MXPA06006616A - Muteins of fibroblast growth factor 21 - Google Patents

Abstract

The present invention relates to novel muteins of human fibroblast growth factor 21 with improved pharmaceutical properties. Both protein and the respective encoding nucleic acid species are disclosed. The invention also embodies vectors and host cells for the propagation of said nucleic acid sequences and the production of said muteins. Also disclosed are methods for treating type 2 diabetes, obesity, metabolic syndrome, and in reducing the mortality and morbidity of critically ill patients.

Description

MUTEINS OF THE FIBROBLASTO GROWTH FACTOR 21 BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to the identification of new fibroblast growth factor muteins 21 having improved pharmaceutical properties.

DESCRIPTION OF THE RELATED ART The fibroblast growth factors are large polypeptides widely expressed in adult and developing tissues (Baird et al., Cancer Cells, 3: 239-243, 1 991) and play crucial roles in the multiple physiological functions that they include angiogenesis, mitogenesis, pattern formation, cell differentiation, metabolic regulation and repair of tissue injury (McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 59: 135-176, 1998). According to published literature, the FGF family now consists of at least twenty-three elements, FGF-1 through FGF-23 (Reuss et al., Cell Tissue Res. 313: 139-157 (2003).) The fibroblast growth factor 21 (FGF-21) has been reported to be preferentially expressed in the liver (Nishimura et al., Biochimica et Biophysica Acta, 1492: 203-206, (2000), WO01 / 36640, and WO01 / 18172) and is described as a treatment for ischemic vascular disease, wound healing and diseases associated with loss of lung, bronchial or alveolar cellular function and numerous other disorders.Most recently, FGF-21 has been shown to stimulate glucose uptake in 3T3-L1 adipocytes of mice in the presence and absence of insulin, and by decreasing blood glucose, triglycerides and glucagon levels in fasting and feeding, in ob / ob and db / db mice and ZDF rats of 8 weeks of age in a dose-dependent manner, like this, provide the basis for the use of FGF-21 as a therapy to treat diabetes and obesity (WO03 / 01121 3). In addition, FGF-21 has been shown to be effective in reducing the mortality and morbidity of critically ill patients (WO03 / 059270). A significant challenge in the development of protein pharmacists, such as FGF-21, is covered with their physical and chemical instabilities. The compositional variety and protein characteristics define specific behaviors such as folding, conformational stability and unfolding / denaturation. Such characteristics should be directed to stabilize proteins when developing pharmaceutical formulation conditions using aqueous protein solutions (Wang, W., Int.J. of Pharmaceutics, 1 8, (1999) .Specifically, in the development of protein Pharmaceutical, anti-microbial preservatives such as phenol, m-cresol, methylparaben, resorcinol and benzyl alcohol, are necessary in parenteral pharmaceutical formulations which are proposed to be a sterile, multi-use formulation.

Unfortunately, these compounds often adversely affect the stability of the protein product, in particular, the association of activation and aggregation (Maa et al., J. of Pharmaceutics 140: 155-168 (1996); Lam et al., Pharm. Res. 14 (6): 725-729 (1997)). The FGF-21 will probably be used as a sterile, multipurpose pharmaceutical formulation. However, it has been determined that condoms, ie, / 77-creso, have an adverse effect on their stability under these conditions. Clearly, there is a need to develop a stable aqueous protein formulation for the therapeutic FGF-21 protein. The present invention overcomes the significant barrier of physical instabilities with the invention of FGF-21 muteins that are more stable than native-type FGF-21 under pharmaceutical formulation conditions. Thus, the FGF-21 muteins of the present invention provide pharmacologically stable protein formulations, which are useful for the treatment of type 2 diabetes, obesity, metabolic syndrome, and in the reduction of mortality and morbidity of critically ill patients.

BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides muteins of human fibroblast growth factor 21, or a biologically active peptide thereof, which comprises substitution with a chd and / or polar amino acid but dischd by one or more of the following: glycine 42, glutamine 54, nine 77, alanine 81, leucine 86, phenylalanine 88, plant 122, histidine 125, nine 126, proline 130, nine 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. A second aspect of the present invention provides muteins of human fibroblast growth factor 21, or a biologically active peptide thereof, which comprises the replacement of a cysteine by two or more of the following: nine 19, tyrosine 20, leucine 21, tyrosine 22, threonine 23, aspartate 24, aspartate 25, alanine 26, glutamine 27, lutamine 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine 41, glycine 42, glycine 43, glutamate 50, glutamine 54, leucine 58 , valine 62, leucine 66, glycine 67, lysine 69, nine 72, phenylalanine 73, glutamine 76, nine 77, aspartate 79, glycine 80, alanine 81, leucine 82, glycine 84, serine 85, proline 90, alanine 92, serine 94, phenylalanine 95, leucine 100, aspartate 102, tyrosine 104, tyrosine 107, serine 109, glutamate 1 10, proline 1 15, histidine 1 17, leucine 1 1 8, proline 1 19, asparagine 121, lysine 122, serine 123, proline 124, histidine 125, nine 126, aspartate 127, alanine 129, proline 1 30, glycine 132, alanine 134, nine 135, leucine 1 37, proline 138 or leucine 139, wherein the numbering of amino acids is based on SEC I D NO: 1. A third aspect of the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, which comprises substitution with any chd and / or polar amino acid but dischd at any of the amino acid positions indicated in the first embodiment of the present invention, in combination with the substitution of a cysteine at two or more amino acid positions indicated in the second embodiment of the invention. Other embodiments are directed to polynucleotides encoding the muteins of the first, second and third embodiments, a vector containing said polynucleotides and a host cell carrying said vector. Another embodiment is directed to processes for producing a polypeptide, to produce cells capable of producing said polypeptide and to produce a vector containing DNA encoding said polypeptide. Yet another embodiment is directed to methods of treating a patient having one or more of obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome, which comprises administering said patient in need of such treatment. A therapeutically effective amount of a human FGF-21 mutein of the first, second or third embodiment or a pharmaceutical composition thereof.

DETAILED DESCRIPTION OF THE INVENTION For purposes of the present invention, as described and claimed herein, the following terms are as defined below.

FGF-21 is an amino acid polypeptide 208 that contains a 27 amino acid leader sequence. Human FGF-21 has -79% amino acid identity to mouse FGF-21 and ~ 80% amino acid identity to FGF-21 of rat. Human FGF-21 is the preferred polypeptide template for the muteins of the present invention, but it is recognized that one skilled in the art can easily make muteins based on an alternative polypeptide sequence of mammalian FGF-21. The amino acid positions of the muteins of the present invention are determined from the FGF-21 polypeptide of mature human amino acid 181 as shown below (SEQ ID NO: 1 10 - 20 His Pro lie Pro Asp Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr '30 40 Leu Tyr Thr Asp Asp Wing Gki Gln Thr Glu Wing His Leu Glu He Arg Glu Asp Gly Thr 50 60 Val Gly Gly Wing Wing Asp Gln Ser Pro Glu Be Leu Leu Gln Leu Lys Wing Leu Lys Pro 70 80 Gly Val He Gln lie Leu Gly Val Lys Thr Ser Axg Phe Leu Cys Gln Arg Pro Asp Gly 90 100 Wing Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Wing Cys Ser Phe Arg Glu Leu Leu Leu 110 120 Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Wing His Gly Leu Pro Leu His Leu Pro Gly 130 140 Asn Lys Ser Pro His Arg Asp Pro Wing Pro Arg Gly Pro Wing Arg Phe Leu Pro Leu Pro 150 160 Gly Leu Pro Pro Wing Leu Pro Glu Pro Pro Gly He Leu Wing Pro Gln Pro Pro Asp Val 170. 180 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Allah Ser The corresponding DNA sequence coding for the human 81 amino acid FGF-21 polypeptide is (SEQ ID NO: 2).

CACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCA GCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATC AGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTC CTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGA CATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCAC TTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAA TGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCÁCCTGCCAGGGAACAAG TCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACC AGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCC CCCGATGTGGGCTCCTCGGCCCCTGAGCATGGTGGGACCTTCCCAGGGCCG AAGCCCCAGCTACGCTTCC One skilled in the art of protein expression will recognize that the methionine or methionine-arginine sequence can be introduced into the N-terminus of the mature sequence (SEQ ID NO: 1) for expression in E. coli and are contemplated within the context of this invention. The amino acids are identified using the three-letter code or alternatively, they can be designated using the standard one-letter code. Mutations are designated by the three letter code for the original amino acid, followed by the amino acid number, followed by the three letter code for amino acid replacement. The numerical designations of each mutein are based on the amino acid sequence 181 of the human, native, mature FGF-21. For example, a substitution for leucine at position 139 (ie Leu 139), with the negatively charged amino acid, glutamate (Glu), is designated as Leu139Glu or L139E. In a similar fashion, the double substitution for isoleucine at position 152 and serine at position 163 (Ile52, Ser163) with the negatively charged amino acid, glutamate (Glu), is designated as He152Glu / Ser163Glu, I 1 52E / S163E or I 152E-S163E. A human FGF-21 mutein is defined as comprising human FGF-21 in which at least one amino acid of the native-type mature protein has been replaced by another amino acid. Generally speaking, a mutein possesses some modified property, structural or functional, of the native type protein. For example, the mutein may have enhanced or enhanced physical stability in concentrated solutions (eg, less hydrophobic mediated aggregation), while maintaining a favorable bioactivity profile. The mutein may possess increased compatibility with pharmaceutical preservatives (eg, / rj-cresol, phenol, benzyl alcohol), thus enabling the preparation of a preserved pharmaceutical formulation that maintains the physicochemical properties and biological activity of the protein during storage. Accordingly, muteins with improved pharmaceutical stability when compared to native-type FGF-21, have improved physical stability in concentrated solutions under both physiological and preserved pharmaceutical formulation conditions, while maintaining biological potency. As used herein, these terms are not limiting, it is entirely possible that a given mutein has one or more modified properties of the native-type protein. A "biologically active peptide", is defined as a peptide of a mutein of the present invention that maintains the modified properties (s) and biological potency of the mutein. A "therapeutically effective amount" is the minimum amount of an active agent necessary to impart therapeutic benefit to a patient. For example, a "therapeutically effective amount" to a patient who presents, suffers or is prone to suffer or to prevent suffering from type 2 diabetes, obesity or metabolic syndrome, is such amount which induces, improves or otherwise causes a improvement in pathological symptoms, progression of the disease, associated physiological conditions, or resistance to succumbing to the aforementioned disorders. For the purposes of the present invention, a "subject" or "patient" is preferably a human, but may also be an animal, more specifically, a companion animal (eg, dogs, cats and the like), farm animals ( for example, cows, sheep, pigs, horses and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). "Type 2 diabetes" is characterized by excess glucose production despite the availability of insulin, and circulating glucose levels remain excessively high, as a result of inadequate glucose separation. "Glucose intolerance" can be defined as an exceptional sensitivity to glucose. "Hyperglycemia" is defined as an excess of sugar (glucose) in the blood. "Hypoglycemia," also called low blood sugar, occurs when the blood glucose level drops too low to provide energy for bodily activities. "Hyperinsulinemia" is defined as a level higher than the normal level of insulin in the blood. "Insulin Resistance" is defined as a state in which a normal amount of insulin produces a subnormal biological response. "Obesity", in terms of the human subject, can be defined as the body weight over 20 percent above the ideal body weight for a given population (R. H. Williams, Texbook of Endocrinology, 1974, p.904-916). "Metabolic syndrome" can be defined as a grouping of at least three of the following signs: abdominal fat - in most men, up to 101.6 cm (40 inches) waist or greater; high blood sugar - at least 1 10 milligrams per deciliter (mg / dl) after fasting; elevated triglycerides - at least 150 mg / dl in the bloodstream; Low HDL-less than 40 mg / dL; and, blood pressure of 1 30/85 or higher. Critically ill patients encompassed by the present invention generally experience an unstable hypermetabolic state. This unstable metabolic state is due to changes in substrate metabolism, which can lead to relative deficiencies in some nutrients. Generally there is an increased oxidation of both fat and muscle. However, critically ill patients are preferably patients who experience systemic inflammatory response syndrome or respiratory distress. A reduction in morbidity means reducing the likelihood that a critically ill patient will develop additional diseases, conditions or symptoms, or reduce the severity of diseases, conditions, or additional symptoms. For example, reducing morbidity may correspond to a decrease in the incidence of bacteremia or sepsis or complications associated with multiple organ failure. "Systemic inflammatory response syndrome (SIRS)" as used herein, describes an inflammatory process associated with a broad number of clinical conditions and includes, but is not limited to, more than one of the following clinical manifestations: (1) a body temperature greater than 38 ° C or less than 36 ° C; (2) a heart rate greater than 90 beats per minute; (3) tachypnea, manifested by a heart rate greater than 20 breaths per minute, or hyperventilation, as indicated by a PaCO2 less than 32 mm Hg; and (4) an alteration in the white blood cell count, such as a count greater than 12,000 / cu mm, a count less than 4,000 / cu mm, or the presence of more than 10% of immature neutrophils. These physiological changes should represent an acute alteration of the baseline in the absence of other known causes for such abnormalities, such as chemotherapy, neutropenia and induced leukopenia. "Sepsis" as used herein, is defined as an origin of SIRS infection. Non-infectious pathogens causing SIRS may include pancreatitis, ischemia, multiple trauma and tissue injury, i.e., crushing injuries or severe burns, haemorrhagic stroke, immune mediated organ injury, and exogenous administration of such putative mediators of inflammatory processes as the factor necrosis of the tumor and other cytokines. Septic apoplexy and multiple organ dysfunction are the main contributors to morbidity and mortality in ICU facilities. Sepsis is associated with and mediated by the activation of a number of host defense mechanisms that include the cytokine network, leukocytes and complement cascade, and coagulation / fibrinolysis systems that include the endothelium. Disseminated intravascular coagulation (DIC) and other degrees of consumption coagulopathy associated with fibrin deposition within the microvasculature of various organs are manifestations of sepsis / septic apoplexy. The downstream effects of host defense response on target organs are an important mediator in the development of multiple organ dysfunction syndrome (MODS) and contribute to poor prognosis of patients with sepsis, severe sepsis and sepsis complicated by stroke. "Respiratory distress" as used herein, denotes a condition in which patients have difficulty breathing due to some types of pulmonary dysfunction. Often these patients have varying degrees of hypoxemia that may or may not be refractory to treatment with supplemental oxygen. Respiratory distress may occur in patients with impaired lung function due to direct lung injury or it may occur due to indirect lung injury, such as in the establishment of a systemic process. further, the presence of multiple predisposition disorders, substantially increases the risk, due to the presence of secondary factors such as chronic alcohol abuse, chronic lung disease and low serum pH. Some causes of direct lung injury include pneumonia, aspiration of gastric contents, lung contusion, fat embolism, near drowning by submersion, inhalation injury, pulmonary reperfusion edema and elevated altitude after lung transplantation or pulmonary embolectomy. Some cases of indirect lung injury include sepsis, severe stroke trauma and multiple transfusions, cardiopulmonary bypass, drug overdose, acute pancreatitis, and transfusions of blood products. A class of lung disorders that cause respiratory distress are associated with the syndrome known as the pulmonary heart. These disorders are associated with chronic hypoxemia that results in elevated pressure within the pulmonary circulation called pulmonary hypertension. The supervening pulmonary hypertension increases the workload of the right ventricle, thus leading to its widening or hypertrophy. The pulmonary heart usually presents as right heart failure defined by a sustained increase in right ventricular pressure and clinical evidence of reduced venous return to the right heart. "Chronic obstructive pulmonary diseases" (COPD), which include emphysema and chronic bronchitis, also cause respiratory distress and are characterized by obstruction to airflow.

COPD is the fourth leading cause of death and demands more than 100,000 lives annually. "Acute Respiratory Distress Syndrome" (ARDS) is generally progressive and is characterized by different stages. The syndrome is usually manifested by the rapid onset of respiratory failure in a patient with a risk factor for the condition. Arterial hypoxemia that is refractory to treatment with complementary oxygen is a characteristic feature. There may be alveolar filling, consolidation and atelectasis that occurs in dependent areas of the lung; however, non-dependent areas may have substantial inflammation. The syndrome may progress to fibroid alveolitis with persistent hypoxemia, increased alveolar dead space, and an additional decrease in lung compliance. Pulmonary hypertension, which results from damage to the pulmonary capillary bed, can also develop. The first preferred aspect of the invention comprises muteins of human FGF-21 in which the substitution means that any charged and / or polar but uncharged amino acid replaces at least one of the following: glycine 42, glutamine 54, arginine 77, alanine 81 , leucine 86, phenylalanine 88, lysine 122, histidine 125, arginine 126, proline 1 30, arginine 131, leucine 1 39, alanine 145, leucine 146, isoleucine 152, alanine 154, glutamine 156, glycine 161, serine 163, glycine 170 or serine 172, wherein the numbering of amino acids is based on SEQ ID NO: 1. A charged amino acid is defined as a positively or negatively charged amino acid. A positively charged amino acid is defined to include histidine, lysine, arginine and analogs thereof that do not originate naturally (e.g., gamma-aminobutyric acid, ornithine, etc.). A negatively charged amino acid is defined as including aspartate, glutamate and analogs thereof that do not originate naturally (eg, aminoadipic acid). A polar but discharged amino acid is defined as including serine, threonine, asparagine, glutamine and analogs thereof that do not originate naturally. The most preferred muteins of the first embodiment are Gln54Glu, Leu139Glu, Ala145Glu, Leu146Glu, lle152Glu, Gln 156Glu, Ser163Glu and He152Glu-Ser163Glu. The second aspect of the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, which comprises the replacement of a cysteine by two or more of the following: arginine 19, tyrosine 20, leucine 21, tyrosine 22, threonine 23, aspartate 24, aspartate 25, alanine 26, glutamine 27, lutamine 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine 41, glycine 42, glycine 43, glutamate 50, glutamine 54, leucine 58, valine 62 , leucine 66, glycine 67, lysine 69, arginine 72, phenylalanine 73, glutamine 76, arginine 77, aspartate 79, glycine 80, alanine 81, leucine 82, glycine 84, serine 85, proline 90, alanine 92, serine 94, phenylalanine 95, leucine 1 00, aspartate 102, tyrosine 104, tyrosine 1 07, serine 109, glutamate 1 10, proline 1 15, histidine 1 17, leucine 1 1 8, proline 1 19, asparagine 121, lysine 122, serine 123, proline 124, histidine 125, arginine 126, aspartate 127, alanine 129, proline 130, glycine 132, alanine 134, arginine 135, leucine 137, proline 138 or leucine 139, where the numbering of the amino acid is based on SEC I D NO: 1. One skilled in the art will also recognize that the native cysteines, cysteine 75 and cysteine 93, may be used as loci to introduce a new disulfide bond that can impart improved properties. Specifically contemplated is the introduction of a substitution of cysteine to serine 85 or phenylalanine 73, coupled with a concomitant change to either cysteine 93 or cysteine 75, respectively, where the latter sites are replaced with any other amino acid. Disulfide bonds that originates naturally, as provided by cysteine residues, generally increase the thermodynamic stability of proteins. Successful examples of increased thermodynamic stability, as measured in the increase in the melting temperature, are multiple mutants linked to disulfides of the lysozyme T4 enzymes (Matsumura, et al., PNAS 86: 6562-6566 (1988)) and barnase (Johnson et al., Mol. Biol. 268: 198-208 (1997)). One aspect of the present invention is the premise that by restricting the loop flexibility of amino acid 1 1 8- 134 of FGF-21 by disulfide bonds, the physical stability of FGF-21 is improved in the presence of a condom, presumably by limiting the access of the condom to the hydrophobic nucleus of the protein. The FGF-21 muteins with engineered disulfide bonds, in addition to those that naturally originate from Cys75-Cys93, are as follows: Gln76Cys-Ser109Cys, Cys75-Ser85Cys, Cys75-Ala92Cys, Phe73Cys-Cys93, Ser123Cys- His125-Cys, Asp102Cys-Tyr104Cys, Asp127Cys-Gly132Cys, Ser94Cys-Glu1 10Cys, Pro1 15Cys-His1 17Cys, Asn121 Cys-Asp127Cys, Leu100Cys-Asp102Cys, Phe95Cys-Tyr107Cys, Arg19Cys-Pro138Cys, Tyr20Cys-Leu139Cys, Tyr22Cys-Leu137Cys, Arg77Cys-Asp79Cys, Pro90Cys-Ala92Cys, Glu50Cys-Lys69Cys, Thr23Cys-Asp25Cys, Ala31 Cys-Gly43Cys, Gln28Cys-Gly43Cys, Thr23Cys-Gln28Cys, Val41 Cys-Leu82Cys, Leu58Cys-Val62Cys, Gln54Cys-Leu66Cys, Ile35Cys-Gly67Cys, Gly67Cys-Arg72Cys, Me35Cys-Gly84Cys, Arg72Cys-Gly84Cys, or Arg77Cys-Ala81 Cys, wherein the numbering of the amino acids is based on SEQ ID NO: 1. Preferred muteins with engineered disulfide bonds are Tyr22Cys-Leu139Cys; Asp24Cys-Arg135Cys; Leu1 18Cys-Gly132Cys; His1 17Cys-Pro130Cys; His1 17Cys-Ala129Cys; Leu82Cys-Pro1 19Cys; GlydOCys-Ala129Cys; Gly43Cys-Pro124Cys; Gly42Cys-Arg126Cys; Gly42Cys-Pro124Cys; Gln28Cys-Pro124Cys; Gln27Cys-Ser123Cys; Ala26Cys-Lys122Cys; or Asp25Cys-Lys122Cys. Most preferred muteins with engineered disulfide bonds are Leu1 18Cys-Ala134Cys; Leu21 Cys-Leu33Cys; Ala26Cys-Lys122Cys; Leu21 Cys-Leu33Cys / Leu1 18Cys-Ala134Cys. The third aspect of the present invention provides muteins of human FGF-21, or a biologically active peptide thereof, which comprises a substitution of any charged and / or polar amino acid but discharged at any of the amino acid positions indicated in the first embodiment of the present invention, combined with the substitution of a cysteine at two or more amino acid positions indicated in the second embodiment of the invention. It is well known in the art that a significant challenge in the development of protein pharmaceutics is ideal with the physical and chemical instabilities of proteins. It is even more apparent when a pharmaceutical formulation of protein is proposed to be of multiple use, the injectable formulation requires a stable, concentrated and preserved solution, while maintaining a favorable bioactivity profile. The detailed biophysical characterization of native-type FGF-21 establishes that a concentrated protein solution (> 5 mg / ml), when exposed to stress conditions, such as elevated temperature or low pH, leads to an accelerated association and aggregation (ie, physical stability and poor biopharmaceutical properties). Exposure of a concentrated protein solution of FGF-21 to pharmacological preservatives (ie, m-cresol) also has a negative impact on physical stability. Therefore, one embodiment of the present invention is to improve the physical stability of concentrated solutions, while maintaining biological potency and chemical stability under both physiological and preserved formulation conditions. It is thought that the association and aggregation may result from hydrophobic interactions, since, a given protein concentration, temperature and ionic strength have considerable impact on physical stability. For the most part, target amino acid residues were exposed to presumed, non-conserved surfaces. The local environment of these residues was analyzed and, those that are not considered structurally important were selected by mutagenesis. One method to initiate specific changes is to further decrease the p / of the protein by introducing glutamic acid residues ("glutamic acid scan"). It is assumed that the introduction of charged substitutes could inhibit hydrophobic mediated aggregation via charge-charge repulsion and potentially improve condom compatibility. Furthermore, a person skilled in the art will also recognize that with sufficient degree of mutagenesis, p / may be changed in a basic pH range by the introduction of positive charge with or without concomitant decrease in negative charge, thus allowing repulsion to load -load. Although the embodiments of the present invention relate to physical and chemical stability under both physiological and preserved pharmaceutical formulation conditions, maintaining the biological potency of the muteins compared to the native-type FGF-21 is an important consideration factor as well. Thus, the biological potency of the muteins of the present invention is defined by the ability of the muteins to affect glucose uptake as measured in the 3T3-L1 cell assay in vitro (Example 4) and / or the decrease in glucose levels plasma, as well as plasma triglycerides, as measured in vivo in the ob / ob mice assay (Example 5). The FGF-21 muteins administered in accordance with this invention can be generated and / or isolated by any means known in the art. The most preferred method for producing the mutein is through recombinant DNA methodologies and is well known to those skilled in the art. Such methods are described in Curren Protocols in Molecular Biology (John Wiley & amp; amp;; Sons, Inc.), which is incorporated herein by reference. Additionally, preferred embodiments include a biologically active peptide derived from the mutein described herein. Such a peptide will contain at least one of the described substitutions and the mutein will possess biological activity. The peptide can be produced by any of the means known to those skilled in the art, examples of which include but are not limited to enzymatic digestion, enzymatic synthesis or recombinant DNA methodologies. It is established in the art, that peptide fragments of certain fibroblast growth factors are biologically active. See, for example, Baird et al., Proc. Nati Acad. Sci (USA) 85: 2324-2328 (1988), and J. Cell. Phys. Suppl. 5: 1 01 -106 (1987). Therefore, the selection of mutein fragments or peptides is based on the criteria known in the art. For example, dipeptidyl peptidase IV (DPP-IV) is known to be a protease of the serine type involved in the inactivation of neuropeptides, endocrine peptides and cytokines (Damme et al., Chem. Immunol., 72: 42-56, (1999)). ). The N-terminus of FGF-21 (HisProllePro), contains two dipeptides that could potentially be subtracted from DPP-IV, resulting in a fragment of FGF-21 truncated at the N-terminus by 4 amino acids. Unexpectedly, this native FGF-21 fragment has been shown to retain biological activity (Table 1), thus, muteins of the present invention, truncated to the N-terminus up to 4 amino acids, are an embodiment of the present invention. invention. The present invention also encompasses polynucleotides that encode the muteins described above, which may be in the form of RNA or in the form of DNA, in which, the DNA includes cDNA, genomic DNA and synthetic DNA. DNA can be double-stranded or single-stranded. The coding sequences encoding muteins of the present invention may vary as a result of the redundancy or degeneracy of the genetic code. The polynucleotides encoding muteins of the present invention may include the following: only the coding sequence for the mutein, the coding sequence for the mutein and additional coding sequence such as a functional polypeptide, or a leader or secretory sequence or a pro sequence -proteins; the coding sequence for the mutein and the non-coding sequence, such as introns or non-coding sequences 5 'and / or 3' of the coding sequence for the mutein. Thus, the term "polynucleotide encoding a mutein" encompasses a polynucleotide, which includes additional coding and / or non-coding sequences. The present invention further relates to variants of the described polynucleotides that encode fragments, analogs and derivatives of the polypeptide that contain the indicated substitutions. The variant of the polynucleotide can be an allelic variant that naturally originates from the human FGF-21 sequence, a variant "that does not originate naturally, or a truncated variant as described above." Thus, the present invention also includes polynucleotides that encode the muteins described above, as well as variants of such polynucleotides, such variants encode a fragment, derivative or analogue of the described mutein Such nucleotide variants include deletion variants, substitution variants, truncated variants and addition or insertion variants , as soon as at least one of the indicated amino acid substitutions of the first or second embodiment is present.The polynucleotides of the present invention will be expressed in hosts after the sequences have been operably linked (i.e., positioned to ensure friction) to an exp control sequence These expression vectors are typically replicable in host organisms either as episomers or as an integral part of the chromosomal DNA of the host. Commonly, the expression vectors will contain selection markers, for example, tetracycline, neomycin, and reductase dihydrofolate, to allow the detection of those cells transformed with the desired DNA sequences. The mutein of FGF-21 can be expressed in mammalian, insect, yeast, bacterial or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from DNA constructs of the present invention. E. coli is a prokaryotic host used particularly to clone the polynucleotides of the present invention. Other microbial hosts suitable for use include Bacillus subtilus, Salmonella typhimurium and several species of Serratia, Pseudomonas, Streptococcus and Staphylococcus, although others may also be used as the material of choice. In these prokaryotic hosts, expression vectors can also be made, which typically contain the expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of well-known promoters can be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a lambda or T7 phage promoter system. Promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, to initiate and complete transcription and translation. One skilled in the art of protein expression will recognize that the methionine or methionine-arginine sequence can be introduced into the N-terminus of the mature sequence (SEQ ID NO: 1) for expression in E. coli and are contemplated within of the context of this invention. Thus, unless stated otherwise, the muteins of the present invention expressed in E. coli have a methionine sequence introduced into the N-terminus. Other microbes, such as yeast or fungi, can also be used for expression. Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia angustia are examples of preferred yeast hosts, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired. Aspergillus niger, Trichoderma reesei; and Schizophyllum commune, are examples of fungi hosts, although others may also be used as a matter of choice. The culture of mammalian tissue cells can also be used to express and produce the polypeptides of the present invention. Eukaryotic cells are currently preferred, because a number of strains of suitable host cells capable of secreting intact muteins have been developed in the art, and include strains of CHO cells, various strains of COS cells, NSO cells, cell lines. of Syrian Hamster Ovary, HeLa cells or strains of human embryonic kidney cells (ie, HEK293, HEK293EBNA). Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer and necessary processing information sites, such as ribosome binding sites, RNA splice sites, sites of polyadenylation and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from SV40, adenovirus, bovine papilloma virus, cytomegalovirus, Raus sarcoma virus and the like. Preferred polyadenylation sites include sequences derived from SV40 and bovine growth hormone. Vectors containing the polynucleotide sequences of interest (eg, the FGF-21 muteins and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly used by prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts. Various methods of protein purification can be employed and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 1 82: 83-9 (1990) and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, NY (1 982). The selected purification step (s) will depend, for example, on the nature of the production process used for the FGF-21 muteins. The compositions containing mutein of FGF-21 should be formulated and dosed in a manner consistent with good medical practice, taking into account the clinical condition of the patient, the site of delivery of the mutein composition of FGF-21, the method of administration , the administration program and other factors known to practitioners. The "therapeutically effective amount" of the FGF-21 mutein for purposes herein is thus determined by such considerations. The pharmaceutical compositions of the FGF-21 muteins and of the present invention can be administered by any of the means that achieve the proposed purpose in general: to treat type 2 diabetes, obesity, metabolic syndrome, or critically ill patients. The term "parenteral" as used herein, refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intramuscular, subcutaneous and intraarticular injection and infusion. The dosage administered will be dependent on the age, health and weight of the recipient, type of concurrent treatment if any, frequency of treatment, and the nature of the desired effect. Compositions within the scope of the invention include all compositions wherein a FGF-21 mutein is present in an amount that is effective to achieve the desired medical effect for the treatment of type 2 diabetes, obesity or metabolic syndrome. While individual needs may vary from one patient to another, the determination of optimal ranges of effective amounts of all components is within the capacity of the ordinary skill specialist. The FGF-21 muteins of the present invention can be formulated in accordance with known methods to prepare pharmaceutically useful compositions. A desired formulation may be one that is a stable lyophilized product that is reconstituted with an appropriate diluent or a high purity aqueous solution with optional pharmaceutically acceptable carriers, preservatives, excipients or stabilizers, [Remington's Pharmaceutical Sciences 16th. Edition (1980)]. The muteins of the present invention can be combined with a pharmaceutically stable buffer, and the pH adjusted to provide acceptable stability, and an acceptable pH for administration. For parenteral administration in one embodiment, the muteins of FGF-21 are formulated in a general manner by mixing one or more of these to the desired degree of purity, in an injectable unit dosage form (solution, suspension or emulsion), with a pharmaceutically acceptable carrier. , that is, one that is not toxic to containers in the dosages and concentrations used and is compatible with other ingredients of the formulation. Preferably, one or more pharmaceutically acceptable anti-microbial agents can be added. Phenol, m-cresol and benzyl alcohol are preferred pharmaceutically acceptable anti-microbial agents.

Optionally, one or more pharmaceutically acceptable salts can be added to adjust the ionic strength or tonicity. One or more excipients may be added to further adjust the isotonicity of the formulation. Glycerin, sodium chloride, and mannitol are examples of an excipient that adjusts isotonicity. Those skilled in the art can readily optimize pharmaceutically effective dosages and administration regimens for therapeutic compositions comprising a mutein of FGF-21, as determined by good medical practice and the clinical condition of the individual patient. A typical dose range for the FGF-21 muteins of the present invention will vary from about 0.01 mg per day to about 1000 mg per day for an adult. Preferably, the dosages range from about 0.1 mg per day, to about 100 mg per day, more preferably from about 1.0 mg / day to about 10 mg / day. More preferably, the dosage is about 1-5 mg / kg. The appropriate dose of an administered FGF-21 mutein, will result in decreased blood glucose levels and increased energy expenditure by faster and more efficient glucose utilization, and thus, is useful in treating type diabetes 2, obesity and metabolic syndrome. In addition, because hyperglycemia and insulin resistance are common in critically ill patients giving nutritional support, some ICUs administer insulin to treat excessive hyperglycemia in the diet of critically ill patients. Indeed, recent studies document the use of exogenous insulin to maintain blood glucose at a level not higher than 10 mg per deciliter, due to morbidity and reduced mortality among critically ill patients in the surgical intensive care unit, with respect to if they have a history of diabetes (Van den Berghe, et al., N. Engl J Med., 345 (19): 1359 (2001)). Thus, muteins of FGF-21 of the present invention are uniquely suited to help restore metabolic stability in critically ill patients metabolically unstable. The muteins of FGF-21 are unique in that they stimulate glucose absorption and improve insulin sensitivity, but do not induce hypoglycemia. In another aspect of the present invention, muteins of FGF-21 are contemplated for use as a medicament for the treatment of type 2 diabetes, obesity, metabolic syndrome, or critically ill patients. Having now described the present invention in detail, it will now be more clearly understood by reference to the following examples, which are included together with this for purposes of illustration only and are not intended to limit the invention. All patents and publications referred to in this document are expressly incorporated by reference.

EXAMPLE 1 Expression and Purification of Muteins from FGF-21 in E. coli The bacterial expression vector pET30a was used for bacterial expression in this example (Novagen, Inc., Madison, Wisconsin)). The resistance gene to the antibiotic kanamycin encodes pET30a and contains a bacterial origin of replication ("ori"), a strong inducible phage-lPTG T7 promoter, a ribosome binding site ("RBS"), and suitable MCS with a number of unique restriction endonuclease cleavage sites. Conveniently for purification purposes, the vector can encode His and S ends for N-terminal peptide fusions, as well as a C-terminal His-end function. However, for purposes of the present invention, the FGF-21 variants encoding the cDNA, are inserted between the Ndel and BamHI restriction sites, respectively, and the resulting construct does not take advantage of any of the described ends. The nucleic acid sequence encoding the FGF-21 mutein, which lacks the leader sequence, but is substituted with a methionine residue, is amplified from a cDNA clone using PCR oligonucleotide primers, which strengthen the 5 'ends and 3 'of the open reading structure. Additional nucleotides, which contain recognition sites for restriction enzymes Ndel and BamHI, are added to the 5 'and 3' sequences, respectively. For cloning, primers by 5 'forward and 3' reverse PCR, have nucleotides corresponding to or complementary to a portion of the coding sequence of the nucleic acid encoding the FGF-21 mutein in accordance with methods known in the art. technique. One of ordinary skill in the art will appreciate that the point in a polynucleotide sequence where the primers start can be varied. The amplified nucleic acid fragments and the pET30a vector are digested with Ndel and BamHI restriction enzymes and the purified digested DNA fragments are then ligated together. The insertion of the DNA encoding the mutein of FGF-21 into the restricted pET30a vector, places the coding region of the mutein polypeptide of FGF-21 including its associated arrest codon, downstream of the IPTG inducible promoter and in structure with a codon. ATG of initiation. The associated stop codon, TAG, prevents the translation of the six histidine codons downstream of the insertion point. The ligation mixture is transformed into competent E. coli cells using standard procedures, such as those described in Current Protocols in Molecular Biology (John Wiley &; Sons, Inc.). The transformation reactions are plated on LB / kanamycin plates and after overnight growth, the transformants are drilled for plasmid preparations or subjected to in situ lysis for PCR selection. The positive recombinant plasmids, which contain the desired FGF-21 variant inserts, are identified by restriction analysis, followed by DNA sequence analysis. These plasmids are subsequently used to transform expression strains and protein production. Strains of E. coli BL21 (DE3), BL21 (DE3) STAR, or BL21 (DE3) RP, are used to express muteins of FGF-21. These strains, which are only some of the many that are suitable for expressing FGF-21 muteins, are commercially available from Novagen, I nc., Invitrogen and Stratagen, respectively. Transformants are identified by their ability to grow on LB plates in the presence of kanamycin. Clones containing the desired constructs are grown overnight (o / n) in liquid culture in LB medium supplemented with kanamycin (30 μg / ml). The o / n culture is used to inoculate a large culture, at a dilution of about 1: 25 to 1: 250. The cells are grown at an optical density of 0.6 ("OD600") at 600 nm. Then isopropyl-b-D-thiogalactopyranoside ("IPTG") is added to a final concentration of 1 mM to induce transcription of the lac repressor responsive promoter, inactivating the lacl repressor. The cells are subsequently further incubated for 3 to 12 hours. The cells are then harvested by centrifugation, pellets washed with 50 mM Tris buffer, pH 8.0 and stored at -20 ° C until purification. Muteins of FGF-21 are expressed in the insoluble fraction ie, inclusion bodies (or granules) of E. coli. Although the level of expression may vary from variant to variant, a level typically observed for the native-type protein (Wl) of FGF-21 is 50 mg / l. The subsequent purification process starts with the solubilization of the granules and the withdrawal of the variants followed by four chromatographic steps. To purify the muteins of E. coli FGF-21, the granules are solubilized in 50 mM Tris, pH 9.0, 7 M Urea and 1 mM DTT through a ramp of pH at pH 1 1 .0, at temperature room for 1 hour with agitation. The protein is then captured on a Q-Sepharose column using the same buffer described above, and eluted with a linear gradient of 0-400 mM NaCl. The Q-Sepharose combination is then treated with 10 mM DTT, for two hours at RT, to reduce all disulfide bonds. The combination is then diluted 10 times, so that the concentration of the buffer is as follows: 50 mM Tris, pH 9.0, 7 M Urea, 10 mM Cysteine, 1 mM DTT with a protein concentration of about 250-500 μg / ml. After another two hours of incubation under reducing conditions at RT, to obtain the protein in a disulfide-free form, the combination is then dialyzed in 20 mM glycine, pH 9.0, for approximately 48 hours, so that they can be formed the correct disulfide bonds. Reverse phase HPLC chromatography, on a Vydac C18 column and 0.1% TFA / 0-50% CH3CN as a mobile phase, was used as an initial purification step. This column is used to concentrate the FGF-21 or the muteins of the FGF-21 and remove the contaminating endotoxin. The next purification step is size exclusion chromatography on a Superdex 35/600 column carried out in 1 X PBS buffer, pH 7.4. In this stage, the muteins of FGF-21 are ~ 95% pure. The last step involves MonoQ chromatography in 50 mM Tris, pH 8.0 and elution with a linear gradient of 0-300 mM NaCl, which usually provides protein > 97% pure. The purification scheme of column step 4 described above was used for all the muteins of FGF-21 and stable preparations produced.

Example 2 Expression and Purification of Muteins of FGF-21 in HEK293EBNA Cells Alternatively, muteins of FGF-21 can be produced in a mammalian cell expression system, such as HEK293EBNA cells (EdgeBiosystems, Gaiethersburg, MD). The FGF-21 muteins are subcloned into the proprietary expression vector representing a modification of commercially available pEAK10 between the Nhel and Xbal restriction sites in the MCS. The cDNA sequence encoding the mature FGF-21 is fused in structure with the leader sequence lg? to improve the secretion of the desired product in the tissue culture medium. The expression is activated by the strong viral CMV promoter. HEK293EBNA cells are temporarily transfected using a standard transfection reagent such as Fugene (Roche Diagnostics, Indianapolis, I N) and the appropriate amount of recombinant plasmid, either as a monolayer or suspension culture, at the appropriate cell density. The cells are incubated at 37 ° C and 5% CO2, in serum-free medium, and the collections are elaborated every day for 5 days. Typically, the expression level in the suspension culture HEK293EBNA is -30 mg / L. The expression of human FGF-21 in mammalian cells, provides the natural sequence to the N-terminus of HPIP, ie, without a residue of methionine in the N-terminus. It was discovered that enzymatically treating the FGF-21 of HEK293EBNA cells with DPP-IV (kidney portion, SIGMA St. Louis), resulted in the truncation of the N-terminus by four amino acids. When tested in the mouse 3T3-L1 adipocyte assay (see Example 4), this truncated variant of FGF-21 stimulates glucose uptake to a level comparable to that of native-type FGF-21 (Table 1). ).

EXAMPLE 3 Expression and Purification of Muteins from FGF-21 in Yeast Still another expression system for the production of muteins of FGF-21 is yeast, such as Pichia pastoris, Pichia methanolica or Sccharomyces cerevisiae. For production in Pichia pastoris a commercially available system (Invitrogen, Carlsbad, CA), uses vectors with the potent AOX1 promoters (alcohol oxidase) to drive high level expression of recombinant proteins. Alternatively, vectors using the GAP gene promoter (glyceraldehyde-3-phosphate dehydrogenase), are available for high level constitutive expression. The vectors of expression of Pichia of multiple copies, allow to obtain strains with multiple copies of the gene of interest integrated in the genome. By increasing the number of copies of the gene of interest in a recombinant Pichia strain, the levels of protein expression can be increased. Yet another yeast expression system is Saccharomyces cerevisiae. Expression vectors contain the promoter and GAL1 gene enhancer sequences. The GAL1 promoter is one of the most widely used yeast promoters, due to its strong transcriptional activity on galactose induction. The analytical characterization (mass spectrum analysis), indicates that the FGF-21 expressed in Pichia pastoris, is truncated (up to by removal of four amino acids [HisProllePro] to the N-term, hereinafter designated as des-HPI P). When tested in the 3T3-L1 adipocyte assay (see Example 4), this truncated variant of FGF-21 stimulates glucose uptake at the same level as native-type FGF-21 (Table 1).

Example 4 Absorption of Glucose in 3T3-L1 Adipocytes in Mouse 3T3-L1 Cells are obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells are grown in growth medium (GM) containing 10% fetal bovine serum enriched with 10% iron in Dulbecco's modified Eagle medium.

For standard adipocyte differentiation, two days after the cells reached confluence (referred to as day 0), the cells are exposed to differentiation medium (DM) containing 10% fetal bovine serum, 10 μg / ml of insulin, 1 μM dexamethasone, and 0.5 μM isobutylmethylxanthine, for 48 hours. The cells are then maintained in post-differentiation medium containing 10% fetal bovine serum and 10 μg / ml insulin. Glucose Transport Assay - The hexose uptake, as tested by the accumulation of 0.1 Mm of 2-deoxy-D- [4C] glucose, is measured as follows: 3T3-L1 adipocytes in 12 cavity plates, are washed twice with KRP buffer (136 mM NaCl, 4.7 mM KCI, 1.0 mM NaPO4, 0.9 mM CaCl2, 0.9 mM MgSO4, pH 7.4), heated at 37 ° C and containing 0.2% BSA, incubated in medium Leibovitz L-15 containing 0.2% BSA for 2 hours at 37 ° C in room air, washed again with KRP containing 0.2% BSA buffer, and incubated in KRP, 0.2% BSA buffer in the absence (only Me2SO) or presence of wortmanin for 30 minutes at 37 ° C in ambient air. The insulin is then added to a final concentration of 100 nM for 15 minutes, and the absorption of 2-deoxy-D- [14 C] glucose is measured for at least 4 minutes. Non-specific absorption, measured in the presence of 10 μM cytochalastin B, is subtracted from all values. Protein concentrations are determined with the Pierce bicinchoninic acid assay. Absorption is routinely measured in triplicate or quadrupled for each experiment.

The in vitro potency is normalized to the in vitro activity of native type FGF-21, which is given a designation of 1.0 and used as a positive control. The in vitro potency of the FGF-21 muteins of the present invention is compared to the native type FGF-21 in Table 1. As indicated in Table 1, the muteins of the present invention maintain biological potency at several degrees compared to native-type FGF-21. Table 1 * truncated by 4 amino acids in the N-term, that is, c / es-HPIP. ** Enzymatically truncated by 4-amino acids to the N-terminus by DPP-IV, ie, des-HPIP Example 5 Mouse model Ob / ob A study was made in an obesity model using male ob / ob mice to monitor the levels of plasma glucose and triglyceride levels after treatment with FGF-21, compared to the insulin and vehicle control groups. Test groups of male ob / ob mice (7 weeks old), were injected with vehicle alone (0.9% NaCl), or mutein of FGF-21 (0.125 mg / kg) subcutaneously (0.1 mL, once daily) for seven days. Blood was collected by bleeding by holding the tail on day 7, one hour after the last injection of the compound and plasma glucose levels were measured using a standard protocol. The ability of the FGF-21 muteins to lower plasma glucose levels compared to vehicle control is shown in Table 2. The data in Table 2 indicate that the muteins of the present invention decrease glucose levels of plasma compared with vehicle control. The ability of the muteins of FGF-21 to lower triglyceride levels compared to vehicle control is shown in Table 3. Table 2 Table 3 Example 6 Pharmaceutical Stability of Muteins of FGF-21 The stability of the muteins of FGF-21 of the present invention was analyzed under physiological and pharmaceutical formulation conditions. To simulate physiological conditions, the mutein was analyzed for stability in PBS at room temperature (RT) at a target protein concentration of 10 mg / ml, pH 7.4. The physical stability / solubility of the muteins in PBS is considered satisfactory if recovery of the protein after preparation results in > 90% recovery to RT as determined by reverse phase chromatography and / or size exclusion. The muteins of the present invention indicated in Tables 4 and 5 suggest this criterion. It is anticipated that the pharmaceutical formulation of a mutein of the present invention will probably be a preservative multiple-use formulation, thus, compatibility with a common condom was analyzed. To test the compatibility of the formulation, a condom, m-cresol, (final concentration of 3 mg / mL, a concentration usually sufficient to suggest the criterion of European Pharmacopoeia B, to determine the effectiveness of the condom under neutral pH conditions) , a solution containing the mutein at about 10 mg / ml in PBS, pH 7.4, was added at room temperature. The physical stability in the presence of the condom was initially assessed by determining the recovery of protein from the main chromatographic peak after reverse phase chromatography and size exclusion at RT. In addition, the extent of aggregation measured by DLS (dynamic light scattering) at 37 ° C, is shown as the average particle diameter in the presence of m-cresol after two hours, compared to native-type FGF-21. A larger average diameter corresponds to an association and / or aggregation of protein of increased degree. The preservative compatibility (as an average diameter of particulate function) of the muteins of the first and second embodiments of the present invention, compared to native-type FGF-21, is shown in Table 4. All the muteins were expressed in E. coli. The muteins of the present invention that are stable in PBS and compatible with condoms are designated to have enhanced or improved pharmaceutical properties compared to native-type FGF-21. As shown in Table 4, the preferred muteins of the present invention having the improved pharmaceutical properties compared to native-type FGF-21 are L139E, A145E, L146E, I152E, Q156E, [I152E, S163E], S163E, Q54E , [L21 C-L33C, L1 18C-A134C], L21 C-L33C, A26C-K122C and L1 1 8C-A134C. TABLE 4 * The average particulate diameter represents a protein solution at a target concentration of 10 mg / ml, t? -cresol at 3 mg / ml, after 2 hours of incubation at 37 ° C.

Claims (41)

1. CLAIMS 1. A mutein of human fibroblast growth factor 21 (FGF-21), or a biologically active peptide thereof, characterized in that it comprises the substitution of a charged and / or polar amino acid but discharged by one or more of the following: glycine 42, glutamine 54, arginine 77, alanine 81, leucine 86, phenylalanine 88, lysine 122, histidine 125, arginine 126, 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.
2. 2. The mutein according to claim 1, characterized in that the negatively charged amino acid is selected from the group consisting of aspartate, glutamate and analogs thereof which do not originate naturally.
3. 3. The mutein according to claim 1, characterized in that the polar but discharged amino acid is selected from the group consisting of serine, threonine, asparagine, glutamine and analogs thereof which do not originate naturally.
4. 4. The mutein according to claim 1, characterized in that said mutein is selected from the group consisting of Leu139Glu, Ala145Glu, Leu146Glu, lle1 52Glu, Gln 156Glu, Ser163Glu, Me152Glu, Ser163Glu and Gln54Glu.
5. 5. The mutein according to claim 4, characterized in that said mutein is truncated to the N-terminus by up to 4 amino acids.
6. 6. A polynucleotide, characterized in that it encodes the mutein according to claim 1.
7. 7. The polynucleotide according to claim 6, characterized in that said polynucleotide is DNA.
8. 8. An expression vector, characterized in that it contains the DNA according to claim 7.
9. 9. A host cell, characterized in that it comprises the expression vector according to claim 8.
10. 10. The host cell according to claim 9 , characterized in that said host cell is a yeast cell. eleven .
11. A process for producing a polypeptide, characterized in that it comprises: (a) expressing said polypeptide from the host cell according to claim 10, and; (b) isolating said polypeptide.
12. 12. A pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of the mutein of the FGF-21 according to claim 1, and an acceptable pharmaceutical carrier, wherein said composition is useful for treating a patient having one or more of the indications from the group consisting of obesity, type 2 diabetes, resistance to insulin, hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome.
13. A method for treating a patient, characterized in that it comprises administering to said patient, a therapeutically effective amount of the FGF-21 mutein according to claim 1, wherein said patient has one or more of the indications from the group which consists of obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome.
14. 14. The method according to claim 13, characterized in that said patient has type 2 diabetes.
15. 1 5. A human FGF-21 mutein, or a biologically active peptide thereof, characterized in that it comprises the replacement of one cysteine by two or more of the following: arginine 19, tyrosine 20, leucine 21, tyrosine 22, threonine 23, aspartate 24, aspartate 25, alanine 26, glutamine 27, glutamine 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine 41, glycine 42, glycine 43, glutamate 50, glutamine 54, leucine 58, valine 62, leucine 66, glycine 67, lysine 69, arginine 72, phenylalanine 73, glutamine 76, arginine 77, aspartate 79, glycine 80, alanine 81, leucine 82 , glycine 84, serine 85, proline 90, alanine 92, serine 94, phenylalanine 95, leucine 100, aspartate 102, tyrosine 104, tyrosine 107, serine 109, glutamate 1 10, proline 1 15, histidine 1 17, leucine 1 18, proline 1 19, asparagine 121, lysine 122, serine 123, proline 124, histidine 125, arginine 126, aspartate 127 , alanine 129, proline 130, glycine 132, alanine 134, arginine 135, leucine 137, proline 138 or leucine 139, wherein the numbering of amino acids is based on SEQ ID NO: 1.
16. 16. The mutein according to claim 1, characterized in that said mutein is selected from the group consisting of Leu21 Cys-Leu33Cys / Leu1 18Cys-Ala134Cys, Leu21Cys / Leu33Cys, Leu118Cys / Ala134Cys or Ala26Cys / Lys122Cys.
17. 17. The mutein according to claim 16, characterized in that said mutein is truncated to the N-terminus by up to 4 amino acids.
18. 1 8. The mutein according to claim 17, characterized in that said mutein is c / es-HPIP-Leu1 18Cys / Ala134Cys.
19. 19. A polynucleotide, characterized in that it encodes the mutein according to claim 15.
20. 20. The polynucleotide according to claim 19, characterized in that said polynucleotide is DNA. twenty-one .
21. An expression vector, characterized in that it contains the DNA according to claim 20.
22. 22. A host cell, characterized in that it comprises the vector according to claim 21.
23. 23. The host cell according to claim 22, characterized in that said host cell is a yeast cell.
24. 24. A process for producing a polypeptide, characterized in that it comprises: (a) expressing said polypeptide from the host cell according to claim 23; and (b) isolating said polypeptide.
25. 25. A pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of the FGF-21 mutein according to claim 15, and an acceptable pharmaceutical carrier, wherein said composition is useful for treating a patient having one or more indications to from the group consisting of obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome.
26. 26. A method for treating a patient, characterized in that it comprises administering to said patient, a therapeutically effective amount of the FGF-21 mutein according to claim 15, wherein said patient has one or more of the indications of the group consisting of of obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome.
27. 27. The method according to claim 26, characterized in that said patient has type 2 diabetes.
28. 28. A mutein of human FGF-21, or a biologically active peptide thereof, characterized in that it comprises substitution with a charged amino acid and / or polar but discharged by one or more of the following: glycine 42, glutamine 54, arginine 77, alanine 81, leucine 86, phenylalanine 88, lysine 122, histidine 125, arginine 126, proline 130, arginine 131, leucine 1 39, alanine 145 , leucine 146, isoleucine 152, alanine 154, glutamine 156, glycine 161, serine 163, glycine 170, or serine 172; in combination with the replacement of a cysteine by two or more of the following amino acids at the positions: arginine 19, tyrosine 20, leucine 21, tyrosine 22, threonine 23, aspartate 24, aspartate 25, alanine 26, glutamine 27, glutamine 28, Alanine 31, Leucine 33, Solucine 35, Leucine 37, Valine 41, Glycine 42, Glycine 43, Glutamate 50, Glutamine 54, Leucine 58, Valine 62, Leucine 66, Glycine 67, Lysine 69, Arginine 72, Phenylalanine 73, Glutamine 76, arginine 77, aspartate 79, glycine 80, alanine 81, leucine 82, glycine 84, serine 85, proline 90, alanine 92, serine 94, phenylalanine 95, leucine 100, aspartate 102, tyrosine 104, tyrosine 107, serine 109, glutamate 1 10, proline 1 15, histidine 1 17, leucine 1 18, proline 1 19, asparagine 121, lysine 122, serine 123, proline 124, histidine 125, arginine 126, aspartate 127, alanine 129, proline 130, glycine 132, alanine 134, arginine 135, leucine 137, proline 138 or leucine 139, where the amino acid numbering s e based on SEC I D NO: 1.
29. 29. A polynucleotide characterized in that it encodes the mutein according to claim 28.
30. 30. The polynucleotide according to claim 29, characterized in that said polynucleotide is DNA.
31. 31 An expression vector, characterized in that it contains the DNA according to claim 30.
32. 32. A host cell characterized in that it comprises the expression vector according to claim 31.
33. 33. The host cell according to claim 32, characterized in that said host cell is a yeast cell.
34. 34. A process for producing a polypeptide, characterized in that it comprises: (a) expressing said polypeptide from the host cell according to claim 33; and (b) isolating said polypeptide.
35. 35. A pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of the FGF-21 mutein according to claim 28, and an acceptable pharmaceutical carrier, wherein said composition is useful for treating a patient having one or more indications to from the group consisting of obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome.
36. 36. A method for treating a patient, characterized in that it comprises administering to said patient, a therapeutically effective amount of the mutein of FGF-21 in accordance with claim 28, wherein said patient presents one or more of the indications of the group consisting of obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome.
37. 37. The method according to claim 36, characterized in that said patient has type 2 diabetes.
38. 38. The mutein according to claim 28, characterized in that said mutein is truncated at the N-terminus by up to 4 amino acids.
39. 39. The use of the FGF-21 mutein according to claim 1, for the manufacture of a medicament for the treatment of one or more of the indications of the group consisting of obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome.
40. 40. The use of the FGF-21 mutein according to claim 1, for the manufacture of a medicament for the treatment of one or more of the indications of the group consisting of obesity, type 2 diabetes, insulin resistance , hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome.
41. 41 The use of the FGF-21 mutein according to claim 28, for the manufacture of a medicament for the treatment of one or more of the indications of the group consisting of obesity, type 2 diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia or metabolic syndrome.