U.S. patent application number 12/349119 was filed with the patent office on 2009-05-07 for muteins of fibroblast growth factor 21.
Invention is credited to John Michael Beals, Christopher Carl Frye, Wolfgang Glaesner, Shun Li, Radmila Micanovic, Radhakrishnan Rathnachalam, Jing Shang, Beth Ann Strifler.
Application Number | 20090118190 12/349119 |
Document ID | / |
Family ID | 34710086 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090118190 |
Kind Code |
A1 |
Beals; John Michael ; et
al. |
May 7, 2009 |
MUTEINS OF FIBROBLAST GROWTH FACTOR 21
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.
Inventors: |
Beals; John Michael;
(Indianapolis, IN) ; Frye; Christopher Carl;
(Bargersville, IN) ; Glaesner; Wolfgang;
(Indianapolis, IN) ; Li; Shun; (Carmel, IN)
; Rathnachalam; Radhakrishnan; (Carmel, IN) ;
Shang; Jing; (Fishers, IN) ; Strifler; Beth Ann;
(Brownsburg, IN) ; Micanovic; Radmila;
(Indianapolis, IN) |
Correspondence
Address: |
ELI LILLY & COMPANY
PATENT DIVISION, P.O. BOX 6288
INDIANAPOLIS
IN
46206-6288
US
|
Family ID: |
34710086 |
Appl. No.: |
12/349119 |
Filed: |
January 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10579510 |
May 16, 2006 |
7491697 |
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PCT/US04/37200 |
Dec 1, 2004 |
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12349119 |
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60528582 |
Dec 10, 2003 |
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Current U.S.
Class: |
514/1.1 ;
530/399 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 31/04 20180101; A61P 11/16 20180101; A61P 3/04 20180101; A61P
5/50 20180101; A61K 38/00 20130101; A61P 3/00 20180101; A61P 3/08
20180101; A61P 29/00 20180101; A61P 3/10 20180101; C07K 14/50
20130101 |
Class at
Publication: |
514/12 ;
530/399 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C07K 14/50 20060101 C07K014/50 |
Claims
1-41. (canceled)
42. A mutein of human fibroblast growth factor 21 (FGF-21),
consisting of human FGF-21 wherein a substitution of a charged
and/or polar but uncharged amino acid for one or more amino acids
at the positions from the group consisting of: glutamine 54,
arginine 77, leucine 139, alanine 145, leucine 146, isoleucine 152,
glutamine 156, glycine 161, serine 163, wherein the numbering of
the amino acids is based on SEQ ID NO:1.
43. The mutein of claim 42 wherein the charged amino acid is
selected from the group consisting of aspartate, glutamate, and
non-naturally occurring analogs thereof.
44. The mutein of claim 42 wherein the polar but uncharged amino
acid is selected from the group consisting of serine, threonine,
asparagine, glutamine, and non-naturally occurring analogs
thereof.
45. The mutein of claim 42, wherein said mutein is selected from
the group consisting of Leu139Glu-human FGF-21, Ala145Glu-human
FGF-21, Leu146Glu-human FGF-21, Ile152Glu-human FGF-21,
Gln156Glu-human FGF-21, Ser163Glu-human FGF-21, Ile152Glu-human
FGF-21, Ser163Glu-human FGF-21, Arg77Glu-human FGF-21, and
Gln54Glu-human FGF-21.
46. A pharmaceutical composition comprising a therapeutically
effective amount of a mutein of claim 45 and a pharmaceutically
acceptable carrier.
47. A biologically active peptide of a mutein of human FGF-21
consisting of a human FGF-21 wherein: (a) a substitution of a
charged and/or polar but uncharged amino acid for one or more amino
acids at the positions from the group consisting of: glutamine 54,
arginine 77, leucine 139, alanine 145, leucine 146, isoleucine 152,
glutamine 156, glycine 161, serine 163, wherein the numbering of
the amino acids is based on SEQ ID NO:1; and (b) one, two, three,
or four amino acids are truncated from the N-terminus.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the identification of new
muteins of fibroblast growth factor 21 that have improved
pharmaceutical properties.
[0003] 2. Description of the Related Art
[0004] Fibroblast growth factors are large polypeptides widely
expressed in developing and adult tissues (Baird et al., Cancer
Cells, 3:239-243, 1991) and play crucial roles in multiple
physiological functions including angiogenesis, mitogenesis,
pattern formation, cellular differentiation, metabolic regulation
and repair of tissue injury (McKeehan et al., Prog. Nucleic Acid
Res. Mol. Biol. 59:135-176, 1998). According to the published
literature, the FGF family now consists of at least twenty-three
members, FGF-1 to FGF-23 (Reuss et al., Cell Tissue Res.
313:139-157 (2003).
[0005] 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 described as a treatment for ischemic vascular
disease, wound healing, and diseases associated with loss of
pulmonary, bronchia or alveolar cell function and numerous other
disorders. More recently, FGF-21 has been shown to stimulate
glucose-uptake in mouse 3T3-L1 adipocytes in the presence and
absence of insulin, and to decrease fed and fasting blood glucose,
triglycerides, and glucagon levels in ob/ob and db/db mice and 8
week old ZDF rats in a dose-dependant manner, thus, providing the
basis for the use of FGF-21 as a therapy for treating diabetes and
obesity (WO03/011213). In addition, FGF-21 has been shown to be
effective in reducing the mortality and morbidity of critically ill
patients (WO03/059270).
[0006] A significant challenge in the development of protein
pharmaceuticals, such as FGF-21, is to cope with their physical and
chemical instabilities. The compositional variety and
characteristics of proteins define specific behaviors such as
folding, conformational stability, and unfolding/denaturation. Such
characteristics must be addressed to stabilize proteins when
developing pharmaceutical formulation conditions utilizing aqueous
protein solutions (Wang, W., Int. J. of Pharmaceutics, 18,
(1999).
[0007] Specifically, in pharmaceutical protein development,
anti-microbial preservative agents such as phenol, m-cresol,
methylparaben, resorcinol, and benzyl alcohol are necessary in
parenteral pharmaceutical formulations that are intended to be a
sterile, multi-use formulation. Unfortunately, these compounds
often adversely affect the stability of the protein product,
triggering association and aggregation, in particular (Maa et al.,
Int. J. of Pharmaceutics 140:155-168 (1996); Lam et al., Pharm.
Res. 14(6):725-729 (1997)).
[0008] FGF-21 will likely be utilized as a multi-use, sterile
pharmaceutical formulation. However, it has been determined that
preservatives, i.e. m-cresol, have an adverse affect on its
stability under these conditions. Clearly, there is a need to
develop a stable aqueous protein formulation for the therapeutic
protein FGF-21. The present invention overcomes the significant
hurdles of physical instabilities with the invention of muteins of
FGF-21 that are more stable than wild-type FGF-21 under
pharmaceutical formulation conditions. Thus, the muteins of FGF-21
of the present invention provide stable pharmacological protein
formulations that are useful for the treatment of type 2 diabetes,
obesity, metabolic syndrome, and in reducing the mortality and
morbidity of critically ill patients.
SUMMARY OF THE INVENTION
[0009] In a first aspect, the present invention provides muteins of
human fibroblast growth factor 21, or a biologically active peptide
thereof, comprising the substitution with a charged and/or polar
but uncharged amino acid for 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.
[0010] A second aspect of the present invention provides muteins of
human fibroblast growth factor 21, or a biologically active peptide
thereof, comprising the substitution of a cysteine for 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 100, aspartate
102, tyrosine 104, tyrosine 107, serine 109, glutamate 110, proline
115, histidine 117, leucine 118, proline 119, 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 the amino acids is based on SEQ ID NO:1.
[0011] A third aspect of the present invention provides muteins of
human FGF-21, or a biologically active peptide thereof, comprising
the substitution with any charged and/or polar but uncharged amino
acid 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.
[0012] Other embodiments are drawn 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 drawn to processes to produce a
polypeptide, to produce cells capable of producing said polypeptide
and to produce a vector containing DNA encoding said
polypeptide.
[0013] Yet another embodiment is drawn to methods of treating a
patient exhibiting one or more of obesity, type 2 diabetes, insulin
resistance, hyperinsulinemia, glucose intolerance, hyperglycemia,
or metabolic syndrome comprising administering to 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
[0014] For purposes of the present invention, as disclosed and
claimed herein, the following terms are as defined below.
[0015] FGF-21 is a 208 amino acid polypeptide containing a 27 amino
acid leader sequence. Human FGF-21 has .about.79% amino acid
identity to mouse FGF-21 and .about.80% amino acid identity to rat
FGF-21. Human FGF-21 is the preferred polypeptide template for the
muteins of the present invention but it is recognized that one with
skill in the art could readily make muteins based on an alternative
mammalian FGF-21 polypeptide sequence.
[0016] The amino acid positions of the muteins of the present
invention are determined from the mature human 181 amino acid
FGF-21 polypeptide as shown below (SEQ ID NO:1):
TABLE-US-00001 1 10 20 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln
Phe Gly Gly Gln Val Arg Gln Arg Tyr 30 40 Leu Tyr Thr Asp Asp Ala
Gln Gln Thr Glu Ala His Leu Gln Ile Arg Glu Asp Gly Thr 50 60 Val
Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu
Lys Pro 70 80 Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe
Leu Cys Gln Arg Pro Asp Gly 90 100 Ala Leu Tyr Gly Ser Leu His Phe
Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu 110 120 Glu Asp Gly
Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly
130 140 Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg
Phe Leu Pro Leu Pro 150 160 Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro
Gly Ile Leu Ala 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 Ala Ser
[0017] The corresponding DNA sequence coding for the mature human
181 amino acid FGF-21 polypeptide is (SEQ ID NO:2):
TABLE-US-00002 CACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCG
GCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGG
AGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAA
AGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGG
AGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATG
GATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTT
GAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCA
CCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAG
CTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACTCCCGGAGCCACCC
GGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAG
CATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCTTCC
[0018] One skilled in the art of expression of proteins will
recognize that methionine or methionine-arginine sequence can be
introduced at 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.
[0019] Amino acids are identified using the three-letter code or
alternatively could 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 the replacement amino acid. The numerical
designations of each mutein is based on the 181 amino acid sequence
of mature, wild-type, human FGF-21. For example, a substitution for
leucine at position 139 (i.e. Leu139) 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 (Ile152, Ser163) with the
negatively charged amino acid, glutamate (Glu) is designated as
Ile152Glu/Ser163Glu, I152E/S163E or I152E-S163E.
[0020] A human FGF-21 mutein is defined as comprising human FGF-21
in which at least one amino acid of the wild-type mature protein
has been substituted by another amino acid. Generally speaking, a
mutein possesses some modified property, structural or functional,
of the wild-type protein. For example, the mutein may have enhanced
or improved physical stability in concentrated solutions (e.g.,
less hydrophobic mediated aggregation), while maintaining a
favorable bioactivity profile. The mutein may possess increased
compatibility with pharmaceutical preservatives (e.g., m-cresol,
phenol, benzyl alcohol), thus enabling the preparation of a
preserved pharmaceutical formulation that maintains the
physiochemical properties and biological activity of the protein
during storage. Accordingly, muteins with enhanced pharmaceutical
stability when compared to wild-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 being entirely possible that a given mutein has one or more
modified properties of the wild-type protein.
[0021] A "biologically active peptide" is defined as a peptide of a
mutein of the present invention that maintains the modified
property(s) and the biological potency of the mutein.
[0022] A "therapeutically-effective amount" is the minimal amount
of an active agent necessary to impart therapeutic benefit to a
patient. For example, a "therapeutically-effective amount" to a
patient exhibiting, suffering or prone to suffer or to prevent it
from suffering from type 2 diabetes, obesity, or metabolic syndrome
is such an amount which induces, ameliorates or otherwise causes an
improvement in the pathological symptoms, disease progression,
physiological conditions associated with or resistance to
succumbing to the afore mentioned disorders. For the purposes of
the present invention a "subject" or "patient" is preferably a
human, but can also be an animal, more specifically, a companion
animal (e.g., dogs, cats, and the like), farm animals (e.g., cows,
sheep, pigs, horses, and the like) and laboratory animals (e.g.,
rats, mice, guinea pigs, and the like).
[0023] "Type 2 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.
[0024] "Glucose intolerance" can be defined as an exceptional
sensitivity to glucose.
[0025] "Hyperglycemia" is defined as an excess of sugar (glucose)
in the blood.
[0026] "Hypoglycemia", also called low blood sugar, occurs when
your blood glucose level drops too low to provide enough energy for
your body's activities.
[0027] "Hyperinsulinemia" is defined as a higher-than-normal level
of insulin in the blood.
[0028] "Insulin resistance" is defined as a state in which a normal
amount of insulin produces a subnormal biologic response.
[0029] "Obesity", in terms of the human subject, can be defined as
that body weight over 20 percent above the ideal body weight for a
given population (R. H. Williams, Textbook of Endocrinology, 1974,
p. 904-916).
[0030] "Metabolic syndrome" can be defined as a cluster of at least
three of the following signs: abdominal fat--in most men, a
40--inch waist or greater; high blood sugar--at least 110
milligrams per deciliter (mg/dl) after fasting; high
triglycerides--at least 150 mg/dL in the bloodstream; low HDL--less
than 40 mg/dl; and, blood pressure of 130/85 or higher.
[0031] The 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 may lead to relative deficiencies in some
nutrients. Generally there is an increased oxidation of both fat
and muscle.
[0032] Moreover, critically ill patients are preferably patients
that experience systemic inflammatory response syndrome or
respiratory distress. A reduction in morbidity means reducing the
likelihood that a critically ill patient will develop additional
illnesses, conditions, or symptoms or reducing the severity of
additional illnesses, conditions, or symptoms. For example reducing
morbidity may correspond to a decrease in the incidence of
bacteremia or sepsis or complications associated with multiple
organ failure.
[0033] "Systemic inflammatory response syndrome (SIRS)" as used
herein describes an inflammatory process associated with a large
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.degree. C. or less than 36.degree. C.;
(2) a heart rate greater than 90 beats per minute; (3) tachypnea,
manifested by a respiratory rate greater than 20 breaths per
minute, or hyperventilation, as indicated by a PaCO.sub.2 of 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% immature neutrophils.
These physiologic changes should represent an acute alteration from
baseline in the absence of other known causes for such
abnormalities, such as chemotherapy, induced neutropenia, and
leukopenia.
[0034] "Sepsis" as used herein is defined as a SIRS arising from
infection. Noninfectious pathogenic causes of SIRS may include
pancreatitis, ischemia, multiple trauma and tissue injury, i.e.
crushing injuries or severe burns, hemorrhagic shock,
immune-mediated organ injury, and the exogenous administration of
such putative mediators of the inflammatory process as tumor
necrosis factor and other cytokines.
[0035] Septic shock and multi-organ dysfunction are major
contributors to morbidity and mortality in the ICU setting. Sepsis
is associated with and mediated by the activation of a number of
host defense mechanisms including the cytokine network, leukocytes,
and the complement cascade, and coagulation/fibrinolysis systems
including 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 shock. The downstream effects of
the host defense response on target organs is an important mediator
in the development of the multiple organ dysfunction syndrome
(MODS) and contributes to the poor prognosis of patients with
sepsis, severe sepsis, and sepsis complicated by shock.
[0036] "Respiratory distress" as used herein denotes a condition
wherein patients have difficulty breathing due to some type of
pulmonary dysfunction. Often these patients exhibit varying degrees
of hypoxemia that may or may not be refractory to treatment with
supplemental oxygen.
[0037] Respiratory distress may occur in patients with impaired
pulmonary function due to direct lung injury or may occur due to
indirect lung injury such as in the setting of a systemic process.
In addition, the presence of multiple predisposing disorders
substantially increases the risk, as does the presence of secondary
factors such as chronic alcohol abuse, chronic lung disease, and a
low serum pH.
[0038] Some causes of direct lung injury include pneumonia,
aspiration of gastric contents, pulmonary contusion, fat emboli,
near drowning, inhalation injury, high altitude and reperfusion
pulmonary edema after lung transplantation or pulmonary
embolectomy. Some causes of indirect lung injury include sepsis,
severe trauma with shock and multiple transfusions, cardiopulmonary
bypass, drug overdose, acute pancreatitis, and transfusions of
blood products.
[0039] One class of pulmonary disorders that causes respiratory
distress are associated with the syndrome known as Cor Pulmonale.
These disorders are associated with chronic hypoxemia resulting in
raised pressure within the pulmonary circulation called pulmonary
hypertension. The ensuing pulmonary hypertension increases the
work-load of the right ventricle, thus leading to its enlargement
or hypertrophy. Cor Pulmonale generally presents as right heart
failure defined by a sustained increase in right ventricular
pressures and clinical evidence of reduced venous return to the
right heart.
[0040] "Chronic obstructive pulmonary diseases" (COPDs), which
include emphysema and chronic bronchitis also cause respiratory
distress and are characterized by obstruction to air flow. COPDs
are the fourth leading cause of death and claim over 100,000 lives
annually.
[0041] "Acute respiratory distress syndrome" (ARDS) is generally
progressive and characterized by distinct stages. The syndrome is
generally 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 supplemental oxygen is a
characteristic feature. There may be alveolar filling,
consolidation, and atelectasis occurring in dependent lung zones;
however, non-dependent areas may have substantial inflammation. The
syndrome may progress to fibrosing alveolitis with persistent
hypoxemia, increased alveolar dead space, and a further decrease in
pulmonary compliance. Pulmonary hypertension, which results from
damage to the pulmonary capillary bed, may also develop.
[0042] The first preferred aspect of the invention comprises
muteins of human FGF-21 in which 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 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. 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 non-naturally occurring analogs thereof
(e.g., gamma aminobutyric acid, ornithine, etc.). A negatively
charged amino acid is defined to included aspartate, glutamate, and
non-naturally occurring analogs thereof (e.g., aminoadipic acid). A
polar but uncharged amino acid is defined to include serine,
threonine, asparagine, glutamine, and non-naturally occurring
analogs thereof. The most preferred muteins of the first embodiment
are Gln54Glu, Leu139Glu, Ala145Glu, Leu146Glu, Ile152Glu,
Gln156Glu, Ser163Glu, and Ile152Glu-Ser163Glu.
[0043] The second aspect of the present invention provides muteins
of human FGF-21, or a biologically active peptide thereof,
comprising the substitution of a cysteine for 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 100, aspartate
102, tyrosine 104, tyrosine 107, serine 109, glutamate 110, proline
115, histidine 117, leucine 118, proline 119, 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 the amino acids is based on SEQ ID NO:1.
[0044] One skilled in the art will also recognize that the native
cysteines, cysteine 75 and cysteine 93, could also be utilized as
loci to introduce a novel disulfide bond that may impart improved
properties. Specifically contemplated is the introduction of a
cysteine substitution at serine 85 or phenylalanine 73, coupled
with a concomitant change at either cysteine 93 or cysteine 75,
respectively, wherein the latter sites are replaced with any other
amino acid.
[0045] Naturally occurring disulfide bonds, as provided by cysteine
residues, generally increase thermodynamic stability of proteins.
Successful examples of increased thermodynamic stability, as
measured in increase of the melting temperature, are multiple
disulfide-bonded mutants of the enzymes T4 lysozyme (Matsumura, et
al., PNAS 86:6562-6566 (1989)) and barnase (Johnson et al., J. Mol.
Biol. 268:198-208 (1997)). An aspect of the present invention is
the premise that constraining the flexibility of the 118-134 amino
acid loop of FGF-21 by disulfide bonds enhances the physical
stability of FGF-21 in the presence of a preservative, presumably
by limiting access of the preservative to the hydrophobic core of
the protein.
[0046] Muteins of FGF-21 with engineered disulfide bonds, in
addition to the naturally occurring one at Cys75-Cys93, are as
follows: Gln76Cys-Ser109Cys, Cys75-Ser85Cys, Cys75-Ala92Cys,
Phe73Cys-Cys93, Ser123Cys-His125-Cys, Asp102Cys-Tyr104Cys,
Asp127Cys-Gly132Cys, Ser94Cys-Glu110Cys, Pro115Cys-His117Cys,
Asn121Cys-Asp127Cys, Leu100Cys-Asp102Cys, Phe95Cys-Tyr107Cys,
Arg19Cys-Pro138Cys, Tyr20Cys-Leu139Cys, Tyr22Cys-Leu137Cys,
Arg77Cys-Asp79Cys, Pro90Cys-Ala92Cys, Glu50Cys-Lys69Cys,
Thr23Cys-Asp25Cys, Ala31Cys-Gly43Cys, Gln28Cys-Gly43Cys,
Thr23Cys-Gln28Cys, Val41Cys-Leu82Cys, Leu58Cys-Val62Cys,
Gln54Cys-Leu66Cys, Ile35Cys-Gly67Cys, Gly67Cys-Arg72Cys,
Ile35Cys-Gly84Cys, Arg72Cys-Gly84Cys, or Arg77Cys-Ala81Cys, 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; Leu118Cys-Gly132Cys; His 117Cys-Pro130Cys; His
17Cys-Ala129Cys; Leu82Cys-Pro119Cys; Gly80Cys-Ala129Cys;
Gly43Cys-Pro124Cys; Gly42Cys-Arg126Cys; Gly42Cys-Pro124Cys;
Gln28Cys-Pro124Cys; Gln27Cys-Ser123Cys; Ala26Cys-Lys122Cys; or
Asp25Cys-Lys122Cys. Most preferred muteins with engineered
disulfide bonds are Leu118Cys-Ala134Cys; Leu21Cys-Leu33Cys;
Ala26Cys-Lys122Cys; Leu21Cys-Leu33Cys/Leu118Cys-Ala134Cys
[0047] The third aspect of the present invention provides muteins
of human FGF-21, or a biologically active peptide thereof,
comprising a substitution of any charged and/or polar but uncharged
amino acid 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.
[0048] It is well known in the art that a significant challenge in
the development of protein pharmaceuticals is to deal with the
physical and chemical instabilities of proteins. This is even more
apparent when a protein pharmaceutical formulation is intended to
be a multiple use, injectable formulation requiring a stable,
concentrated and preserved solution, while maintaining a favorable
bioactivity profile. Detailed biophysical characterization of
wild-type FGF-21 established that a concentrated protein solution
(>5 mg/ml), when exposed to stress conditions, such as high
temperature or low pH, lead to accelerated association and
aggregation (i.e., poor physical stability and biopharmaceutical
properties). Exposure of a concentrated protein solution of FGF-21
to pharmaceutical preservatives (e.g., m-cresol) also had a
negative impact on physical stability.
[0049] Therefore, an embodiment of the present invention is to
enhance physical stability of concentrated solutions, while
maintaining chemical stability and biological potency, under both
physiological and preserved formulation conditions. It is thought
that association and aggregation may result from hydrophobic
interactions, since, at a given protein concentration, temperature,
and ionic strength have considerable impact on physical stability.
For the most part, non-conserved, presumed surface exposed amino
acid residues were targeted. The local environment of these
residues was analyzed and, those that were not deemed structurally
important were selected for mutagenesis. One method to initiate
specific changes is to further decrease the pI of the protein by
introducing glutamic acid residues ("glutamic acid scan"). It is
hypothesized that the introduction of charged substitutes would
inhibit hydrophobic-mediated aggregation via charge-charge
repulsion and potentially improve preservative compatibility. In
addition, one skilled in the art would also recognize that with
sufficient degree of mutagenesis the pI could be shifted into a
basic pH range by the introduction of positive charge with or
without concomitant decrease in negative charge, thus allowing for
charge-charge repulsion.
[0050] Although the embodiments of the present invention concern
the physical and chemical stability under both physiological and
preserved pharmaceutical formulation conditions, maintaining the
biological potency of the muteins as compared to wild-type FGF-21
is an important factor of consideration as well. Therefore, 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 in vitro 3T3-L1 cell assay (Example 4) and/or the
lowering of plasma glucose levels, as well as, plasma
triglycerides, as measured in vivo in the ob/ob mouse assay
(Example 5).
[0051] The muteins of FGF-21 administered according to this
invention may 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 Current Protocols
in Molecular Biology (John Wiley & Sons, Inc.), which is
incorporated herein by reference.
[0052] Additionally, the preferred embodiments include a
biologically active peptide derived from the mutein described
herein. Such a peptide will contain at least one of the
substitutions described and the mutein will possess biological
activity. The peptide may be produced by any and all means known to
those skilled in the art, examples of which included but are not
limited to enzymatic digestion, chemical synthesis or recombinant
DNA methodologies.
[0053] It is established in the art that fragments of peptides of
certain fibroblast growth factors are biologically active. See for
example, Baird et al., Proc. Natl. Acad. Sci. (USA) 85:2324-2328
(1988), and J. Cell. Phys. Suppl. 5:101-106 (1987). Therefore, the
selection of fragments or peptides of the mutein is based on
criteria known in the art. For example, it is known that dipeptidyl
peptidase IV (DPP-IV) is a serine type protease involved in
inactivation of neuropeptides, endocrine peptides, and cytokines
(Damme et al. Chem. Immunol 72: 42-56, (1999)). The N-terminus of
FGF-21 (HisProIlePro) contains two dipeptides that could
potentially be substrates to DPP-IV, resulting in a fragment of
FGF-21 truncated at the N-terminus by 4 amino acids. Unexpectedly,
this fragment of wild-type FGF-21 has been demonstrated to retain
biological activity (Table 1), thus, muteins of the present
invention truncated at the N-terminus by up to 4 amino acids, is an
embodiment of the present invention.
[0054] The present invention also encompasses polynucleotides
encoding the above-described muteins that may be in the form of RNA
or in the form of DNA, which DNA includes cDNA, genomic DNA, and
synthetic DNA. The DNA may be double-stranded or single-stranded.
The coding sequences that encode the muteins of the present
invention may vary as a result of the redundancy or degeneracy of
the genetic code.
[0055] The polynucleotides that encode for the 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-protein sequence; the coding
sequence for the mutein and non-coding sequence, such as introns or
non-coding sequence 5' and/or 3' of the coding sequence for the
mutein. Thus the term "polynucleotide encoding a mutein"
encompasses a polynucleotide that may include not only coding
sequence for the mutein but also a polynucleotide, which includes
additional coding and/or non-coding sequence.
[0056] The present invention further relates to variants of the
described polynucleotides that encode for fragments, analogs and
derivatives of the polypeptide that contain the indicated
substitutions. The variant of the polynucleotide may be a naturally
occurring allelic variant of the human FGF-21 sequence, a
non-naturally occurring variant, or a truncated variant as
described above. Thus, the present invention also includes
polynucleotides encoding the muteins described above, as well as
variants of such polynucleotides, which variants encode for a
fragment, derivative or analog of the disclosed mutein. Such
nucleotide variants include deletion variants, substitution
variants, truncated variants, and addition or insertion variants as
long as at least one of the indicated amino acid substitutions of
the first or second embodiments is present.
[0057] The polynucleotides of the present invention will be
expressed in hosts after the sequences have been operably linked to
(i.e., positioned to ensure the functioning of) an expression
control sequence. These expression vectors are typically replicable
in the host organisms either as episomes or as an integral part of
the host chromosomal DNA. Commonly, expression vectors will contain
selection markers, e.g., tetracycline, neomycin, and dihydrofolate
reductase, to permit detection of those cells transformed with the
desired DNA sequences. The FGF-21 mutein can be expressed in
mammalian cells, 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.
[0058] E. coli is a prokaryotic host useful particularly for
cloning the polynucleotides of the present invention. Other
microbial hosts suitable for use include Bacillus subtilus,
Salmonella typhimurium, and various species of Serratia,
Pseudomonas, Streptococcus, and Staphylococcus, although others may
also be employed as a matter of choice. In these prokaryotic hosts,
one can also make expression vectors, which will typically contain
expression control sequences compatible with the host cell (e.g.,
an origin of replication). In addition, any of a number of
well-known promoters may be present, such as the lactose promoter
system, a tryptophan (trp) promoter system, a beta-lactamase
promoter system, or a promoter system from phages lambda or T7. The
promoters will typically control expression, optionally with an
operator sequence, and have ribosome binding site sequences and the
like, for initiating and completing transcription and
translation.
[0059] One skilled in the art of expression of proteins will
recognize that methionine or methionine-arginine sequence can be
introduced at 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. Thus, unless otherwise noted, muteins of the
present invention expressed in E. coli have a methionine sequence
introduced at the N-terminus.
[0060] Other microbes, such as yeast or fungi, may also be used for
expression. Pichia pastoris, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Pichia angusta 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 employed as a matter of
choice.
[0061] Mammalian tissue cell culture may also be used to express
and produce the polypeptides of the present invention. Eukaryotic
cells are actually preferred, because a number of suitable host
cell lines capable of secreting intact muteins have been developed
in the art, and include the CHO cell lines, various COS cell lines,
NS0 cells, Syrian Hamster Ovary cell lines, HeLa cells, or human
embryonic kidney cell lines (i.e. HEK293, HEK293EBNA).
[0062] 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, polyadenylation sites,
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.
[0063] The vectors containing the polynucleotide sequences of
interest (e.g., the muteins of FGF-21 and expression control
sequences) can be transferred into the host cell by well-known
methods, which vary depending on the type of cellular host. For
example, calcium chloride transfection is commonly utilized for
prokaryotic cells, whereas calcium phosphate treatment or
electroporation may be used for other cellular hosts.
[0064] Various methods of protein purification may be employed and
such methods are known in the art and described, for example, in
Deutscher, Methods in Enzymology 182: 83-9 (1990) and Scopes,
Protein Purification: Principles and Practice, Springer-Verlag, NY
(1982). The purification step(s) selected will depend, for example,
on the nature of the production process used for the muteins of
FGF-21.
[0065] The FGF-21 mutein-containing compositions should be
formulated and dosed in a fashion consistent with good medical
practice, taking into account the clinical condition of the
patient, the site of delivery of the FGF-21 mutein composition, the
method of administration, the scheduling of administration, and
other factors known to practitioners. The "therapeutically
effective amount" of the FGF-21 mutein for purposes herein is thus
determined by such considerations
[0066] The pharmaceutical compositions of the FGF-21 muteins and of
the present invention may be administered by any means that achieve
the generally intended purpose: 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, intrasternal,
subcutaneous, and intraarticular injection and infusion. The dosage
administered will be dependent upon the age, health, and weight of
the recipient, kind of concurrent treatment, if any, frequency of
treatment, and the nature of the effect desired. Compositions
within the scope of the invention include all compositions wherein
an FGF-21 mutein is present in an amount that is effective to
achieve the desired medical effect for treatment type 2 diabetes,
obesity, or metabolic syndrome. While individual needs may vary
from one patient to another, the determination of the optimal
ranges of effective amounts of all of the components is within the
ability of the clinician of ordinary skill.
[0067] The muteins of FGF-21 of the present invention can be
formulated according to known methods to prepare pharmaceutically
useful compositions. A desired formulation would be one that is a
stable lyophilized product that is reconstituted with an
appropriate diluent or an aqueous solution of high purity with
optional pharmaceutically acceptable carriers, preservatives,
excipients or stabilizers [Remington's Pharmaceutical Sciences 16th
edition (1980)]. The muteins of the present invention may be
combined with a pharmaceutically acceptable buffer, and the pH
adjusted to provide acceptable stability, and a pH acceptable for
administration.
[0068] For parenteral administration, in one embodiment, the FGF-21
muteins are formulated generally by mixing one or more of them at
the desired degree of purity, in a unit dosage injectable form
(solution, suspension, or emulsion), with a pharmaceutically
acceptable carrier, i.e., one that is non-toxic to recipients at
the dosages and concentrations employed and is compatible with
other ingredients of the formulation. Preferably, one or more
pharmaceutically acceptable anti-microbial agents may be added.
Phenol, m-cresol, and benzyl alcohol are preferred pharmaceutically
acceptable anti-microbial agents.
[0069] Optionally, one or more pharmaceutically acceptable salts
may 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 isotonicity adjusting excipient.
[0070] Those skilled in the art can readily optimize
pharmaceutically effective dosages and administration regimens for
therapeutic compositions comprising an FGF-21 mutein, 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 range from about 0.01 mg per day to
about 1000 mg per day for an adult. Preferably, the dosage ranges
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. Most preferably, the
dosage is about 1-5 mg/day. The appropriate dose of an FGF-21
mutein administered will result in lowering blood glucose levels
and increasing energy expenditure by faster and more efficient
glucose utilization, and thus is useful for treating type 2
diabetes, obesity and metabolic syndrome.
[0071] In addition, because hyperglycemia and insulin resistance
are common in critically ill patients given nutritional support,
some ICUs administer insulin to treat excessive hyperglycemia in
fed critically ill patients. In fact, recent studies document the
use of exogenous insulin to maintain blood glucose at a level no
higher than 110 mg per deciliter reduced morbidity and mortality
among critically ill patients in the surgical intensive care unit,
regardless of whether they had 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 metabolically unstable critically
ill patients. Muteins of FGF-21 are unique in that they stimulate
glucose uptake and enhances insulin sensitivity but do not induce
hypoglycemia.
[0072] In another aspect of the present invention, muteins of
FGF-21 for use as a medicament for the treatment of type 2
diabetes, obesity, metabolic syndrome, or critically ill patients
is contemplated.
[0073] Having now described the present invention in detail, the
same will be more clearly understood by reference to the following
examples, which are included herewith for purposes of illustration
only and are not intended to be limiting of the invention.
[0074] All patents and publications referred to herein are
expressly incorporated by reference.
EXAMPLE 1
Expression and Purification of FGF-21 Muteins in E. coli
[0075] The bacterial expression vector pET30a is used for bacterial
expression in this example. (Novagen, Inc., Madison, Wis.)). pET30a
encodes kanamycin antibiotic resistance gene and contains a
bacterial origin of replication ("ori"), a strong T7 phage-IPTG
inducible promoter, a ribosome binding site ("RBS"), and suitable
MCS with a number of unique restriction endonuclease cleavage
sites. Conveniently for purification purpose, the vector can encode
His- and S-tags for N-terminal peptide fusions, as well as, a
C-terminal His-tag fusion. However, for purposes of the present
invention, the cDNA encoding FGF-21 variants is inserted between
restriction sites NdeI and BamHI, respectively, and the resulting
construct does not take advantage of either of the described
tags.
[0076] The nucleic acid sequence encoding the FGF-21 mutein,
lacking the leader sequence but substituted with a methionine
residue, is amplified from a cDNA clone using PCR oligonucleotide
primers, which anneal to the 5' and 3' ends of the open reading
frame. Additional nucleotides, containing recognition sites for
restriction enzymes NdeI and BamHI, are added to the 5' and 3'
sequences, respectively.
[0077] For cloning, the 5' forward and 3' reverse PCR primers have
nucleotides corresponding or complementary to a portion of the
coding sequence of the FGF-21 mutein-encoding nucleic acid
according to methods known in the art. One of ordinary skill in the
art would appreciate that the point in a polynucleotide sequence
where primers begin can be varied.
[0078] The amplified nucleic acid fragments and the vector pET30a
are digested with NdeI and BamHI restriction enzymes and the
purified digested DNA fragments are then ligated together.
Insertion of FGF-21 mutein-encoding DNA into the restricted pET30a
vector places the FGF-21 mutein polypeptide coding region including
its associated stop codon downstream from the IPTG-inducible
promoter and in-frame with an initiating ATG codon. The associated
stop codon, TAG, prevents translation of the six-histidine codons
downstream of the insertion point.
[0079] 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.).
[0080] Transformation reactions are plated on LB/Kanamycin plates
and after an overnight growth transformants are picked for plasmid
preparations or lysed in situ for screening by PCR. Positive
recombinant plasmids, containing desired FGF-21 variant inserts,
are identified by restriction analysis followed by DNA sequence
analysis. Those plasmids are subsequently used to transform
expression strains and protein production.
[0081] E. coli strains BL21(DE3), BL21(DE3)STAR or BL21(DE3) RP,
are used for expressing FGF-21 muteins. These strains, which are
only some of many that are suitable for expressing FGF-21 muteins,
are available commercially from Novagen, Inc., Invitrogen and
Stratagen, respectively. Transformants are identified by their
ability to grow on LB plates in the presence of kanamycin.
[0082] Clones containing the desired constructs are grown overnight
(o/n) in liquid culture in LB media supplemented with kanamycin (30
.mu.g/ml). The o/n culture is used to inoculate a large culture, at
a dilution of approximately 1:25 to 1:250. The cells are grown to
an optical density of 0.6 ("OD600") at 600 Mn.
Isopropyl-b-D-thiogalactopyranoside ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 12 hours. Cells
are then harvested by centrifugation, pellets washed with 50 mM
Tris buffer, pH 8.0 and stored at -20.degree. C. until
purification. The FGF-21 muteins are expressed in the insoluble
fraction i.e inclusion bodies (or granules) of E. coli. Although
the expression level may vary from variant-to-variant, a typically
observed level for the wild-type (WT) FGF-21 protein is 50 mg/L.
The subsequent purification process starts with solubilization of
the granules and refolding of the variants followed by four
chromatographic steps.
[0083] To purify the FGF-21 muteins from E coli, the granules are
solubilzed in 50 mM Tris, pH 9.0, 7M Urea and 1 mM DTT through a pH
ramp to pH 11.0, at room temperature for 1 hour with stirring. 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 pool is then treated with 10 mM DTT, for
two hours, at RT, to reduce all disulfide bonds. The pool is then
diluted 10-fold so that the buffer concentration is as follows: 50
mM Tris, pH 9.0, 7 M Urea, 10 mM Cysteine, 1 mM DTT with a protein
concentration of approximately 250-500 .mu.g/ml. After another
two-hour incubation under reducing conditions at RT, to obtain the
protein in a free disulfide form, the pool is then dialyzed into 20
mM glycine, pH 9.0 for approximately 48 hours so that the correct
disulfide bonds can be formed.
[0084] Reversed-phase HPLC chromatography, on a Vydac C18 column
and 0.1% TFA/0-50% CH.sub.3CN as a mobile phase is used as an
initial purification step. This column is used to concentrate
FGF-21 or the FGF-21 muteins and removes contaminating
endotoxin.
[0085] The following purification step is size exclusion
chromatography on a Superdex 35/600 column performed in 1.times.PBS
buffer, pH7.4. At this step FGF-21 muteins are .about.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
yields >97% pure protein.
[0086] The above described 4-column step purification scheme was
used for all the FGF-21 muteins and produced stable
preparations.
EXAMPLE 2
Expression and Purification of FGF-21 Muteins in HEK293EBNA
Cells
[0087] Alternatively, FGF-21 muteins can be produced in a mammalian
cell expression system such as HE 93EBNA cells (EdgeBiosystems,
Gaiethersburg, Md.). FGF-21 muteins are subcloned in the
proprietary expression vector representing a modification of
commercially available pEAK10, between NheI and XbaI restriction
sites in the MCS. The cDNA sequence encoding mature FGF-21 is fused
in frame with the Ig.kappa. leader sequence to enhance secretion of
the desired product in the tissue culture media. The expression is
driven by the strong viral CMV promoter. HEK93EBNA cells are
transiently transfected using a standard transfection reagent such
as Fugene (Roche Diagnostics, Indianapolis, Ind.) and the
appropriate amount of recombinant plasmid, either as a monolayer or
suspension culture, at the adequate cell density. Cells are
incubated at 37.degree. C. and 5% CO.sub.2, in serum free media,
and collections are made every day for 5 days. Typically the
expression level in the HEK239EBNA suspension culture is 30 mg/L.
The expression of human FGF-21 in mammalian cells yields the
natural N-terminus sequence of HPIP, i.e. without a methionine
residue at the N-terminus. It was discovered that enzymatically
treating FGF-21 from HEK239EBNA cells with DPP-IV (porcine kidney,
SIGMA St Louis) resulted in truncation of the N-terminus by four
amino acids. When assayed in the mouse 3T3-L1 adipocyte assay (see
Example 4), this truncated variant of FGF-21 stimulates glucose
uptake at a comparable level to that of wild-type FGF-21 (Table
1).
EXAMPLE 3
Expression and Purification of FGF-21 Muteins in Yeast
[0088] Yet another expression system for production of FGF-21
muteins is yeast, such as Pichia pastoris, Pichia methanolica or
Saccharomyces cerevisiae. For production in Pichia pastoris a
commercially available system (Invitrogen, Carlsbad, Calif.) uses
vectors with the powerful AOX1 (alcohol oxidase) promoters to drive
high-level expression of recombinant proteins. Alternatively,
vectors that use the promoter from the GAP gene
(glyceraldehyde-3-phosphate dehydrogenase) are available for high
level constitutive expression. The multi-copy Pichia expression
vectors allows one to obtain strains with multiple copies of the
gene of interest integrated into the genome. Increasing the number
of copies of the gene of interest in a recombinant Pichia strain
can increase protein expression levels. Yet another yeast
expression system is Saccharomyces cerevisiae. Expression vectors
contain the promoter and enhancer sequences from the GAL1 gene. The
GAL1 promoter is one of the most widely used yeast promoters
because of its strong transcriptional activity upon induction with
galactose.
[0089] Analytical characterization (mass spectrum analyses)
indicates that the FGF-21 expressed in Pichia pastoris is truncated
(up to four amino acid removal [His ProIlePro] at the N-terminus,
designated hereinafter as des-HPIP). When assayed in the mouse
3T3-L1 adipocyte assay (see Example 4), this truncated variant of
FGF-21 stimulates glucose uptake at the same level as wild-type
FGF-21 (Table 1).
EXAMPLE 4
Glucose Uptake in Mouse 3T3-L1 Adipocytes
[0090] 3T3-L1 cells are obtained from the American Type Culture
Collection (ATCC, Rockville, Md.). Cells are cultured in growth
medium (GM) containing 10% iron-enriched fetal bovine serum in
Dulbecco's modified Eagle's medium. For standard adipocyte
differentiation, two days after cells reached confluency (referred
as day 0), cells are exposed to differentiation medium (DM)
containing 10% fetal bovine serum, 10 .mu.g/ml of insulin, 1 .mu.M
dexamethasone, and 0.5 .mu.M isobutylmethylxanthine, for 48 h.
Cells then are maintained in post differentiation medium containing
10% fetal bovine serum, and 10 .mu.g/ml of insulin.
[0091] Glucose Transport Assay--Hexose uptake, as assayed by the
accumulation of 0.1 mM 2-deoxy-D-[.sup.14C]glucose, is measured as
follows: 3T3-L1 adipocytes in 12-well plates are washed twice with
KRP buffer (136 mM NaCl, 4.7 mM KCl, 10 mM NaPO.sub.4, 0.9 mM
CaCl.sub.2, 0.9 mM MgSO.sub.4, pH 7.4) warmed to 37.degree. C. and
containing 0.2% BSA, incubated in Leibovitz's L-15 medium
containing 0.2% BSA for 2 h at 37.degree. C. in room air, washed
twice again with KRP containing, 0.2% BSA buffer, and incubated in
KRP, 0.2% BSA buffer in the absence (Me.sub.2SO only) or presence
of wortmannin for 30 min at 37.degree. C. in room air. Insulin is
then added to a final concentration of 100 nM for 15 min, and the
uptake of 2-deoxy-D-[.sup.14C]glucose is measured for the last 4
min. Nonspecific uptake, measured in the presence of 10 .mu.M
cytochalasin B, is subtracted from all values. Protein
concentrations are determined with the Pierce bicinchoninic acid
assay. Uptake is measured routinely in triplicate or quadruplicate
for each experiment.
[0092] In vitro potency is normalized to the in vitro activity of
wild-type FGF-21, which is given a designation of 1.0 and used as a
positive control. The in vitro potency of muteins of FGF-21 of the
present invention is compared to wild-type FGF-21 in Table 1.
[0093] As indicated in Table 1, the muteins of the present
invention maintained biological potency to various degrees compare
to wild-type FGF-21.
TABLE-US-00003 TABLE 1 Expression Expression In vitro FGF-21 Mutein
System Potency Wild-type E. coli 1.0 Truncated Wild-type* Yeast 0.9
Truncated Wild-Type** HEK293EBNA 1.3 Wild-type HEK293EBNA 0.7 R77E
HEK293EBNA 1.1 L139E E. coli 0.1 L146E E. coli 0.8 Q156E E. coli
0.6 S163E E. coli 1.3 I152E/S163E E. coli 0.9 A145E E. coli 0.5
I152E E. coli 1.2 L118C/A134C E. coli 0.4 des-HPIP-L118C/A134C
Yeast 0.3 *truncated by 4 amino acids at the N-terminus, i.e.
des-HPIP **enzymatically truncated by 4-amino acids at the
N-terminus by DPP-IV, i.e. des-HPIP
EXAMPLE 5
Ob/ob Mouse Model
[0094] A study in an obesity model using male ob/ob mice was done
to monitor plasma glucose levels and triglyceride levels after
treatment with FGF-21, compared to vehicle and insulin control
groups. The test groups of male ob/ob mice (7 weeks old) were
injected with vehicle alone (0.9% NaCl), or FGF-21 mutein (0.125
mg/kg) subcutaneously (0.1 mL, once daily) for seven days. Blood
was collected by tail clip bleeding on day 7, one hour after the
last compound injection and plasma glucose levels were measured
using a standard protocol. The ability of the FGF-21 muteins to
lower plasma glucose levels as compared to the vehicle control is
shown in Table 2. The data in Table 2 indicates that muteins of the
present invention lowered plasma glucose levels as compared to
vehicle control. The ability of the FGF-21 muteins to lower
triglyceride levels as compared to the vehicle control is shown in
Table 3.
TABLE-US-00004 TABLE 2 Plasma Glucose levels FGF-21 Mutein as % of
Control Wild-type 60% R77E 63% Q156E 65% S163E 60% A145E 81% I152E
82% G161E 78% L118C-A134C 80%
TABLE-US-00005 TABLE 3 FGF-21 Mutein Triglyceride Levels (mg/dL)
Experiment #1 Vehicle Control 200 Wild-type 145 R77E 125 Experiment
#2 Vehicle Control 165 Wild-type 90 Q156E 80 S163E 70 Experiment #3
Vehicle Control 100 Wild-type 75 A145E 70 I152E 60 G161E 70
L118C-A134C 75
EXAMPLE 6
Pharmaceutical Stability of FGF-21 Muteins
[0095] The stability of the FGF-21 muteins of the present invention
was analyzed under simulated 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.
Solubility/physical stability of the muteins' in PBS is considered
satisfactory if recovery of protein following preparation resulted
in >90% recovery at RT as determined by size-exclusion and/or
reversed-phase chromatography. The muteins of the present invention
indicated in Tables 4 and 5 meet this criteria.
[0096] It is anticipated that pharmaceutical formulation of a
mutein of the present invention will likely be a preserved
multi-use formulation, thus, compatibility with a common
preservative was analyzed. To test for formulation compatibility, a
preservative, m-cresol, (3 mg/mL final concentration, a
concentration usually sufficient to meet European Pharmacopia B
criteria for preservative effectiveness under neutral pH
conditions), was added at room temperature to a solution containing
the mutein at approximately 10 mg/ml in PBS, pH 7.4. Physical
stability in the presence of preservative was initially accessed by
determining protein recovery of the main chromatographic peak after
reversed-phase and size exclusion chromatography at RT.
Furthermore, the extent of aggregation as measured by DLS (dynamic
light scattering) at 37.degree. C. is shown as the average diameter
of particles in the presence of m-cresol after two hours, compared
to wild-type FGF-21. A larger average diameter corresponds to an
increased degree protein association and/or aggregation. The
preservative compatibility (as a function average diameter of
particulates) of the muteins of the first and second embodiments of
the present invention compared to wild-type FGF-21 is shown in
Table 4. All muteins were expressed in E. coli.
[0097] Muteins of the present invention that are stable in PBS and
compatible with preservative are designated to have enhanced or
improved pharmaceutical properties as compared to wild-type FGF-21.
As shown in Table 4, the preferred muteins of the present invention
that have enhanced pharmaceutical properties as compared to
wild-type FGF-21 are L139E, A145E, L146E, 1152E, Q156E, [1152E,
S163E], S163E, Q54E, [L21C-L33C, L118C-A134C], L21C-L33C,
A26C-K122C, and L118C-A134C.
TABLE-US-00006 TABLE 4 Average Particulate FGF-21 Mutein Diameter
(nm)* Experiment #1 Wild-type FGF-21 1356 Q54E 210 L139E 234 A145E
223 L146E 248 I152E 76 Q156E 353 I152E, S163E 179 S163E 154
Experiment #2 Wild-type FGF-21 813 L21C, L33C, L118C, 10 A134C
L21C-L33C 10 L118C-A134C 7 A26C-K122C 7 *Average Particulate
diameter represents a protein solution at a target conc. of 10
mg/ml, m-cresol at 3 mg/ml, after 2 hours incubation at 37.degree.
C..
Sequence CWU 1
1
21181PRThuman 1His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly
Gly Gln Val1 5 10 15Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln
Thr Glu Ala His 20 25 30Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly
Ala Ala Asp Gln Ser 35 40 45Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu
Lys Pro Gly Val Ile Gln 50 55 60Ile Leu Gly Val Lys Thr Ser Arg Phe
Leu Cys Gln Arg Pro Asp Gly65 70 75 80Ala Leu Tyr Gly Ser Leu His
Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95Glu Leu Leu Leu Glu Asp
Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110Gly Leu Pro Leu
His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 115 120 125Ala Pro
Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135
140Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp
Val145 150 155 160Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser
Gln Gly Arg Ser 165 170 175Pro Ser Tyr Ala Ser 1802543DNAhuman
2caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac
60ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg
120gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc
cttgaagccg 180ggagttattc aaatcttggg agtcaagaca tccaggttcc
tgtgccagcg gccagatggg 240gccctgtatg gatcgctcca ctttgaccct
gaggcctgca gcttccggga gctgcttctt 300gaggacggat acaatgttta
ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360aacaagtccc
cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca
420ggcctgcccc ccgcactccc ggagccaccc ggaatcctgg ccccccagcc
ccccgatgtg 480ggctcctcgg accctctgag catggtggga ccttcccagg
gccgaagccc cagctacgct 540tcc 543
* * * * *