) and low high-density lipoprotein cholesterol (HDL-C); (3) hypertension; (4) insulin resistance and/or abnormal glucose tolerance. Some criteria also include microalbuminuria, hyperuricemia, increased pro-inflammatory state (C-reactive protein, CRP), and increased pro-thrombotic state (increased fibrinogen and plasminogen inhibitor-1, PAI-1).
“Dyslipidemia” refers to a disorder of lipoprotein metabolism, including lipoprotein overproduction or deficiency. Dyslipidemias may be manifested by an increase in the total cholesterol, low-density lipoprotein (LDL) cholesterol and triglyceride concentrations, and a decrease in high-density lipoprotein (HDL) cholesterol concentration in the blood.
“Non-alcoholic fatty liver disease (NAFLD)” is a spectrum of liver disease characterized by hepatic steatosis, in the absence of excessive alcohol consumption.
“Non-alcoholic steatohepatitis (NASH)” is a spectrum of liver disease that includes simple steatosis, lobular inflammation, progressing to fibrosis, in the absence of excessive alcohol consumption.
) fragment in the PCDNA3.1 expression vector according to an embodiment of the present invention, consisting of a α1 microglobulin lead peptide (1-19, marked as ______), hFGF21 (20-200, having P instead of L), a flexible peptide linker (201-227, marked as
), CTP1 (228-255, marked as
), and vFc (256-478).
The present invention is further illustrated below in combination with specific embodiments. It should to be understood that the embodiments are only illustrative and are not intended to limit the scope of the present invention. In the following examples, experimental methods in which conditions are not specifically indicated are usually carried out according to conventional conditions, such as conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conditions suggested by the manufacturer.
The fusion proteins of the present invention are typically prepared by biosynthetic methods. Based on 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, for example, but not limited to, PCR, DNA synthesis, etc. For specific methods, see J. Sambrook, Molecular Cloning: A Laboratory Manual. As an embodiment of the present invention, the nucleic acid sequence of the present invention can be constructed by segmentally synthesizing nucleotide sequences and then performing overlap extension PCR.
The present invention also provides an expression vector comprising a sequence encoding a fusion protein of the present invention and an expression regulatory sequence operably linked thereto. By “operably link” or “operably linked to” is meant a condition in which some portions of a linear DNA sequence are capable of regulating or controlling the activity of other portions of the same linear DNA sequence. For instance, a promoter is operably linked to a coding sequence if the promoter controls the transcription of the 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 in a eukaryotic cell expression system. One skilled in the art can select a suitable expression vector based on the host cell.
The coding sequence of the fusion protein of the present invention may be introduced into suitable restriction sites by one skilled in the art by restriction enzyme cleavage and splicing according to a conventional method based on the restriction enzyme map of the known empty expression vector, to produce the recombinant expression vector of the present invention.
The invention also provides a host cell expressing a fusion protein of the invention comprising a coding sequence of a fusion protein of the present 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. In a preferred embodiment of the present invention, the cell is a CHO cell which can well express the fusion protein of the present invention to obtain a fusion protein having good binding activity and good stability.
The present invention also provides a method for producing a fusion protein of the present invention by using recombinant DNA, including the steps of:
1) providing a nucleic acid sequence encoding a fusion protein;
2) inserting the nucleic acid sequence of 1) into a suitable expression vector to obtain a recombinant expression vector;
3) introducing the recombinant expression vector of 2) into a suitable host cell;
4) growing the transformed host cell under conditions suitable for expression;
5) collecting the supernatant and purifying the fusion protein product.
The coding sequence can be introduced into a host cell by various techniques known in the art such as, but not limited to, calcium phosphate precipitation, protoplast fusion, lipofection, electroporation, microinjection, reverse transcription, phage transduction and method using alkali metal ions.
For the culture and expression of host cells, see Olander R M Dev Biol Stand, 1996, 86:338. The cells and debris in the suspension can be removed by centrifugation and the supernatant is collected. Identification can be performed by agarose gel electrophoresis.
The fusion protein obtained as described above can be purified to a substantially uniform nature, for example, showing a single band on SDS-PAGE electrophoresis. For example, when the recombinant protein is expressed by secretion, a commercially available ultrafiltration membrane such as products from Millipore, Pellicon, etc. can be used to separate the protein.
The supernatant is firstly to be concentrated. The concentrated supernatant may be further purified by gel chromatography or by ion exchange chromatography, such as 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. Finally, the purified product may be further finely purified by methods such as hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and the like. All of the above purification steps can be used in different combinations to ultimately obtain proteins with a substantially uniform purity.
The expressed fusion protein can be purified by using an affinity chromatography column containing an antibody, receptor or ligand specific for the fusion protein. Depending on the nature of the affinity column used, the fusion polypeptide bound to the affinity column can be eluted by using conventional methods such as high salt buffer, pH change, and the like. Alternatively, the fusion protein may also contain one or more polypeptide fragments as a protein tag at the amino terminus or carboxyl terminus. Any suitable label can be used in the present invention. For example, the label may be FLAG, HA, HAL c-Myc, 6-His or 8-His, and the like. These tags can be used to purify the fusion protein.
Gene sequences encoding the α1 microglobulin leader peptide and mature hFGF21 or its analog, flexible peptide linker, CTP rigid unit and human IgG Fc variant were artificially-optimized, CHO cell-biased codons. The full-length sequence was obtained by chemical synthesis. A restriction site, SpeI or EcoRI respectively, were present at each of the 5′-end and 3′-end of the synthesized fragment to facilitate insertion of the target fragment into a specific site of the expression vector. The verified gene of the fusion protein of hFGF21 or its analog was digested with SpeI and EcoRI, and then inserted into corresponding restriction sites in PCDNA3.1-modified plasmid PXY1A1 to obtain an expression plasmid of the fusion gene. This plasmid contains a cytomegalovirus early promoter which is an enhancer required for mammalian cells to express foreign genes at high levels. This plasmid also contains selectable markers which may confer kanamycin resistance to bacteria and G418 resistance to mammalian cells. In addition, the PXY1A1 expression vector contains the mouse dihydrofolate reductase (DHFR) gene, thus the fusion gene and DHFR gene may be co-amplified in the presence of methotrexate (MTX) in host cells deficient in expressing a DHFR gene (See U.S. Pat. No. 4,399,216).
As shown in Table 1, the present invention constructed a series of fusion proteins of hFGF21, which comprised wild-type hFGF21 and its analogs, flexible peptide linkers (Linkers) with different lengths and Fc variant (vFc) elements of different IgG subtypes. In addition, the position and length of the CTP rigid unit were also different. In order to verify that fusion proteins comprising at least one CTP rigid unit with different lengths have a high biological activity, we constructed fusion proteins FP4A, FP4B, FP4C, FP4D, FP4E, FP4F, FP4G, FP4H and FP4I; as well as FP4J comprising no CTP rigid unit. The nucleotide sequence and translated amino acid sequence of FP4I are shown in
In the present example, the wild-type hFGF21 had the amino acid sequence as shown in SEQ ID NO: 1 (expressed as hFGF21), and it had an equivalent form, L174PhFGF21, wherein the Leu at position 174 of SEQ ID NO: 1 was replaced by Pro. The hFGF21 analog preferably comprises a truncated wild-type hFGF21 polypeptide, hFGF21(HPIP−), wherein the HPIP at the N-terminus of the wild-type hFGF21 mature protein has been deleted (having an amino acid sequence of amino acids 33-209 of SEQ ID NO: 1). The hFGF21 analog also preferably comprises variants in which an amino acid has been substituted/replaced, such as variant Q55C, which is obtained by replacing Gln at position 55 of SEQ ID NO: 1 with Cys, and several other variants including A109T, L126R K150R, P199G and G202A which are obtained by replacing the amino acid at the corresponding site of SEQ ID NO: 1.
P199GhFGF21-L1-CTP1-vFcγ2-3
K150RhFGF21-L3-CTP5-vFcγ1
G202AhFGF21-L4-CTP4-vFcγ4
A109ThFGF21-L1-CTP3-CTP3-CTP3-vFcγ2-2
Q55ChFGF21-L4-CTP3-vFcγ2-2
L126RhFGF21-L6-CTP2-vFcγ4
L174PhFGF21-L3-CTP1-vFcγ2-3
L174PhFGF21-L3-vFcγ2-3
The recombinant expression vector plasmid was transfected into a mammalian host cell line to express the hFGF21 fusion protein. DHFR enzyme-deficient CHO cells are preferred host cell line for stable expression at high levels (See U.S. Pat. No. 4,818,679). CHO-derived cell line DXB11 was used as a host cell in this example. A preferred method of transfection was electroporation, and other methods including calcium phosphate co-deposition and liposome transfection might also be used. In electroporation, Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, Calif.) was used at a voltage of 300 V and a capacitance of 1500 μFd, and 50 μg of high-purity expression plasmid was added to 5×107 cells in the cuvette. Two days after the transfection, the medium was replaced with a growth medium containing 0.6 mg/mL of G418. Transfectants resistant to the selected drug were screened by an ELISA assay against human IgG Fc. Quantification of the expression of the fusion protein can also be performed by using an ELISA assay against hFGF21. The wells producing high levels of Fc fusion protein were subcloned by limiting dilutions on 96-well culture plates.
To achieve higher levels of fusion protein expression, co-amplification utilizing the DHFR gene that can be inhibited by an MTX drug is preferred. The transfected gene of the fusion protein was co-amplified with the DHFR gene in growth media containing increasing concentrations of MTX. Subclones with positive DHFR expression were subjected to limiting dilution, and transfectants capable of growing in media containing up to 6 μM of MTX were screened by increasing the selection pressure gradually. The transfectants were measured for secretion rates and cell lines yielding high levels of exogenous protein were screened. Cell lines with a secretion rate of more than about 30, preferably about 50 μg/106 [i.e. million] cells/24 h, were adapted to suspension culture by using serum-free growth media. Conditioned media was then used to purify the fusion protein.
The conditioned media 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 Protein A column that had been equilibrated with phosphate buffered saline (PBS). After the fusion protein was bound onto the Protein A column, the effluent fractions were 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 K2HPO4, and fractions containing the purified protein were combined, dialyzed against PBS, and then filtered through a 0.22 micron nitrocellulose filter and stored at 4° C. The protein product was identified and analyzed by SDS-PAGE under non-reducing and reducing conditions. The results of SDS-PAGE electrophoresis for purified FP4I protein were shown in
32 male C57BL/6J mice aged 7 weeks (purchased from SLAC Laboratory Animal Co., Ltd, Shanghai, SPF-grade) were housed in a controlled environment: temperature of 22-25° C., relative humidity of 45-65%, and 12 h light/dark cycle. After a week of adaption, the mice were fed with high-fat diet (D12492 diet, a product from Research Diets, USA) for 12 weeks. The mice were divided into 4 groups according to the body weight: control group, FP4I-2.5 mg/kg (low-dose group), FP4I-5 mg/kg (middle-dose group) and FP4I-10 mg/kg (high-dose group). The mice in the drug-administration groups were subcutaneously injected with the corresponding FP4I solutions, and the mice in the control group were subcutaneously injected with PBS buffer. Thereafter, all the mice in each group were fasted for 16 hours followed by a glucose tolerance test. The fasting blood glucose level was measured. The mice were then intraperitoneally injected with 2 g/kg of glucose solution and the blood glucose levels at 15 min, 30 min, 60 min, 90 min and 120 min after injection were measured. The incremental area under the curve and above the baseline (iAUC) was calculated by trapezoidal method. 48 hours after drug administration, glucose tolerance test was carried out once more as described above. Data were expressed as mean±standard error of mean (mean±SEM) and analyzed using SPSS 18.0 statistical software. In normal distribution, differences between the means of multiple groups were analyzed by one-way ANOVA, followed by LSD test for homogeneous variances or Dunnett's T3 test for heterogeneous variances. Abnormal distribution was examined by non-parametric test. P values of less than 0.05 were considered statistically significant.
As show in
The metabolic activities of other hFGF21 fusion proteins in vivo were evaluated by glucose tolerance test as described above. The obese mice induced by high-fat diet were randomly divided into 10 groups (n=6 each group): FP4A, FP4B, FP4C, FP4D, FP4E, FP4F, FP4G, FP4H, FP4J, and control group. Mice in drug-administration groups were subcutaneously injected with 10 mg/kg of corresponding protein solutions while the mice in the control group were subcutaneously injected with PBS buffer. Glucose tolerance tests were performed at 16 h and 48 h after injection, respectively. The results showed that FP4A, FP4B, FP4C, FP4D, FP4E, FP4F, FP4G, FP4H and FP4J significantly improved glucose utilization 16 h after drug-administration (P<0.05) (
Six male SD rats weighted 180±10 g (purchased from SLAC Laboratory Animal Co., Ltd, Shanghai, SPF-grade) were housed in the following environment: temperature of 22-25° C., relative humidity of 45-65%, and 12 h light/dark cycle. After one week of adaption, 3 mg/kg of FP4I and FP4J were injected into the tail vein. 0.3 ml of blood was collected from the orbit respectively before administration, and at 1 h, 4 h, 7 h, 24 h, 48 h, 72 h, 96 h, 120 h, 149 h, 168 h, 192 h, 216 h and 240 h after administration. The whole blood was allowed to stand, and then centrifuged at 2000×g for 15 minutes to obtain serum. The sandwich ELISA assay was performed to detect drug concentrations in serum. The mouse anti-hFGF21 antibody (R&D, Cat. No. MAB25373-100) was used as capturing antibody while HRP-labeled goat anti-human IgG-Fc mAb (Jackson, Inc., Cat. No. 109-035-098) was used as detecting antibody. The software PKSOLVER was applied to calculate the pharmacokinetic parameters: T1/2 and AUC(0-t).
The results showed that the circulating half-life T1/2 of FP4I at the dosage of 3 mg/kg in SD rats was 29.81±1.56 h, and the AUC(0-t) thereof was 1716711±201507 ng/ml·h, the circulating half-life T1/2 of FP4J at the dosage of 3 mg/kg in SD rats was 22.43±1.45 h, and the AUC(0-t) thereof was 1210604±191426 ng/ml·h, while the circulating half-life of native FGF-21 in rats was 1-1.5 h. In summary, FP4I had a significantly increased half-life in vivo, and it had a longer circulating half-life in vivo and higher bioavailability than FP4J.
Twenty-four 7-week-old C57BL/6J male mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd) were housed in a controlled environment: temperature of 22-25° C., relative humidity of 45-65%, and 12 h light/dark cycle. After a week of adaption, the mice were divided into 3 groups according to their body weight: low-fat diet (LFD) group, high-fat diet (HFD) group, and high-fat diet plus 3.6 mg/kg hFGF21 fusion protein FP4I (HFD+FP4I 3.6 mg/kg) group. The mice in the LFD group and HFD groups were fed with D12450B diet and D12492 diet (Research diets, USA) respectively. Mice in the HFD+FP4I 3.6 mg/kg group were subcutaneously injected with 3.6 mg/kg of FP4I every four days, and mice in LFD and HFD groups were subcutaneously injected with PBS solution. The study was conducted for 116 days. At the end of the experiment, all the mice were fasted for 16 hours, and the fasting blood glucose levels and body weights were measured. Whole blood samples were collected by removing eyeballs, and then centrifuged at 2000×g for 15 min to separate and obtain the serum. The liver tissue was excised, washed with physiological saline, cleared the remaining fluid with filter paper and then weighed. Two parts of liver tissue were taken from the same site, wherein one was fixed in 10% formalin solution for HE staining, and the other was rapidly frozen in liquid nitrogen and stored at −80° C. for liver index analysis. The adipose tissue around the epididymis on one side was collected and fixed in a 10% formalin solution for HE staining.
The mice were weighed every 4 days, and muscle, fat and body fluid contents of the mice were analyzed by time-domain nuclear magnetic resonance on day 104 after the start of the experiment. Changes between body weight before and after the experiment were calculated according to the following formulae: body weight gain=the body weight of the mouse at the end of the experiment—the body weight of the mouse at the beginning of the experiment.
ALT, AST, HDL-c, LDL-c, TG and TC concentrations in serum were measured with automatic biochemical analyzer (Erba XL-200). The specific procedure was performed according to the instrument manual.
At day 118, the mice in each group were fasted for 6 h (10:00 am-4:00 pm) and then the basal blood glucose levels and body weight were measured. 0.75 IU/kg of insulin solution was injected intraperitoneally to the mice. The blood glucose levels were measured at 15, 30, 60, 90, 120 min after injection to draw an insulin tolerance curve. The AUC was calculated by trapezoidal method. The fasting serum insulin content in mice was measured by ELISA assay, and the insulin resistance index was calculated according to the following formula: insulin resistance index=fasting blood glucose level (mmol/L)×fasting insulin content (mIU/L)/22.5.
50 mg of liver tissue was accurately excised for each mouse. The liver TG contents were measured following the method described by Floch and the results were expressed as the TG content per mg liver tissue.
Mouse liver and adipose tissues which were collected from the same site and preserved in 10% formalin solution were stained with HE for morphopathological observation.
Data were expressed as mean±standard error of mean (mean±SEM) and analyzed by using SPSS 18.0 statistical software. In normal distribution, differences between the means of multiple groups were analyzed by one-way ANOVA, followed by LSD test for homogeneous variances or Dunnett's T3 test for heterogeneous variances. Abnormal distribution was examined by non-parametric test. P<0.05 indicated significant statistical difference.
4.1. Effect of the hFGF21 Fusion Protein on Body Weight and Fat Content in Mice Fed with High-Fat Diet
Compared with the low-fat diet group, mice in the high-fat diet group had significantly increased body weight, body weight gain and body fat content, showing obvious obesity symptoms. The hFGF21 fusion protein significantly reduced the body weight gain and body fat content induced under high-fat diet feeding conditions. The results are shown in
4.2. Effect of the hFGF21 Fusion Protein on Liver Function and Hepatic Steatosis in Mice Fed with High-Fat Diet
As shown in
4.3. Effect of hFGF21 Fusion Protein on Insulin Resistance in Mice Fed with High-Fat Diet.
The insulin tolerance test showed that mice fed high-fat diet showed significant symptoms of insulin resistance. Results of the serum insulin test further indicated that mice developed hyperinsulinemia and insulin resistance. The hFGF21 fusion protein significantly alleviated the insulin resistance symptoms and the increase in fasting blood glucose in mice fed with high-fat diet. The results are shown in
4.4 Effect of hFGF21 Fusion Protein on Hypercholesterolemia in Mice Fed with High-Fat Diet
As shown in
As shown in
Based on the above results, it was confirmed that hFGF21 fusion protein can effectively control the body weight, improve insulin resistance, reverse liver steatosis and hypercholesterolemia in obese mice induced by high-fat diet.
All documents mentioned in the present invention are hereby incorporated by reference to the same extent as if each of the documents is individually recited for reference. It is to be understood that various changes and modifications may be made by those skilled in the art upon reading the above teachings of the present invention, which also fall within the scope of the claims appended hereto.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201610694914.1 | Aug 2016 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/079871 | 4/10/2017 | WO | 00 |
