WO2015103782A1

WO2015103782A1 - Fgf21 mutant and conjugate thereof

Info

Publication number: WO2015103782A1
Application number: PCT/CN2014/070506
Authority: WO
Inventors: Zhifeng Huang; Xiaokun Li; Lintao SONG; Jianlou NIU
Original assignee: Wenzhou Medical College Biological Pharmaceuticals And Nature Products Ltd., Co; Hangzhou Receptz Biotech, Co., Ltd.
Priority date: 2014-01-13
Filing date: 2014-01-13
Publication date: 2015-07-16

Abstract

Provided are a stable FGF21 mutant, fusion polypeptide and conjugate comprising the mutant, as well as a use of the mutant in the preparation of medicaments for treating metabolic disorders. The mutant comprises only one amino acid substitution at position 59 or 71.

Description

FGF21 MUTANT AND CONJUGATE THEREOF

FIELD OF THE INVENTION

The present invention relates to a stable FGF21 mutant, fusion polypeptide, conjugate, pharmaceutical composition and pharmaceutical use thereof.

The study of the invention is supported by grants obtained from the Natural Science Foundation of China (81102486 to Z.H. and 81273421 to H.W.) and Zhejiang Key Group Project in Scientific Innovation (2010R10042-01 to Z.H.).

BACKGROUND OF THE INVENTION

Fibroblast growth factors (FGFs) are widely expressed in fetal and adult tissues, and play critical roles in multiple physiological functions, including angiogenesis, mitogenesis, pattern formation, cell differentiation, metabolic regulation and tissue injury repair. The 209-amino acid fibroblast growth factor 21 (FGF21) comprises 28-amino acid leader peptide and 181-amino acid mature FGF21. FGF21 was originally identified in a screen for molecules modulating glucose uptake in 3T3-L1 adipocyte, and has since been examined for its potential use in the treatment of metabolic disorders such as type 2 diabetes.

The PCT application WO2005/061712 discloses a mutein of human fibroblast growth factor 21, comprising the substitution for one or more of the following: glycine 42, glutamine 54, arginine 77, alanine 81, leucine 86, phenylalanine 88, lysine 122, histidine 125, arginine 125, proline 130, arginine 131, leucine 139, alanine 145, leucine 146, isoleucine 152, alanine 154, glutamine 156, glycine 161, serine 163, glycine 170, or serine 172, wherein the numbering of the amino acids is based on SEQ ID NO: 1 (of the 181-amino acid mature FGF21). The application also teaches a human FGF-21, comprising the substitution of a cysteine for two or more of the following: arginine 19, tyrosine 20 ... alanine 31 ... glycine 43 ... proline 138, or leucine 139. The substitution of a cysteine for two or more amino acid residues is explicitly utilized as loci to introduce a novel disulfide bond, so the application does not teach the substitution of a cysteine for a single amino acid residue such as alanine 31 or glycine 43.

The disclosure of the PCT application WO2006/028595 is similar to that of WO2005/061712, so the substitution of a cysteine for a single amino acid residue such as alanine 31 or glycine 43 is not taught either.

The PCT application WO2008/121563 discloses a modified fibroblast growth factor 21 polypeptide comprising non-naturally encoded amino acids.

The PCT application WO2009/149171 discloses an FGF21 mutant comprising an amino acid sequence of SEQ ID NO: 4 (of the 181-amino acid mature FGF21), further comprising the substitution of any amino acid for: the alanine residue at position 45, the leucine residue at position 86, the leucine residue at position98, the alanine residue at position 111, the alanine residue at position 129, the glycine residue at position 170, the proline residue at position 171, or the serine residue at position 172, and combinations thereof.

The PCT application WO2010/042747 discloses an FGF21 mutant comprising an amino acid sequence of SEQ ID NO: 4 (of the 181-amino acid mature FGF21) having at least one amino acid substitution that is a cysteine residue at one or more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and 179. Therefore, the application does not teach the substitution of a cysteine residue at position 31 or 43.

The PCT application WO2011/154349 discloses a [-1A, L166F, M168L, G174V, Y179F] FGF21 analogue containing one or more of amino acid substitutions of 71C, 121Q, 173A and/or desl81. However, the numbering of the amino acids in the application is based on the 181-amino acid mature FGF21, so position 71 is equivalent to position 99 of the 209-amino acid FGF21.

After massive research, the inventors surprisingly found that the substitution of a cysteine residue at just one of positions 59 and 71 of the 209-amino acid FGF21 can increase stability of the FGF21 mutant without significantly losing bioactivity (even with similar bioactivity of wild-type FGF21). Together with our development of fusion polypeptide strategy and solid-phase PEGylation process, the inventors obtained mono-PEGylated FGF21 mutant with further improved biostability and increased in vivo half-life.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a stable FGF21 mutant comprising only one amino acid substitution at position 59 or 71. The mutant is suitable for the construction of PEG conjugate with a long in vivo half-life.

In the first aspect, the present invention provides an isolated polypeptide, which is an FGF21 mutant and comprises only one amino acid substitution for the amino acid residue at position 59 or 71. The substitution is preferably a substitution for the glycine residue at position 71, e.g., a substitution of a cysteine residue for the glycine residue at position 71. In the invention, the numbering of the position is based on SEQ ID NO: 1.

In one embodiment, the FGF21 is the full-length FGF21 having an amino acid sequence of SEQ ID NO: 1, or the FGF21 is the mature FGF21 having amino acids 29-209 of SEQ ID NO: 1.

In another embodiment, the substitution is preferably a substitution of a cysteine residue, more preferably the substitution A59C or G71C, most preferably the substitution G71C.

In the second aspect, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of the first aspect.

In the third aspect, the present invention provides a vector comprising the nucleic acid of the second aspect. The vector is preferably an expression vector.

In the fourth aspect, the present invention provides a host cell comprising the vector of the third aspect. The host cell may be a eukaryotic cell or a prokaryotic cell, preferably a prokaryotic cell, e.g., E. coli cell.

In the fifth aspect, the present invention provides a process of producing the polypeptide of the first aspect comprising culturing the host cell of the fourth aspect under suitable conditions to express the polypeptide, and optionally isolating the polypeptide.

In the sixth aspect, the present invention provides a fusion polypeptide comprising the polypeptide of the first aspect and one or more (preferably one) heterologous polypeptides covalently linked thereto.

In one embodiment, the heterologous polypeptide is preferably SUMO (small ubiquitin-related modifier), more preferably His-tagged SUMO.

In another embodiment, the heterologous polypeptide is covalently linked to the N-terminus of the polypeptide of the first aspect.

In the seventh aspect, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of the sixth aspect.

In the eighth aspect, the present invention provides a vector comprising the nucleic acid of the seventh aspect. The vector is preferably an expression vector.

In the ninth aspect, the present invention provides a host cell comprising the vector of the eighth aspect. The host cell may be a eukaryotic cell or a prokaryotic cell, preferably a prokaryotic cell, e.g., E. coli cell.

In the tenth aspect, the present invention provides a process of producing the fusion polypeptide of the sixth aspect comprising culturing the host cell of the ninth aspect under suitable conditions to express the polypeptide, and optionally isolating the fusion polypeptide.

In the eleventh aspect, the present invention provides a conjugate comprising the polypeptide of the first aspect and one or more (preferably one) polymers covalently linked thereto.

In one embodiment, the polymer is preferably a water-soluble polymer, e. g., polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols, or polyvinyl alcohol, most preferably PEG.

In another embodiment, the polymer is covalently linked to a cysteine residue of the polypeptide.

In a preferable embodiment, the present invention provides a mono-PEGylated conjugate consisting of

an isolated polypeptide, which is an FGF21 mutant and comprises only one amino acid substitution G71C,

and a PEG moiety covalently linked to a cysteine residue of the polypeptide.

In the twelfth aspect, the present invention provides a process of producing the conjugate of the eleventh aspect comprising the steps of PEGylation of the fusion polypeptide with SUMO of the sixth aspect in solid phase, digestion of the SUMO moiety, and isolation of the conjugate. Preferably the fusion polypeptide is a fusion polypeptide with His-tagged SUMO, and is PEGylated in Ni-NTA affinity chromatography. Additionally the SUMO moiety is cleaved by SUMO protease.

In the thirteenth aspect, the present invention provides a pharmaceutical composition comprising the polypeptide of the first aspect or the conjugate of the eleventh aspect.

Generally, the pharmaceutical composition further comprises a pharmaceutically acceptable formulation agent, e.g., carrier, adjuvant, solubilizer, stabilizer, and/or anti-oxidant.

In the fourteenth aspect, the present invention provides a use of the polypeptide of the first aspect or the conjugate of the eleventh aspect in the preparation of medicaments for treating a metabolic disorder. Specifically the metabolic disorder is obesity, diabetes (preferably type II diabetes), insulin-resistance, hyperinsulinemia, glucose-intolerance or hyperglycemia.

In the fifteenth aspect, the present invention provides a method for treating a metabolic disorder comprising administering a therapeutically effective amount of the polypeptide of the first aspect, the conjugate of the eleventh aspect, or the pharmaceutical composition of the thirteenth aspect to a patient in need thereof. Specifically the metabolic disorder is obesity, diabetes (preferably type II diabetes), insulin-resistance, hyperinsulinemia, glucose-intolerance or hyperglycemia.

In the sixteenth aspect, the present invention provides an antibody or fragment thereof that specifically binds to the polypeptide of the first aspect. The antibody and fragment thereof can be used for the detection of the polypeptide.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of cell-based functional characterization and pharmacokinetics of FGF21 mutants: (A) Cellular glucose uptake stimulated by FGF21^WT, FGF21 mutants (FGF21^A59C, FGF21^G71C and FGF21^{A59C G71C}) on 3T3-L1 adipocytes, as measured using a Wallac 1450 MicroBeta counter (Perkin Elmer). * p< 0.05, and ** p< 0.01 vs. vehicle control; n=3. (B) Comparison of the half-life of FGF21^WT, FGF21 mutants (FGF21^A59C, FGF21^G71C and

FGF21 A^/"59^yC"' G^u'1¹C") in SD rats. Normal male SD rats were injected intravenously with 0.5 mg/kg FGF21^WT, FGF21^A59C, FGF21^G71C and FGF21^{A59C G71C}. Blood samples were collected at the indicated time points. The amount of FGF21 was measured using the human FGF21 immunoassay ELISA Kit. * p< 0.05, ** p< 0.01 vs. the corresponding FGF21^WT group; n=3.

Figure 2 shows the results of process, analysis and identification of PEGylated FGF21^G71C: (A) The elution profile of PEG-SUM0-FGF21^G71C from Ni-NTA affinity column following PEGylation. (B) The elution chromatogram of PEG-FGF21^G71C from Q-Sepharose Fast Flow column after cleavage. (C) MALDI-TOF mass spectrometry of PEGylated FGF21^G71C showing the molecular mass of PEGylated FGF21^G71C (39310 Da). (D) MALDI-TOF mass spectrometry of non-PEGylated FGF21^G71C showing the molecular mass of non-PEGylated FGF21^G71C (19312 Da).

Figure 3 shows the results of cell-based functional characterization of PEG-FGF21^G71C: (A) Cellular glucose uptake stimulated by FGF21 , FGF21 and PEG-FGF21 on 3T3-L1 adipocytes, as measured using the Wallac 1450 MicroBeta counter (Perkin Elmer). * p< 0.05 vs. vehicle control; n=3. (B) The levels of p-AKT were examined using western blotting analyses with GAPDH as the loading control. * p< 0.05 vs. vehicle control; n=3.

Figure 4 shows the results of thermostability and pharmacokinetic study of PEG-FGF21^G71C in vitro and in vivo: (A) Thermal stability of FGF21^G71C and PEGylated FGF21^G71C incubated in mouse serum at 37 °C for the indicated times. Subsequently, the serum-incubated proteins were added onto 3T3-L1 adipocytes for which the glucose uptake was measured to determine the functional integrity of each FGF21 variants. (B) Normal male SD rats were injected intravenously with 0.5 mg/kg FGF21^WT, FGF21^G71C and PEG-FGF21^G71C. Blood samples were collected at the indicated time points. The amount of FGF21 was measured using the human FGF21 immunoassay ELISA Kit. A standard curve was made for each FGF21, n = 5. Values were expressed as the mean + SD. (C). Comparison of half-lives of FGF21^WT, FGF21^G71C and PEG-FGF21^G71C. * p< 0.05, ** p< 0.01 vs. the corresponding FGF21 group; n=3. (D) After treatment, PEG-FGF21 was distributed in various tissues. Figure 5 shows the results of anti-diabetic effects of PEG-FGF21^G71C in ob/ob mice. Nine-week-old ob/ob mice were SC administered with PEGylated FGF21^G71C, FGF21^G71C and FGF21^WT (0.5 mg/kg) once daily for 7 days. All FGF21 variants significantly lowered plasma glucose and triglyceride levels (A and B), and reduced body weight (C). Remarkably, after the cessation of the 7-day treatment, the plasma glucose and triglyceride levels remained at significantly lower levels in mice treated with PEG-FGF21^G71C (A and B). (D) Changes in the fat droplet intensity from the liver sections of ob/ob mice on day 7 after treatment cessation with FGF21^WT, FGF21^G71C or PEG-FGF21^G71C, scale bar is ΙΟΟμηι. (E) PEG-FGF21^G71C improves insulin signaling in the livers of ob/ob mice. Cleared tissue lysate phospho-AKT analysis was performed using western blotting analyses as described in the Material and Methods section. * p< 0.05 vs. respective vehicle control, ** p< 0.05 vs. the corresponding FGF21^WT group, ^# p< 0.05 between indicated groups; n = 6.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "a" or "an" means one or more unless specifically indicated otherwise. The term "isolated" molecule refers to a molecule substantially free from any other contaminating molecules or other contaminants that are found in its natural environment that would interfere with its use in production or its therapeutic, diagnostic, prophylactic or research use. So generally the isolated polypeptide, fusion polypeptide, and/or conjugate of the invention is a purified one with at least 80 % pure, preferably at least 90 % pure, more preferably at least 95 % pure, and particularly with pharmaceutical purity, i.e. at least 98 % pure, and pathogen-free and pyrogen-free. Preferably, the isolated polypeptide, fusion polypeptide, and/or conjugate of the invention may substantially comprise no other polypeptides, especially those derived from animals.

The term "FGF21" is the abbreviation of fibroblast growth factor 21, preferably human fibroblast growth factor 21. The full-length FGF21 consists of 209 amino acid residues shown in SEQ ID NO: 1, in which the 181-amino acid mature FGF21 follows 28-amino acid leader peptide located at the amino-terminus of the full-length FGF21.

Although the mature FGF21 is often used for the numbering of positions of FGF21 in prior art, yet for the purpose of academic preciseness, the full-length FGF21 is used herein for the numbering. The numbering as used herein is easily transformed to the numbering based on the mature FGF21. For example, position 59 or 71 used herein is equivalent to position 31 or 43 of the mature FGF21 respectively.

All symbols of the polypeptides, amino acids, and amino acid residues used herein are well known in the art. The abbreviations of amino acids or amino acid residues are defined in Table 1, and these abbreviations may represent L-amino acids, or may represent D-amino acids, preferably represent L-amino acids. The symbols of the mutations used herein are also well known in the art. For example, the mutation Gly71Cys or G71C refers to the substitution of a cysteine residue for the glycine residue at position 71.

Table 1 the Abbreviations of Amino Acids

Amino Acid One Letter Code Three Letter Code Amino Acid One Letter Code Three Letter Code alanine Ala A leucine Leu L

arginine Arg R lysine Lys K

asparagine Asn N methionine Met M

aspartic acid Asp D phenylalanine Phe F

cysteine Cys C proline Pro P glutamine Gin Q serine Ser S glutamic acid Glu E threonine Thr T

glycine Gly G tryptophan Trp w

histidine His H tyrosine Tyr Y

isoleucine He I valine Val V

The term "nucleic acid" as used herein refers to a single stranded or double stranded polymer of deoxyribonucleotides or ribonucleotides, with the sequence reading from 5' end to 3' end, including RNA and DNA. It may be prepared by isolation from the natural source, in vitro synthesis, or recombinant expression.

The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding nucleic acid to a host cell.

The term "expression vector" as used herein refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present. At present, many expression vectors, e.g. pET series, are commercially available.

The term "host cell" is used to refer to a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing a selected gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present.

The term "pharmaceutically acceptable carrier" as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of an FGF21 mutant.

The terms "therapeutically effective amount" refers to the amount of an FGF21 mutant used to support an observable level of one or more biological activities of the wild-type FGF21, such as the ability to lower blood glucose, insulin, triglyceride, or cholesterol levels; reduce body weight; or improve glucose tolerance, energy expenditure, or insulin sensitivity. In the present invention, a "patient" is preferably a human, but can also be an animal, preferably a mammal such as dog, pig, cow, horse, rat, mouse, guinea pig, and the like.

"Obesity" in terms of the human subject, can be defined as that body weight over 20% above the ideal body weight for a given population (R. H. Williams. Text-book of Endocrinology, 1974, 904-906).

"Diabetes" is marked by excessive discharge of urine and persistent thirst, including two types. Type II diabetes is characterized by excess glucose production in spite of the availability of insulin, and circulating glucose levels remain excessively high as a result of inadequate glucose clearance.

The term "insulin-resistance" refers to a state in which normal amount of insulin produces a subnormal biologic response.

The term "hyperinsulinemia" is defined as a higher-than-normal level of insulin in the blood.

The term "glucose-intolerance" as used herein is defined as exceptional sensitivity to glucose.

The term "hyperglycemia" is defined as an excess of sugar (glucose) in the blood.

In one aspect, the present invention provides an isolated polypeptide, which is an FGF21 mutant and comprises only one amino acid substitution for the amino acid residue at position 59 or 71. The isolated polypeptide maintains one or more biological activities of the wild-type FGF21.

In the aspect, only one amino acid (especially one naturally occurring amino acid) substitution is introduced. In a preferable embodiment, the FGF21 mutant refers to an FGF21 polypeptide in which a wild-type FGF21 amino acid sequence (e.g., a full-length FGF21 having an amino acid sequence of SEQ ID NO: 1, or a mature FGF21 having amino acids 29-209 of SEQ ID NO: 1) has been modified by only one amino acid substitution at position 59 or 71.

The preferable substitutions A59C and G71C found by the inventors are non-conservative. Generally the cysteine residue belongs to the class of residues having neutral hydrophilic side chain, while the alanine or glycine residue respectively belongs to the class of residues having hydrophobic side chain or having no side chain. Although the mutation A59C decreases the activity of wild-type FGF21, yet FGF21^A59C was more stable in vivo than FGF21^WT and the in vitro activity of FGF21^A59C was higher than most of FGF21 mutants taught in WO2010/042747. Thus, the mutant is still beneficial. The FGF21 mutant can be prepared as described in Example 1. A person skilled in the art, familiar with standard molecular biology techniques, can employ that knowledge, coupled with the instant disclosure, to prepare the FGF21 mutant of the present invention. Standard techniques, including but not limiting to site-directed mutagenesis, recombinant DNA, oligonucleotide synthesis, transformation (e.g., electroporation or lipofection), cell and/or tissue culture, and protein isolation and the like, can be used (Sambrook et al. Molecular Cloning: A Laboratory Manual).

In another aspect, the present invention provides a fusion polypeptide comprising the polypeptide of the first aspect and one or more (preferably one) heterologous polypeptides covalently linked thereto.

The fusion polypeptide can be used for pharmaceutical purposes. For example, the FGF21 mutant of the invention can be fused to a heterologous polypeptide that can enhance properties such as an increased half-life. The heterologous polypeptide may be human albumin, bovine albumin, or Fc portion of IgG molecule. Albumin can be genetically coupled the FGF21 mutant of the invention to prolong its half-life. And human albumin is the most prevalent naturally occurring blood protein in the human circulatory system, persisting in circulation in the body for over 20 days. Research has shown that therapeutic proteins genetically fused to human albumin have longer half-lives. And other research has shown that the resultant fusion protein fused with Fc portion may have an increased circulating half-life (US patents US5750375A, US5843725A, and US6291646; Barouch et al. Journal of Immunology, 61:1875-1882; Barouch et al. Proc. Natl. Acad. Sci. USA, 97(8):4192-4197; and Kim et al. Transplant Proc, 30(8):4031-4036).

Preferably, the heterologous polypeptide is used for the preparation, isolation, and/or purification of the FGF21 mutant of the invention. In one embodiment, the heterologous polypeptide is SUMO, preferably His-tagged SUMO. His-tagged protein can be purified by Ni-NTA affinity chromatography, and SUMO is useful for enhancing expression and secretion levels of the SUMO-fused polypeptide in a host cell (especially a prokaryotic cell) and easily cleaved from the polypeptide.

The heterologous polypeptide can be covalently linked to the N- or C-terminus of the FGF21 mutant. In the embodiment, the His-tagged SUMO is covalently linked to the N-terminus of the FGF21 mutant.

The fusion polypeptide can be prepared as described in Example 1. A person skilled in the art, familiar with standard molecular biology techniques, can employ that knowledge, coupled with the instant disclosure, to prepare the fusion polypeptide of the present invention. Standard techniques, including but not limiting to recombinant DNA, polymerase chain reaction (PCR), transformation (e.g., electroporation or lipofection), cell and/or tissue culture, and protein isolation and the like, can be used (Sambrook et al. Molecular Cloning: A Laboratory Manual).

In another aspect, the present invention provides a conjugate comprising the polypeptide of the first aspect and one or more (preferably one) polymers covalently linked thereto.

Exemplary polymers each can be of any molecular weight and can be branched or unbranched. The polymers each typically have an average molecular weight of between about 2 kDa to about 100 kDa (the term "about" indicating that in preparations of a water-soluble polymer, some molecules will weigh more and some less than the stated molecular weight). The average molecular weight of each polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 10 kDa and about 40 kDa, and most preferably between about 10 kDa and about 30 kDa, for example lOkDa, 20kDa, or 30kDa.

Suitable water-soluble polymers or mixtures thereof include, but are not limited to, carbohydrates, polyethylene glycol (PEG) (including the forms of PEG that have been used to derivatize proteins, including mono-(Ci-Cio), alkoxy-, or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran (such as low molecular weight dextran of, for example, about 6 kD), cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol. Also encompassed by the present invention are bifunctional crosslinking molecules that can be used to prepare covalently attached the FGF21 mutant multimers.

In some embodiments, the FGF21 mutant is covalently linked to one or more water-soluble polymers, including, but not limited to, polyethylene glycol (PEG), polyoxyethylene glycol, or polypropylene glycol (US patents US4640835A, US4496689A, US4301144A, US4670417A, US4791192A and US4179337A).

In some embodiments, the FGF21 mutant comprises one or more polymers, including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose, another carbohydrate -based polymer, poly-(N- vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, or mixtures of such polymers.

PEG is most preferably useful for the conjugation in the invention. The PEG groups may generally be attached to the FGF21 mutant via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, NHS, or maleimide, vinylsulfone, alkylhalide) to a reactive group on the FGF21 mutant (e.g., an amino or thiol group). PEGylation reactions are described, for example, in the following references: Zalipsky. Bioconjugate Chemistry, 6: 150-165; European patents EP0154316 and EP0401384; US patent US4179337, and the like. For example, when the target residue is a lysine residue (i.e., a residue with a reactive amine group), PEGylation can be carried out via an acylation reaction or an alkylation reaction with an amino-reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer). For the acylation reactions, a selected polymer can have a single reactive ester group. For reductive alkylation, a selected polymer can have a single reactive aldehyde group. A reactive aldehyde is, for example, polyethylene glycol propionaldehyde, which is water stable, or mono CI -CIO alkoxy or aryloxy derivatives thereof (US patent US5252714).

In the aspect, the polymer (e.g., PEG) is preferably covalently linked to a cysteine residue of the polypeptide. When the target residue is a cysteine residue (i.e., a residue with a reactive sulfhydryl group), PEGylation can be carried out via standard maleimide chemistry. For this reaction, the selected polymer can contain one or more reactive maleimide groups or other thiol reactive moiety such as vinylsulfone, orthopyridyl-disulphide or iodoacetamide (Pasut & Veronese. Adv. Polym. Sci., 192:95-134; Zalipsky. Bioconjugate Chemistry, 6: 150-165; and Hermanson, Bioconjugate Techniques, 2nd Ed., Academic Press, 2008).

In a preferable embodiment, the conjugate is a mono-PEGylated conjugate consisting of an isolated polypeptide, which is an FGF21 mutant and comprises only one amino acid substitution G71C, and a PEG moiety covalently linked to a cysteine residue of the polypeptide. The cysteine residue is preferably one at position 71.

Preferably, the fusion polypeptide with SUMO (especially with His-tagged SUMO) is used for the preparation of the conjugate. The SUMO moiety of the fusion polypeptide is useful for enhancing expression and secretion levels of polypeptide the in a host cell (especially a prokaryotic cell) and easily cleaved by SUMO protease. His-tag is useful for the purification via Ni-NTA affinity chromatography. In one embodiment, a process of producing the conjugate comprises PEGylating the fusion polypeptide with SUMO in solid phase (e.g., in Ni-NTA affinity chromatography column), digesting the SUMO moiety and isolating the conjugate. More preferably, the conjugate can be prepared as described in Example 2. Under the optimized conditions, fewer reagents are consumed and the desired product-to-side product ratio is increased.

In another aspect, the present invention provides a pharmaceutical composition comprising the FGF21 mutant, the fusion polypeptide and/or the conjugate thereof. Generally, the pharmaceutical composition further comprises a pharmaceutically acceptable formulation agent.

A pharmaceutically acceptable formulation agent is preferably nontoxic to recipients at the dosages and concentrations employed.

The pharmaceutical composition can contain one or more pharmaceutically acceptable formulation agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation agents include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides -preferably sodium or potassium chloride - or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, A.R. Gennaro, ed., Mack Publishing Company).

The primary vehicle or carrier in the pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water or physiological saline solution. In addition, the FGF21 mutant and conjugate thereof can be formulated as a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical composition of the invention can be administered by any routes well known to a person skilled in the art, for example, oral, rectal, sublingual, intrapulmonary, transdermal, iontophoretic, vaginal, and intranasal administration. Preferably, the pharmaceutical composition of the invention is administered by parenteral routes, such as subcutaneous, intramuscular or intravenous injection.

The pharmaceutical composition for parenteral administration in this invention can be in the form of a pyrogen-free, parenterally acceptable, and aqueous solution comprising the FGF21 mutant and/or conjugate thereof in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the FGF21 mutant and/or conjugate thereof is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

The dosage to be administered will vary depending upon the formulation, the desired time-course and the patient to be treated, and a physician can readily determine the feasible dosage in the therapy based on the practical situation (e.g., the condition of the patient, body weight and so on). For a general adult, the dosage of the pharmaceutical composition of the invention may be lng - lOmg of the FGF21 mutant or conjugate thereof per kg body weight of the adult. For the route of injection, the dosage preferably is lOng - lmg per kg body weight, more preferably lOOng - lOC^g per kg body weight, further more preferably ^g - lC^g per kg body weight, e.g., 2μg, 5μg, 7μg, or 8μg.

In another aspect, the present invention provides an antibody or fragment thereof that specifically binds to the polypeptide of the first aspect. The antibody or fragment thereof does not bind to wild-type FGF21.

The antibody can be polyclonal, including monospecific polyclonal; monoclonal (mAb); recombinant; chimeric; humanized, such as complementarity-determining region (CDR)-grafted; human; single chain; and/or bispecific; as well as fragments; or chemically modified antibody thereof. Examples of antibody fragments include Fab, F(ab'), or Fv fragments.

The antibody or fragment thereof can be used for the detection or quantitation of the FGF21 mutant. The antibody or fragment thereof of the invention can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Sola. CRC Press, Inc., 1987. Monoclonal Antibodies: A Manual of Techniques, 147-158).

The publications cited in the application are used to illustrate the invention, the contents of which are incorporated herein by reference, as if they have been written down herein.

For a better understanding of the invention, it will now be described in greater detail by reference to specific Examples. It should be noted that the examples only exemplify the invention, and should not be construed as limiting the scope of the invention. According to the description of the application, various modifications and alterations of the invention are obvious to a skilled person in the art.

EXAMPLES

The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and should not be construed as limiting the scope of the invention in any way. Materials used in the Examples are commercially available. For example, the PCR purification kit, gel extraction kit, bicinchoninic acid (BCA) kit, QuikChange site-directed mutagenesis kit, plasmid miniprep kit, Pyrobest^® DNA Polymerase and restriction enzymes were purchased from TaKaRa Company (Japan); 20kDa PEG-Maleimides (mPEG-MAL), Isopropyl-l-thio-P-d-galactopyranoside (JPTG) were purchased from Sigma-Aldrich (St. Louis, MO, USA); the Ni-NTA resin column and Q-Sepharose FF column, and AKTA purifier were purchased from GE Healthcare (Piscataway, NJ, USA). Dulbecco's modified Eagle medium (DMEM) was purchased from Invitrogen (Carlsbad, CA).

Example 1. Expression and characterization of FGF21, mutants, and intermediate fusion polypeptides thereof

According to the manuscript of QuikChange site-directed mutagenesis kit, Ala59Cys (FGF21^A59C, GCC→TGC), Gly71Cys (FGF21^G71C, GGC→TGC), and Ala59Cys/Gly71Cys

(FGF21 A59C ' G71 C ) mutations were respectively introduced into a construct of FGF21 ^VT which is the mature FGF21 having amino acids 29-209 of SEQ JO NO: 1.

Essentially according to the construction method of SUMO-FGF21 described in Chinese patent CN101250547A, SUMO-FGF21^WT construct, SUMO-FGF21^A59C construct, SUMO-FGF21^A59C construct, and SUM0-FGF21^{A59C G71C} construct were respectively constructed, and the intermediate fusion polypeptides His-tagged SUMO-FGF21^WT, His-tagged SUMO-FGF21^A59C, His-tagged SUMO-FGF21^A59C, and His-tagged SUM0-FGF21^{A59C G71C}, as well as the final polypeptides FGF21^WT, FGF21^A59C, FGF21^G71C, and FGF21^{A59C G71C} were purified. The amino acids of His-tagged SUMO-FGF21^WT is shown in SEQ ID NO: 3, which is the same as SEQ ID NO: 2 in CN101250547A. The His-tagged SUMO-FGF21^WT is encoded by the nucleotide sequence of SEQ ID NO: 2, which is the same as SEQ JD NO: 1 in CN101250547A.

To evaluate the effect of the mutation on the in vitro activity of FGF21, we tested the mutants in glucose uptake assays in 3T3-L1 adipocytes. Briefly, 3T3-L1 adipocytes were serum- starved overnight, and then stimulated with different concentrations of FGF21^WT, FGF21^A59C, FGF21^G71C, or FGF21^{A59C G71C} for 24 hours, and then washed twice with KRP buffer (15 mM HEPES, pH 7.4, 118 mM NaCl, 4.8 mM KC1, 1.2 mM MgS0₄, 1.3 mM CaCl₂, 1.2 mM KH₂PO₄, and 0.1% BSA), and 100 μΐ. of KRP buffer containing 2-deoxy-D-[¹⁴C]glucose (2-DOG) (0.1 μθϊ, 100 μΜ) was added to each well. Furthermore, the control wells contained 100 μΐ. of KRP buffer with 2-DOG (0.1 μθ, 100 μΜ) to monitor for nonspecificity. The uptake reaction was performed at 37 °C for 1 h, terminated by the addition of cytochalasin B (20 μΜ), and measured using the Wallac 1450 MicroBeta counter (Perkin Elmer, Waltham, MA, USA).

The in vivo half -life of the polypeptides were analyzed by intravenously (i.v.) injecting a single dose of 0.5 mg/kg of FGF21^WT, FGF21^A59C, FGF21^G71C, or FGF21^{A59C G71C} in Male Sprague Dawley (SD) adult rats (220-250 g), and measurements of the dynamic levels of proteins in blood using the human FGF21 immunoassay ELISA kit (R&D, MN, USA). The pharmacokinetic parameters of the test proteins were determined using the Drug and Statistics Software (DAS, v 2.0; Mathematical Pharmacology Professional Committee of China). The elimination half-life (ti/₂) was calculated using the formula, t = 0.693/K_e ( _e stands for elimination rate constant).

The results are shown in Figure 1. The in vitro activity of FGF21^G71C was similar to that of FGF21^WT, and FGF21^G71C was far more stable in vivo than FGF21^WT. Thus, we selected the FGF21^G71C variant for subsequent experiments.

Although the in vitro activity of FGF21^A59C was lower than that of FGF21^WT, yet FGF21^A59C was more stable in vivo than FGF21^WT and the in vitro activity of FGF21^A59C was higher than most of FGF21 mutants taught in WO2010/042747. Thus, the mutant is worthy of further research.

Both of the in vitro activity and in vivo stability of FGF21^{A59C G71C} taught by the PCT applications WO2005/061712 and WO2006/028595 are lower than those of FGF21^A59C.

Example 2. PEGylation, digestion, and purification of FGF21 mutant

For the production of PEGylated SUM0-FGF21^G71C, we adopted solid-phase PEGylation for the cysteine residue site-specific modification of SUM0-FGF21^G71C. Briefly, the process was performed: (1) 2 mL of 0.1 mM His-tagged SUM0-FGF21^G71C in HEPES buffer (20 mM HEPES, pH 7.5) was applied and bound on Ni-NTA affinity chromatography, and a solution containing 4.8 mL of 0.5 mM mPEG-MAL was circulated in the column at low flow rate (0.01 niL/min) for specific reaction time at 4 °C; (2) then 20 mL of equilibrium buffer (20 mM HEPES, 25 mM NaCl, pH 7.5) was used to wash the non-reacted mPEG-MAL; (3) the reaction complex was completely eluted by applying 200 mM imidazole in HEPES buffer (pH 7.5) at a rate of 1 mL/min. The eluate was collected and confirmed using 12% SDS-PAGE and stored at -20 °C for subsequent experiments.

To determine the optimal conditions for the site-specific PEGylation of SUM0-FGF21^G71C. the effects of different mPEG-MAL/SUMO-FGF21^G71c molar ratios (ranging from 5/1 to 30/1) and reaction times (ranging from 2 to 12 hours) were examined. Our data showed that the optimized yield of mono -PEGylation was achieved when the reaction was performed at 4 °C for 12 hours using the mPEG-MAL/SUMO-FGF21^G71c molar ratio of 12/1 (Figure 2A). Our SDS-PAGE and scanning densitometry analysis showed, under optimal conditions, 48.9 % of the SUM0-FGF21^G71C was successfully PEGylated.

Next, the pre-purified PEGylated SUM0-FGF21^G71C (PEG-SUM0-FGF21^G71C) was diluted and cleaved by SUMO protease. Briefly, the eluted PEG-SUM0-FGF21^G71C was concentrated and diluted to a concentration of 1 mg/mL. Ten units of SUMO protease were added to the dilution and the mixture was incubated in HEPES buffer (20 mM HEPES, pH 7.5) for 1 h at 4 °C. Following incubation, the mixture was loaded onto the Q-Sepharose Fast Flow column, The column was further washed with 10 column volumes (CVs) of Tris-HCl buffer (20 mM Tris-HCl, pH 8.0), and then eluted with Tris-HCl buffer containing different concentrations of NaCl. All elution fractions were collected and analyzed using 12% SDS-PAGE. PEG-FGF21^G71C was further concentrated and desalted by ultrafiltration at 4 °C.

As demonstrated in Figure 2B, fractions containing PEG-FGF21^G71C were finally eluted off the Q Sepharose Fast Flow column using 20 mM Tris-HCl (pH 8.0) containing 80 mM NaCl.

To confirm that FGF21^G71C was mono-PEGylated, MALDI-TOF mass spectrometry (MALDI-TOF-MS) was employed by using Applied Biosystems Voyager System DE PRO MALDI-TOF mass spectrometer (Carlsbad, CA, USA). Our data showed that the PEGylated FGF21^G71C had a molecular weight of 39.3 kDa (Figure 2C), which indicated that a single 20 kDa PEG molecule was conjugated to non-PEGylated FGF21^G71C (19.3 kDa, Figure 2D).

Example 3. Cell-based functional characterization of PEGylated FGF21 mutant

We tested FGF21^WT, FGF21^G71c and PEG-FGF21^G71C in glucose uptake assays in 3T3-L1 adipocytes as mentioned in Example 1.

Furthermore, to compare insulin signaling using phospho-AKT as a marker, 3T3-L1 adipocytes were starved for 12 h, stimulated with FGF21^WT, FGF21^G71c and PEG-FGF21^G71C (100 μg /ml) for 15 min, and then lysed for Western blotting analysis. Briefly, the lysates were separated using 10% SDS-PAGE and electrotransferred onto a nitrocellulose membrane. Each membrane was pre-incubated for 1 h at room temperature in Tris-buffered saline, pH 7.6, containing 0.05% Tween 20 and 5% non-fat milk. Each nitrocellulose membrane was incubated with phospho-Akt (Santa Cruz, sc-7985, 1:500) and GAPDH (Santa Cruz, 1:5,000). The immunoreactive bands were then detected by incubating with IgG-HRP secondary antibody (Santa Cruz, sc-2004, 1:300) conjugated with horseradish peroxidase and visualizing using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA). The amount of the proteins were then analyzed using Image J analysis software version 1.38e (NIH, Bethesda, MD, USA) and normalized against their respective control.

The results are shown in Figure 3. Glucose uptake assays showed that the in vitro biological activity of FGF21 GT 1 C and PEGylated FGF21 GT 1 C were essentially consistent with FGF21^WT (Figure 3A). Western blotting analysis of the activation of cellular pathways also supported the fact that most of FGF21-induded signaling was preserved by FGF21^G71C and PEGylated FGF21^G71C (Figure 3B).

Example 4. Thermostability and pharmacokinetic study of PEGylated FGF21 mutant

To determine the effect of site-directed mutation and PEGylation on the thermal stability of FGF21 at a physiologically relevant temperature, FGF21^WT, FGF21^G71C, and PEG-FGF21^G71C were incubated at a concentration of 0.01 mM at 37 °C in mouse serum at specific time periods. The samples were then subjected to the glucose uptake assay as mentioned in Example 1.

The in vivo half-life of non-PEGylated and PEGylated FGF21^G71C were analyzed by intravenously (i.v.) injecting a single dose of 0.5 mg/kg of FGF21 ^VT , FGF21 GT 1 C , or PEG-FGF21^G71C in Male Sprague Dawley (SD) adult rats (220-250 g) as mentioned in Example 1. Furthermore, PEG-FGF21^G71C in various tissues were also quantified using the human FGF21 immunoassay ELISA Kit.

The results are shown in Figure 4. The capacity of stimulating glucose uptake was reduced for all proteins after incubation with serum in a time-dependent manner. After 120 hours of incubation, FGF21^WT and FGF21^G71C retained 34.3% and 45.1% of the original cellular bioactivity, respectively, while PEG-FGF21^G71C preserved 60.6% (Figure 4A). Thus both of the mutation and PEGylation can increase the thermostability of FGF21 while preserving the bioactivity.

The mutation G71C prolonged the in vivo half-life of FGF21 to 59.8 min compared with the 23.7 min half-life of FGF21^WT while both of the mutation and PEGylation increased the half-life of FGF21 to 211.3 min, which is nearly 9-fold higher compared to FGF21^WT (Figure 4B and 4C), and is also 9-fold higher compared to LY2405319, a FGF21 mutant currently in clinical trials (A. Kharitonenkov, et al. Rational design of a fibroblast growth factor 21 -based clinical candidate, LY2405319. PLoS One, 8 (2013) e58575). The PEGylated form of the FGF21 mutant was also more prone to accumulate in target tissues such as the liver, pancreas, and subcutaneous fat (Figure 4D), which are known to co-express the principal receptor (FGFRlc) and coreceptor (β-klotho) of FGF21(H. Kurosu, et al. Tissue-specific expression of betaKlotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF 19 and FGF21. J Biol Chem, 282 (2007) 26687-26695; S. Ito, et al. Molecular cloning and expression analyses of mouse betaklotho, which encodes a novel Klotho family protein. Mech Dev, 98 (2000) 115-119).

Example 5. Anti-diabetic effects of PEGylated FGF21 mutant in ob/ob mice

Adult (aged 11-12 weeks) obese Lep^ob/ob C57BL/6 (ob/ob) mice and normal control C57BL/6 mice (aged 8-12 weeks) were purchased from the Model Animal Research Center of Nanjing University, China. All mice were housed in a temperature-controlled environment with a 12 h light/dark cycle, had free access to water, and were fed with a standard chow diet containing 60% carbohydrate, 13% fat and 27% protein on a caloric basis. The animal care and experiments were performed according to the Guide for the Care and Use of Laboratory Animals provided by U.S. National Institutes of Health and was approved by the Animal Care and Use Committee of Wenzhou Medical University, China. The ob/ob mice were randomly divided into four groups (n=6): where three groups of mice were treated with 0.5 mg/kg FGF21^WT, FGF21^G71C, or PEG-FGF21^G71C and one group was treated with 0.9% physiological saline and served as the negative sham. In addition, normal control C57BL/6 mice treated with 0.9% physiological saline (n=6).

The animals were subcutaneously injected with FGF21^WT, FGF21^G71C, or PEG-FGF21^G71C at a dose of 20 nmol/kg once daily for 7 days. The glucose, body weight, and food consumption were monitored after the commencement of treatment. On days 3 and 7 of treatment, the animals were tail bled (by tail snip) 1 h after injection for the examination of plasma glucose and triglyceride levels. In addition, the long-lasting anti-diabetic effects of PEG-FGF21^G71C were compared by examining the plasma glucose and triglyceride levels in ob/ob mice at 3 and 7 days after cessation of the 7-day treatment. The glucose and plasma triglyceride levels were measured using the Precision G Blood Glucose Testing System (Abbott Laboratories, Abbott Park, IL, USA) and the Hitachi 912 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, IN, USA), respectively.

At 7 days after cessation of the 7 -day treatment, the livers of male ob/ob mice was collected and lysed for Western blotting analysis as mentioned in Example 3.

Additionally, the livers were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections (5 mm) were stained with haematoxylin and eosin (H&E). Liver tissue staining was performed as previously described (Y. Hotta, et al. Fibroblast growth factor 21 regulates lipolysis in white adipose tissue but is not required for ketogenesis and triglyceride clearance in liver, Endocrinology, 150 (2009) 4625-4633). To estimate the extent of damage, the specimen was observed under a light microscope (400x amplification; Nikon). For immunofluorescence, 5 mm liver sections were treated with 3% Η₂0₂ for 10 min and with 1% BSA in PBS for 30 min. The slides were incubated overnight at 4°C with anti-CD68 antibody (Santa Cruz, sc-9139, 1:100) then incubated with IgG-PE secondary antibody (Santa Cruz, sc-3745, 1:100) for 2 h at room temperature. Next, the cell nuclei were stained with Hoechst for 5 min, and the images were viewed using a fluorescence microscope (400x amplification; Nikon).

The results are shown in Figure 5. the treatment of FGF21 ^VT , FGF21 G^UT' 1 C , and PEG-FGF21^G71C results in glucose and triglyceride-lowering activity. Especially at day 3 after treatment, the treatment of FGF21 GT 1 C , and PEG-FGF21 GT 1 C significantly lowered more blood glucose and triglyceride levels than that of FGF21^WT (Figure 5A and 5B). Surprisingly, after cessation of the 7-day treatment, the plasma glucose and triglyceride levels gradually returned to approximately vehicle treatment levels, although FGF21 GT 1 C and PEG-FGF21 GT 1 C afforded a better glucose-lowering effect compared to FGF21 . The plasma glucose and triglyceride levels remained at significantly lower levels in ob/ob mice treated with PEG-FGF21^G71C, even after 14 days post-treatment.

Throughout the treatment period, ob/ob mice treated with the FGF21, FGF21^G71C and PEG-FGF21^G71C showed reduction of body weight (Figure 5C).

To demonstrate that the metabolic effects of FGF21 occurred in an insulin- sensitive state, we profiled phosphorylated AKT levels in the liver, a major metabolic tissue. As shown in Figure 5E, AKT signaling was not only present, but was increased when treated with FGF21, _FGF21G7ic _and p_EG._FGF2i^G71c, especially PEG-FGF21^G71C.

At day 7 after the cessation of 7-day treatment, histological examination of liver sections obtained from vehicle-treated ob/ob mice showed the extensive existence of micro- and macro-vesicular hepatocyte vacuolation; in contrast, hepatocellular vacuolation was significantly reduced in the liver sections of ob/ob mice treated with FGF21, FGF21^G71C and PEG-FGF21^G71C, especially PEG-FGF21^G71C even 7 days post-treatment cessation (Figure 5D).

Claims

What is claimed is:

1. An isolated polypeptide, which is an FGF21 mutant and comprises only one amino acid substitution for the amino acid residue at position 59 or 71 (preferably the glycine residue at position 71).

2. The polypeptide of claim 1, wherein the FGF21 is the full-length FGF21 having an amino acid sequence of SEQ ID NO: 1, or the FGF21 is the mature FGF21 having amino acids 29-209 of SEQ ID NO: 1.

3. The polypeptide of claim 1, wherein the amino acid substitution is A59C or G71C (preferably G71C).

4 An isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of any one of claims 1-3.

5. A vector comprising the nucleic acid of claim 4.

6. A host cell comprising the vector of claim 5.

7. A process of producing the polypeptide of any one of claims 1-3 comprising culturing the host cell of claim 6 under suitable conditions to express the polypeptide, and optionally isolating the polypeptide.

8. A fusion polypeptide comprising the polypeptide of any one of claims 1-3 and one or more (preferably one) heterologous polypeptides covalently linked thereto.

9. The fusion polypeptide of claim 8, wherein the heterologous polypeptide is SUMO, preferably His-tagged SUMO.

10. The fusion polypeptide of claim 8, wherein the heterologous polypeptide is covalently linked to the N-terminus of the polypeptide of any one of claims 1-3.

11. An isolated nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of any one of claims 8-10.

12. A vector comprising the nucleic acid of claim 11.

13. A host cell comprising the vector of claim 12.

14. A process of producing the fusion polypeptide of any one of claims 8-10 comprising culturing the host cell of claim 13 under suitable conditions to express the fusion polypeptide, and optionally isolating the fusion polypeptide.

15. A conjugate comprising the polypeptide of any one of claims 1-3 and one or more (preferably one) polymers covalently linked thereto.

16. The conjugate of claim 15, wherein the polymer is a water-soluble polymer.

17. The conjugate of claim 16, wherein the water-soluble polymer is polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols, or polyvinyl alcohol.

18. The conjugate of claim 17, wherein the water-soluble polymer is PEG.

19. The conjugate of claim 15, wherein the polymer is covalently linked to a cysteine residue of the polypeptide.

20. A mono-PEGylated conjugate consisting of an isolated polypeptide, which is an FGF21 mutant and comprises only one amino acid substitution G71C, and a PEG moiety covalently linked to a cysteine residue of the polypeptide.

21. A process of producing the conjugate of any one of claims 15-20 comprising the steps of PEGylation of the fusion polypeptide of claim 9 in solid phase, digestion of the SUMO moiety, and isolation of the conjugate.

22. A pharmaceutical composition comprising the polypeptide of any one of claims 1-3 or the conjugate of any one of claims 15-20.

23. The pharmaceutical composition of claim 22, further comprising a pharmaceutically acceptable formulation agent.

24. The pharmaceutical composition of claim 23, wherein the pharmaceutically acceptable formulation agent is a carrier, adjuvant, solubilizer, stabilizer, and/or anti-oxidant.

25. A use of the polypeptide of any one of claims 1-3 or the conjugate of any one of claims 15-20 in the preparation of medicaments for treating a metabolic disorder.

26. The use of claim 25, wherein the metabolic disorder is obesity, diabetes (preferably type II diabetes), insulin-resistance, hyperinsulinemia, glucose-intolerance or hyperglycemia.

27. A method for treating a metabolic disorder comprising administering a therapeutically effective amount of the polypeptide of any one of claims 1-3, the conjugate of any one of claims 15-20, or the pharmaceutical composition of any one of claims 22-24 to a patient in need thereof.

28. The method of claim 27, wherein the metabolic disorder is obesity, diabetes (preferably type II diabetes), insulin-resistance, hyperinsulinemia, glucose-intolerance or hyperglycemia.

29. An antibody or fragment thereof that specifically binds to the polypeptide of any one of claims 1-3.