U.S. patent application number 13/043852 was filed with the patent office on 2011-06-30 for methods and compositions for treating asthma in human and nonhuman primates.
This patent application is currently assigned to AEROVANCE, INC.. Invention is credited to Jeffrey Tepper, Adrian Tomkinson.
Application Number | 20110158939 13/043852 |
Document ID | / |
Family ID | 38257043 |
Filed Date | 2011-06-30 |
United States Patent
Application |
20110158939 |
Kind Code |
A1 |
Tepper; Jeffrey ; et
al. |
June 30, 2011 |
Methods and Compositions for Treating Asthma in Human and Nonhuman
Primates
Abstract
The present invention relates generally to methods and compounds
for treating pulmonary disorders, and more specifically to the
inhalation administration and use of hIL-4 mutant proteins to treat
asthma.
Inventors: |
Tepper; Jeffrey; (San
Carlos, CA) ; Tomkinson; Adrian; (El Cerrito,
CA) |
Assignee: |
AEROVANCE, INC.
|
Family ID: |
38257043 |
Appl. No.: |
13/043852 |
Filed: |
March 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11652868 |
Jan 11, 2007 |
|
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13043852 |
|
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60758442 |
Jan 11, 2006 |
|
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60841583 |
Aug 30, 2006 |
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Current U.S.
Class: |
424/85.2 ;
435/29; 436/116 |
Current CPC
Class: |
A61P 11/06 20180101;
A61K 9/0019 20130101; Y10T 436/177692 20150115; A61K 9/0073
20130101; A61P 43/00 20180101; A61K 9/0078 20130101; A61K 38/2026
20130101 |
Class at
Publication: |
424/85.2 ;
435/29; 436/116 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61P 11/06 20060101 A61P011/06; C12Q 1/02 20060101
C12Q001/02; G01N 33/00 20060101 G01N033/00 |
Claims
1. A method of monitoring a therapeutic regimen for treating a
subject having asthma comprising determining a change in pulmonary
function and inflammation during therapy, wherein an increase in
pulmonary function or a decrease in inflammatory cells is
indicative of positive therapeutic efficacy.
2. The method of claim 1, wherein an increase in pulmonary function
is determined by an improved immediate asthmatic response, an
improved late asthmatic response, or a decrease in airway
hyperreactivity.
3. The method of claim 1, further comprising determining a change
in BAL eosinophil concentration during therapy, wherein a decrease
in BAL eosinophil concentration is indicative of positive
therapeutic efficacy.
4. The method of claim 1, further comprising determining a change
in exhaled nitric oxide during therapy, as compared to exhaled
nitric oxide prior to therapy, wherein a decrease in nitric oxide
concentration is indicative of positive therapeutic efficacy.
5. The method of claim 1, wherein the therapy comprises the
treatment of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a divisional application of U.S.
application Ser. No. 11/652,868 filed Jan. 11, 2007, now pending;
which claims the benefit under 35 USC .sctn.119(e) to U.S.
Application Ser. No. 60/841,583 filed Aug. 30, 2006, now expired
and to U.S. Application Ser. No. 60/758,442 filed Jan. 11, 2006,
now expired. The disclosure of each of the prior applications is
considered part of and is incorporated by reference in the
disclosure of this application.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to methods and
compounds for treating pulmonary disorders, and more specifically
to the use of hIL-4 mutant proteins to treat asthma.
[0004] 2. Background Information
[0005] Interleukin-4 (IL-4) and Interleukin-13 (IL-13) are
pleiotropic cytokines with a broad spectrum of biological effects
on several target cells important in the pathogenesis of atopy and
asthma. IL-4 is increasingly appreciated as the pivotal cytokine
initiating the "Th2-type" inflammatory response forming the
underling milieu necessary for the development of atopy and asthma.
IL-4 effects include activation, proliferation and differentiation
of T and B cells. During proliferation of B-lymphocytes, IL-4 acts
as a differentiation factor by regulating class switching from IgG
to the IgE thus, encouraging the development of allergic reactions.
IL-13 is now appreciated as the more probable downstream effector
cytokine. IL-13 dominate effects include induction of airways
hyperresponsiveness (AHR) and goblet cell hyperplasia, both
cardinal features of asthma. However, there is considerable
redundancy in the effects of these two cytokines.
[0006] The redundancy in effects associated with the binding and
signaling of these two cytokines can be explained by their sharing
of common receptors. The IL-4 receptor alpha chain (IL-4R.alpha.)
has two binding partners with which it can associate and signal.
IL-4R.alpha. polypeptide associates with the cytokine common
receptor gamma chain (.gamma.c) to form the type 1 IL-4R
heterodimer. IL-4R polypeptide can also form a heterodimer with the
IL-13 receptor alpha 1 chain to create the type 2 IL-4R (aka
IL-13R). IL-4 activates both the type 1 and type 2 receptors
whereas IL-13 only activates the type 2 receptor heterodimer. Both
receptors, when activated, signal through the transcription factor
signal transducer and activator of transcription 6 (STAT6).
Although IL-4 may uniquely initiate the T-helper 2 (Th2) pathway,
since only type 1 receptors are localized to T lymphocytes, IL-13
may be both more abundant and more potent. Thus, inhibition of both
cytokines is important in disease states regulated and controlled
by the production of these two cytokines.
[0007] Recently, certain antagonistic and partially antagonistic
properties have been observed in human IL-4 (hIL-4) mutant proteins
in which the amino acid(s) occurring naturally in the wild type at
one or more of positions 120, 121, 122, 123, 124, 125, 126, 127 or
128 have been replaced with one or more natural amino acids. Thus,
these hIL-4 muteins have been described as valuable therapeutic
agents for use as medicaments in treating overshooting or falsely
regulated immune reactions and autoimmune diseases.
SUMMARY OF THE INVENTION
[0008] The present invention is based, in part, on the finding that
mutant IL-4 proteins are useful in treating subjects having asthma.
The invention is based in part on the finding that a mutant IL-4
protein having substitutions of R121D and Y124D can be administered
in a pharmaceutical composition to antagonize the binding of
wild-type hIL-4 and wild-type hIL-13 to receptors.
[0009] Accordingly, in one embodiment, the present invention
provides methods of treating asthma by administration of mutant
IL-4 proteins. In one embodiment, the method for treating asthma
includes administering to a subject in need thereof, a
pharmaceutical composition containing a therapeutically effective
amount of an IL-4 mutant protein having the amino acid sequence of
wild-type hIL-4 with substitutions R121D and Y124D numbered in
accordance with the wild-type hIL-4. In one aspect, the composition
is aerosolized prior to administration, and thus may be
administered via inhalation once or twice per day. Typical amounts
of the mutant IL-4 protein per dose are greater than or equal to
0.5 mg nominal dose in the nebulizer. The subject may be a mammal,
such as a human.
[0010] A pharmaceutical composition containing the mutant IL-4
protein of the invention typically contains a pharmaceutically
acceptable carrier, such as saline. In other embodiments, the
mutant IL-4 protein is conjugated to a non-protein polymer.
Non-protein polymers useful in the invention include, but are not
limited to, hydrophilic polymers, such as polyvinylpyrrolidone, and
hydrophobic polymers, such as polyethylene glycol.
[0011] The present invention further relates to methods of
monitoring a therapeutic regimen for treating a subject having
asthma. In one embodiment, the method of monitoring a therapeutic
regimen for treating a subject having asthma includes determining a
change in lung function, including the immediate and late asthmatic
response, airway hyperreactivity and markers of inflammation found
in the bronchoalveolar lavage (BAL), serum or exhaled breath. The
monitoring is accomplished by detecting a change in the influx of
inflammatory cells and mediators in the subject's airway or blood.
In another embodiment, the method of monitoring a therapeutic
regimen for treating a subject having asthma includes determining a
change in BAL eosinophil concentration during therapy. A decrease
in BAL eosinophil concentration during therapy is indicative of
positive therapeutic effect. In a further embodiment, the method of
monitoring includes determining the expired nitric oxide
concentration during therapy in comparison to expired nitric oxide
prior to therapy. A decrease in expired nitric oxide concentration
during or following therapy is indicative of positive therapeutic
effect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graphical representation showing the primate
asthma model therapeutic intervention protocol.
[0013] FIGS. 2A and 2B are graphical representations showing
results of therapeutic intervention with subcutaneous delivery of
IL-4RA on allergen-induced airway hyperresponsiveness and airway
eosinophilia in non-human primates.
[0014] FIG. 3 is a graphical representation showing results of
daily subcutaneous IL-4RA effect on antigen challenge response
before treatment (Screening Visit 2) and at the end of treatment in
asthmatic patients (Day 28).
[0015] FIG. 4 is a graphical representation showing results of
daily subcutaneous IL-4RA effect on adverse events (AEs) requiring
.beta.-agonists in asthmatic patients.
[0016] FIG. 5 is a graphical representation showing bioactivity
evaluation in a TF-1/IL-4 proliferation activity assay of IL-4RA
(lactate formulation) pre and post nebulization in a Aerogen
Aeroneb nebulizer.
[0017] FIG. 6 is a graphical representation showing the effects of
inhaled IL-4RA delivered twice daily (BID) on antigen-induced
airway hyperresponsiveness in nonhuman primates.
[0018] FIG. 7 is a graphical representation showing the effects of
inhaled IL-4RA delivered twice daily (BID) on antigen-induced
airway (BAL) eosinophilia in nonhuman primates.
[0019] FIGS. 8A and 8B are graphical representations showing
Bioactivity evaluation in a TF-1/IL-4 proliferation activity assay
of IL-4RA (lactate formulation) pre and post nebulization in a Pari
LC Plus nebulizer.
[0020] FIG. 9 is a graphical representation showing results of
twice daily inhaled IL-4RA effect on antigen challenge response
before treatment (Screening) and at the end of treatment (Day 27)
in asthmatic patients.
[0021] FIG. 10 is a graphical representation showing the log of
nitric oxide concentration versus drug treatment on Screening Day 2
versus Day 27 in asthmatic patients.
[0022] FIG. 11 is a graphical representation showing that local
delivery of inhaled IL-4RA achieves plasma concentrations below the
plasma concentrations achieved in the subcutaneous IL-4RA clinical
trial.
[0023] FIG. 12 is a graphical representation showing that IL-4RA
was more efficacious at lower plasma concentrations when delivered
by inhalation, as compared to subcutaneous delivery in non-human
primates.
[0024] FIGS. 13A and 13B show the nucleic acid and amino acid
sequences for wt IL-4 and a mutant IL-4, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention is based on the finding that hIL-4
muteins are useful for treating asthma. Thus, the present invention
discloses methods and compositions of treating asthma with
therapeutically effective amounts of mutant IL-4 proteins and
pharmaceutical compositions containing the muteins.
[0026] The present invention is not limited to the particular
methodology, protocols, cell lines, vectors, reagents, etc.,
described herein, as these may vary. It is also to be understood
that the terminology used herein is used for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention. As used herein and in the
appended claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates otherwise.
[0027] The term "asthma" is used herein to generally describe a
chronic respiratory disease, often arising from allergies, that is
characterized by sudden recurring attacks of labored breathing,
chest constriction, and coughing. In a typical asthmatic reaction,
IgE antibodies predominantly attach to mast cells that lie in the
lung interstitium in close association with the bronchioles and
small bronchi. An antigen entering the airway will thus react with
the mast cell-antibody complex, causing release of several
substances, including, but not limited to interleukin cytokines,
chemokines and arachodonic acid derived mediators, resulting in
bronchoconstriction, airway hyperreactivity, excessive mucus
secretion and airway inflammation. Thus, in certain embodiments of
the invention, the treatment of asthma may include the treatment of
airway hyperreactivity and/or the treatment of lung
inflammation.
[0028] The term "antigen" as used herein refers to any substance
that when introduced into the body stimulates the production of an
antibody. Antigens include insect, animal and plant proteins,
toxins, bacteria, foreign blood cells, and the cells of
transplanted organs. "Allergens" refer to any substances that cause
an allergic immune reaction in a subject. Typically, allergens are
from foods, plants, insects or animals that inflame the airway and
cause mucus production and bronchoconstriction.
[0029] The term "subject" as used herein refers to any individual
or patient to which the subject methods are performed. Generally
the subject is human, although as will be appreciated by those in
the art, the subject may be an animal. Thus, other animals,
including mammals such as rodents (including mice, rats, hamsters
and guinea pigs), cats, dogs, rabbits, farm animals including cows,
horses, goats, sheep, pigs, etc., and nonhuman primates (including
monkeys, chimpanzees, orangutans and gorillas) are included within
the definition of subject.
[0030] As used herein, the terms "mutant human IL-4 protein,"
"modified human IL-4 receptor antagonist," "mhIL-4," "IL-4 mutein,"
"IL-4 antagonist," and equivalents thereof are used interchangeably
and are within the scope of the invention. These polypeptides and
functional fragments thereof refer to polypeptides wherein specific
amino acid substitutions to the mature human IL-4 protein have been
made. These polypeptides include the mIL-4 compositions of the
present invention, which are administered to a subject in need of
treatment for asthma. In particular, the mhIL-4 of the present
invention, include at least the R121D/Y124D pair of substitutions
("IL-4RA") (FIG. 13B).
[0031] As used herein, a "functional fragment" is a polypeptide
which has IL-4 antagonistic activity, including smaller peptides.
These and other aspects of mhIL-4 of modification of hIL-4 are
described in U.S. Pat. Nos. 6,335,426; 6,313,272; and 6,028,176,
the entire contents of which are incorporated herein by
reference.
[0032] As used herein, "wild type IL-4" or "wtIL-4" and equivalents
thereof are used interchangeably and mean human Interleukin-4,
native or recombinant, having the 129 normally occurring amino acid
sequence of native human IL-4, as disclosed in U.S. Pat. No.
5,017,691, incorporated herein by reference. Further, the modified
human IL-4 receptor antagonists described herein may have various
insertions and/or deletions and/or couplings to a non-protein
polymer, and are numbered in accordance with the wtIL-4, which
means that the particular amino acid chosen is that same amino acid
that normally occurs in the wtIL-4. Accordingly, one skilled in the
art will appreciate that the normally occurring amino acids at
positions, for example, 121 (arginine), 124 (tyrosine), and/or 125
(serine), may be shifted in the mutein. Thus, an insertion of a
cysteine residue at amino acid positions, for example, 38, 102
and/or 104 may be shifted on the mutein. However, the location of
the shifted Ser (S), Arg (R), Tyr (Y) or inserted Cys (C) can be
determined by inspection and correlation of the flanking amino
acids with those flanking Ser, Arg, Tyr or Cys in wtIL-4.
[0033] Further, the DNA sequence encoding human IL-4 may or may not
include DNA sequences that encode a signal sequence. Such signal
sequence, if present, should be one recognized by the cell chosen
for expression of the IL-4 mutein. It may be prokaryotic,
eukaryotic or a combination of the two. It may also be the signal
sequence of native IL-4. The inclusion of a signal sequence depends
on whether it is desired to secrete the IL-4 mutein from the
recombinant cells in which it is made. If the chosen cells are
prokaryotic, it generally is preferred that the DNA sequence not
encode a signal sequence but include an N-terminal methionine to
direct expression. If the chosen cells are eukaryotic, it generally
is preferred that a signal sequence be encoded and most preferably
that the wild-type IL-4 signal sequence be used, as disclosed in
U.S. Pat. No. 6,028,176, incorporated herein by reference. In one
illustrative example, a mutant human IL-4 protein of the invention
includes the amino acid sequence of wild-type hIL-4 with
modifications, wherein a first modification is replacement of one
or more of the amino acids occurring in the wild-type hIL-4 protein
at positions 121, 124 or 125 with another natural amino acid, and
further optionally comprising an N-terminal methionine. In another
example, the mutant protein further includes a second modification
selected from the group consisting of:
[0034] i) the modification of the C-terminus therein;
[0035] ii) the deletion of potential glycosylation sites
therein;
[0036] iii) the coupling of the protein to a non-protein polymer,
and any combination thereof.
In another example, the mutant protein includes a first
modification of the protein that includes substitutions R121D and
Y124D, numbered in accordance with the wild-type hIL-4.
[0037] As used herein, "mutein" refers to any protein arising as a
result of a natural mutation or a site-directed amino acid
substitution to any protein created by a person skilled in the art.
"Glycosylation" refers to the addition of glycosyl groups to a
protein to form a glycoprotein. As such, the term includes both
naturally occurring glycosylation and synthetic glycosylation, such
as the linking of a carbohydrate skeleton to the side chain of an
asparagine residue ("N-glycosylation") or the coupling of a sugar,
preferably N-acetylgalactosamine, galactose or xylose to serine,
threonine, 4-hydroxyproline or 5-hydroxylysine
(O-glycosylation).
[0038] Accordingly, the present invention relates to compositions
comprising one or more hIL-4 muteins that are antagonists of the
human interleukin-4 and/or the human interleukin-13 by interfering
with the binding of these two interleukins to the type 1 and type 2
IL-4R. Such compositions are useful for treating subjects having
asthma or asthmatic-related symptoms. The hIL-4 muteins may further
include modifications in addition to the replacement(s) at
positions 121, 124 or 125. These modifications are carried out in
order to increase the stability of the hIL-4 muteins, in order to
extend the biological half life or in order to facilitate the
preparation and purification process. As used herein, the term
"agonist" refers to an agent or analog that binds productively to a
receptor and mimics its biological activity. The term "antagonist"
refers to an agent that binds to receptors but does not provoke the
normal biological response and blocks or partially blocks the
activity of the agonist.
[0039] Antagonists to IL-4 have been reported in the literature.
Mutants of IL-4 that function as antagonists include the IL-4
antagonist mutein IL-4/Y124D (Kruse, N., Tony, H. P., Sebald, W.,
Conversion of human interleukin-4 into a high affinity antagonist
by a single amino acid replacement, Embo J. 11:3237-44, 1992) and a
double mutein IL-4[R121D/Y124D] (Tony, H., et al., Design of Human
Interleukin-4 Antagonists in Inhibiting Interleukin-4-dependent and
Interleukin-13-dependent responses in T-cells and B-cells with high
efficiency, Eur. J. Biochem. 225:659-664 (1994)). The single mutein
is a substitution of tyrosine by aspartic acid at position 124 in
the D-helix. The double mutein is a substitution of Arginine by
Aspartic Acid at position 121, and of tyrosine by aspartic acid at
position 124 in the D-helix, as disclosed in U.S. Pat. Nos.
6,313,272 and 6,028,176, incorporated herein by reference.
Variations in this section of the D helix positively correlate with
changes in interactions at the second binding region of the IL-4RA
chain.
[0040] In one embodiment, the mutein is coupled to a non-protein
polymer at various amino acid residues, in particular, at positions
28, 36, 37, 38, 102, 104, 105 or 106. The amino acid positions are
numbered according to the wild type IL-4 (i.e., human
interleukin-4) amino acid sequence (see U.S. Pat. No. 5,017,691
which is incorporated herein by reference). Non-protein polymers
include, for example polyethylene glycol, polypropylene glycol or
polyoxyalkylenes, as described in U.S. Pat. Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, the entire
contents of which are incorporated herein by reference.
[0041] Accordingly, one of skill in the art will be able to
determine suitable variants of the polypeptide and functional
fragment thereof as set forth herein using well-known techniques.
As such, the skilled artisan may identify (1) suitable areas of the
polypeptide that may be changed without destroying activity by
targeting regions not believed to be important for activity (see
Kreitman et al. (1994) Biochemistry 33:11637-11644, incorporated
herein by reference); (2) residues and portions of the polypeptides
that are conserved among similar polypeptides; and (3) areas that
may be important for biological activity or for structure that can
still be subject to conservative amino acid substitutions without
destroying the biological activity or without adversely affecting
the polypeptide structure.
[0042] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, the skilled artisan can predict the
importance of amino acid residues in a protein that correspond to
amino acid residues important for activity or structure in similar
proteins. One skilled in the art may opt for chemically similar
amino acid substitutions for such predicted important amino acid
residues.
[0043] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of such
information, one skilled in the art may predict the alignment of
amino acid residues of a polypeptide with respect to its three
dimensional structure. In certain embodiments, one skilled in the
art may choose to not make radical changes to amino acid residues
predicted to be on the surface of the protein, since such residues
may be involved in important interactions with other molecules.
Moreover, one skilled in the art may generate test variants
containing a single amino acid substitution at each desired amino
acid residue. The variants can then be screened using activity
assays known in the art. Such variants could be used to gather
information about suitable variants. For example, if one discovered
that a change to a particular amino acid residue resulted in
destroyed, undesirably reduced, or unsuitable activity, variants
with such a change can be avoided. In other words, based on
information gathered from such routine experiments, one skilled in
the art can readily determine the amino acids where further
substitutions should be avoided either alone or in combination with
other mutations.
[0044] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult, 1996, Curr. Op. in
Biotech. 7:422-427; Chou et al, 1974, Biochemistry 13:222-245; Chou
et al, 1974, Biochemistry 113:211-222; Chou et al, 1978, Adv.
Enzymol Relat. Areas Mol. Biol. 47:45-148; Chou et al, 1979, Ann.
Rev. Biochem. 47:251-276; and Chou et al, 1979, Biophys. J.
26:367-384. Moreover, computer programs are currently available to
assist with predicting secondary structure. One method of
predicting secondary structure is based upon homology modeling. For
example, two polypeptides or proteins that have a sequence identity
of greater than about 30%, or similarity greater than 40% often
have similar structural topologies. The recent growth of the
protein structural database has provided enhanced predictability of
secondary structure, including the potential number of folds within
a polypeptide's or protein's structure. See Holm et al, 1999, Nucl.
Acid. Res. 27:244-247. It has been suggested (Brenner et al, 1997,
Curr. Op. Struct. Biol. 7:369-376) that there are a limited number
of folds in a given polypeptide or protein and that once a critical
number of 5 structures have been resolved, structural prediction
will become dramatically more accurate.
[0045] In one embodiment, the invention provides methods useful as
part of a treatment regimen for asthma. The methods include
administration of a pharmaceutical composition comprising a
therapeutically effective amount of a mutein of the invention, in
which amino acid 121 (arginine) and amino acid 124 (tyrosine) are
replaced with aspartic acid (IL-4RA; see FIG. 2). As provided
herein, further modification of the mutein may include one or more
of the following: the N terminus and/or C terminus of the molecule
being modified, one or more polyethylene glycol molecules being
covalently bonded to the molecule, and glycosylation sites which
are present in the molecule being partially or completely
deleted.
[0046] The terms "administration" or "administering" are defined to
include an act of providing a compound or pharmaceutical
composition of the invention to a subject in need of treatment. The
term "therapeutically effective amount" or "effective amount" means
the amount of a compound or pharmaceutical composition that will
elicit the biological or medical response of a tissue, system,
animal or human that is being sought by the researcher,
veterinarian, medical doctor or other clinician.
[0047] In one embodiment, the pharmaceutical composition includes a
mutein having an N-terminus modification that is an insertion of an
amino acid, at amino acid position+2. In another embodiment, the
mutein has a C-terminus modification that is a deletion of at least
one, at least two, at least three, at least four and at least five
amino acids. However, deletions of greater than five amino acids
from the C-terminus may affect the activity of the mutein. Activity
of the mutein from any of the modifications mentioned above and
herein can be determined by using any of the methods described
previously in related applications and/or patents, and methods
described herein (e.g., the Bimolecular Interaction Analysis (BIA)
and proliferative assays as described in U.S. application Ser. No.
10/820,559, the entire contents of which is incorporated herein by
reference.
[0048] Muteins useful in the methods of the invention may further
include glycosylation variants wherein the number and/or type of
glycosylation site has been altered compared to the amino acid
sequences of the parent polypeptide. In certain embodiments,
protein variants comprise a greater or a lesser number of N-linked
glycosylation sites than the native protein. An N-linked
glycosylation site is characterized by the sequence: Asn-X-Ser or
Asn-X-Thr, wherein the amino acid residue designated as X may be
any amino acid residue except proline. The substitution of amino
acid residues to create this sequence provides a potential new site
for the addition of an N-linked carbohydrate chain. Alternatively,
substitutions that eliminate this sequence will remove an existing
N-linked carbohydrate chain. Also provided is a rearrangement of
N-linked carbohydrate chains wherein one or more N-linked
glycosylation sites (typically those that are naturally occurring)
are eliminated and one or more new N-linked sites are created.
[0049] Additional variants include cysteine variants wherein one or
more cysteine residues are added to, deleted from or substituted
for another amino acid (e.g., serine) compared to the parent amino
acid sequence. Cysteine variants may be useful when proteins must
be refolded into a biologically active conformation such as after
the isolation of insoluble inclusion bodies. In one embodiment,
cysteine variants will have fewer cysteine residues than the native
protein, and an even number of cysteines to minimize interactions
resulting from unpaired cysteines. In another embodiment, the
cysteine variants will permit site-specific coupling of at least
one non-protein polymer, such as a polyethylene glycol (PEG)
molecule, to the mutein.
[0050] Further variants include, but are not limited to, mutations
such as substitutions, additions, deletions, or any combination
thereof, and are typically produced by site-directed mutagenesis
using one or more mutagenic oligonucleotide(s) according to methods
described herein, as well as according to methods known in the art
(see, for example, Sambrook et al, MOLECULAR CLONING: A LABORATORY
MANUAL, 3rd Ed., 2001, Cold Spring Harbor, N.Y. and Berger and
Kimmel, METHODS IN ENZYMOLOGY, Volume 152, Guide to Molecular
Cloning Techniques, 1987, Academic Press, Inc., San Diego, Calif.,
which are incorporated herein by reference).
[0051] Amino acid substitutions of the invention include those
substitutions that: (1) reduce susceptibility to proteolysis, (2)
reduce susceptibility to oxidation, (3) alter binding affinity for
forming protein complexes, (4) alter binding affinities, and/or (5)
confer or modify other physicochemical or functional properties on
such polypeptides. According to certain embodiments, single or
multiple amino acid substitutions (and in some cases, conservative
amino acid substitutions) may be made in the naturally occurring
sequence (e.g., in the portion of the polypeptide outside the
domain(s) forming intermolecular contacts).
[0052] Thus, a conservative amino acid substitution typically does
not substantially change the structural characteristics of the
nucleotide sequence (e.g., a replacement amino acid should not tend
to break a helix that occurs in the nucleotide sequence, or disrupt
other types of secondary structure that characterizes the
nucleotide sequence). Examples of art-recognized polypeptide
secondary and tertiary structures are described in PROTEINS,
STRUCTURES AND MOLECULAR PRINCIPLES, (Creighton, Ed.), 1984, W. H.
Freeman and Company, New York; INTRODUCTION TO PROTEIN STRUCTURE
(C. Branden and J. Tooze, eds.), 1991, Garland Publishing, New
York, N.Y.; and Thornton et al., 1991, Nature 354:105, each of
which are incorporated herein by reference.
[0053] Peptide analogs are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of
the template peptide. These types of non-peptide compound are
termed "peptide mimetics" or "peptidomimetics". See Fauchere, 1986,
Adv. Drug Res. 15:29; Veber & Freidinger, 1985, TINS p. 392;
and Evans et al., 1987, J. Med. Chem. 30:1229, which are
incorporated herein by reference. Such compounds are often
developed with the aid of computerized molecular modeling. Peptide
mimetics that are structurally similar to therapeutically useful
peptides may be used to produce a similar therapeutic or
prophylactic effect. Generally, peptidomimetics are structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a
biochemical property or pharmacological activity), such as a human
antibody, but have one or more peptide linkages optionally replaced
by a linkage selected from: --CH2-NH--, --CH2-S--, --CH2-CH2-,
--CH.dbd.CH-(cis and trans), --COCH2-, --CH(OH)CH2-, and --CH2SO--,
by methods well known in the art. Systematic substitution of one or
more amino acids of a consensus sequence with a D-amino acid of the
same type (e.g., D-lysine in place of L-lysine) may be used in
certain embodiments to generate more stable peptides. In addition,
constrained peptides comprising a consensus sequence or a
substantially identical consensus sequence variation may be
generated by methods known in the art (Rizo & Gierasch, 1992,
Ann. Rev. Biochem. 61:387, incorporated herein by reference); for
example, by adding internal cysteine residues capable of forming
intramolecular disulfide bridges which cyclize the peptide.
[0054] In another embodiment, the pharmaceutical composition
includes a mutein (e.g., IL-4RA) having additional amino acid
substitutions, including those substitutions that enable the
site-specific coupling of at least one non-protein polymer, such as
polypropylene glycol, polyoxyalkylene, or polyethylene glycol (PEG)
molecule to the mutein. Site-specific coupling of PEG, for example,
allows the generation of a modified mutein which possesses the
benefits of a polyethylene-glycosylated (PEGylated) molecule,
namely increased plasma half life (e.g., at least 2 to 10-fold
greater, or 10 to 100-fold greater than that of unmodified IL-4RA)
while maintaining greater potency over non-specific PEGylation
strategies such as N-terminal and lysine side-chain PEGylation.
Methods providing for efficient PEGylation are described in U.S.
application Ser. No. 10/820,559, which is incorporated herein by
reference. The IL-4 mutein must be purified properly to allow
efficient PEGylation. Purification is described in U.S. application
Ser. No. 10/820,559 (see Example 2).
[0055] The Ki of modified IL-4 mutein receptor antagonists to the
IL-4 receptor can be assayed using any method known in the art,
including technologies such as real-time Bimolecular Interaction
Analysis (BIA) as described in U.S. application Ser. No. 10/820,559
(see Example 4). The capacity of modified IL-4 mutein receptor
antagonists to inhibit the proliferative response of immune cells
can be assessed using proliferative assays as described in U.S.
application Ser. No. 10/820,559.
[0056] A number of modified IL-4 mutein receptor antagonists with
the characteristics described above have been identified in U.S.
application Ser. No. 10/820,559, by screening candidates with the
above assays. In one embodiment, a non-protein polymer (e.g.,
polyethylene glycol) is coupled to at least amino acid residue
positions 38, 102 and/or 104.
[0057] In another embodiment, the invention provides methods of
selecting the specific sites of amino acid substitution of hIL-4
that enables proper folding of the polypeptide following
expression. Modified IL-4 mutein receptor antagonists bind to IL-4
and IL-13 receptors with an affinity loss not greater than 100-fold
relative to that of unmodified IL-4RA. Modified IL-4 mutein
receptor antagonists inhibit IL-4 and IL-13 mediated activity with
a loss of potency not greater than 10-fold relative to that of
unmodified IL-4RA. In addition, modified IL-4 mutein receptor
antagonists possess a plasma half-life which is at least 2 to
10-fold greater than that of unmodified IL-4RA.
[0058] The above polypeptide variants are illustrative of the types
of modified human IL-4 polypeptides to be used in the methods
claimed herein, but are not exhaustive of the types of variations
of the claimed invention which may be embodied by the invention.
Derivatives of the above polypeptide which fit the criteria of the
claims should also be considered. All of the polypeptides and
functional fragments thereof can be screened for efficacy following
the methods taught herein and in the examples.
[0059] hIL-4 can be produced as a recombinant protein (rhIL-4) by
genetic manipulation, for example in E. coli. Host cells suitable
for the recombinant production of rhIL-4 are known to those skilled
in the art including prokaryotic cells such as strains of E. coli,
Bacillus or Pseudomonas (Kung, H.-F., M. Boublik, V. Manne, S.
Yamazaki and E. Garcia, Curr. Topics in Cell. Reg. 26: 531-542,
1995) or unicellular eukaryotic cells as yeast Saccharomyces
cerevisiae (Bemis, L. T., F. J. Geske and R. Strange, Methods Cell
Biol., 46: 139-151, 1995). Host cells for recombinant production
may also be derived from multicellular eukaryotes comprising
invertebrates as insects (Spodoptera frugiperda SD cells) (Altmann
F., E. Staudacher, I B Wilson, and L Marz, Glycoconj J., 16:
109-123, 1999) and vertebrate cells, including numerous mammalian
cell lines comprising mouse fibroblasts, Chinese hamster ovary
cells (CHO/-DHFR) (Urlaub and Chasin, Proc Nat Acad Sci 77:4216,
1980), baby hamster kidney (9BHK, ATCC CCL 10); monkey kidney CV1
line transformed by SV40 (COS-7, ATCC CRL 1651) and human embryonic
kidney cell line 293 (Tartaglia et al., Proc Nat Acad Sci 88:
9292-9296, 1991 and Pennica et al., J. Biol. Chem. 267:
21172-21178, 1992).
[0060] Any suitable host may be used to produce the IL-4 muteins of
the current invention, including bacteria, fungi (including
yeasts), plant, insect, mammal, or other appropriate animal cells
or cell lines, as well as transgenic animals or plants. As such,
these hosts may include well known eukaryotic and prokaryotic
hosts, such as strains of E. coli, Pseudomonas, Bacillus,
Streptomyces, fungi, yeast, insect cells such as Spodoptera
frugiperda (SF9), animal cells such as Chinese hamster ovary (CHO)
and mouse cells such as NS/O, African green monkey cells such as
COS 1, COS 7, BSC 1, BSC 40, and BNT 10, and human cells, as well
as plant cells in tissue culture. Although bacteria and yeast have
been the standard recombinant host cells for many recombinant
polypeptides, including rhIL-4, recently transformed cells from
higher plants are able to express human recombinant proteins as
antibodies (Hiatt, A. T. and J. K. Ma, Int. Rev. Immunol., 10:
139-152, 1993) and hemoglobin (Theisen, M. in Chemicals Via Higher
Plant Bioengineering, F. Shahidi et al., eds, Plenum Publishers,
NY, p. 211-220, 1999). Plant biotechnology offers many advantages
for efficient production of heterologous polypeptides, and this
approach may be useful for production of the modified human IL-4
receptor antagonists described herein, including mhIL-4 and its
derivatives (see also Plant Technology: New Products and
Applications, John Hammond, et al., eds., Springer, N.Y.,
1999).
[0061] The formation of the stable complex of IL-4 or IL-13 to the
type 1 or type 2 IL-4 receptor allows receptor signaling and
resultant downstream events are believed to cause asthmatic
symptoms in subjects. Thus, in one embodiment, the invention
provides a method of administering a therapeutically effective
amount of modified human IL-4 receptor antagonist, including
IL-4RA, to a subject to ameliorate symptoms associated with asthma.
Data suggests that IL-4RA binds to the IL-4 receptor .alpha. chain
with similar on and off rates as wtIL-4, which inhibits assembly of
either .gamma.c (type 1) or IL-13R.alpha. (type 2) into receptor
complexes that signal downstream events (see Table 1). Thus, IL-4RA
blocks recruitment of either IL-13R.alpha.1 or .gamma.c to form a
stable heterodimeric complex with the IL-4 receptor .alpha. chain
(A. L. Andrews et al., ATS 2004).
TABLE-US-00001 TABLE 1 Biacore binding of IL-4RA to the immobilized
IL-4 receptor .alpha. chain. Affinity K.sub.on 10.sup.6/Ms
K.sub.off 10.sup.-3/s (nM) IL-4RA 16 1.7 0.14 .+-. 0.05 (n = 7)
[0062] Although the invention describes various dosages, it will be
understood by one skilled in the art that the specific dose level
and frequency of dosage for any particular subject in need of
treatment may be varied and will depend upon a variety of factors.
These factors include the activity of the specific polypeptide or
functional fragment thereof, the metabolic stability and length of
action of that compound, the age, body weight, general health, sex,
diet, mode and time of administration, rate of excretion, drug
combination, the severity of the particular condition, and the host
undergoing therapy. Generally, however, dosage will approximate
that which is typical for known methods of administration of the
specific compound. Thus, a typical dosage of IL-4RA will be about
0.1 to 1 mg/kg. For example, for administration of IL-4RA, an
approximate dosage by aerosol inhalation would be about 0.3 mg to
60 mg. Approximate dosages include, but are not limited to about
0.3 mg, about 0.5 mg, about 1.0 mg, about 3.0 mg, about 20 mg,
about 30 mg or about 60 mg, to a subject, with dosages administered
one or more times per day or week. In another illustrative example,
an approximate dosage for administration of IL-4RA by subcutaneous
injection includes, but is not limited to, about 25 mg. Treatment
by administration of IL-4RA may span days, weeks, years, or
continue indefinitely, as symptoms persist. Hence, an appropriate
dose and treatment regimen can be determined by one of ordinary
skill in the art using routine procedures such as those provided
herein.
[0063] The compositions and formulations of the invention can be
administered systemically or locally, i.e., topically, inter alia
as an aerosol, such as a nebulized inhalation spray or a dry powder
aerosol. As used herein, "systemic administration" or "administered
systemically" refers to compositions or formulations that are
introduced into the blood stream of a subject, and travel
throughout the body of the subject to reach the part of the
subject's body in need of treatment at an effective dose before
being degraded by metabolism and excreted. Systemic administration
of compositions or formulations can be achieved, for example, by
oral application (e.g., syrups, tablets, capsules and the like),
needle injection, transdermal delivery (e.g., a composition
incorporated into a skin patch), and subdermal delivery (e.g., a
formulation in a metabolizable matrix placed beneath the skin to be
released. As used herein, "local administration" or "administered
locally" refers to compositions or formulations that are introduced
directly to part of the subject's body in need of treatment.
Compositions or formulations can be delivered locally, for example,
by injection (e.g., injection of anesthetic into a patient's gums)
or topically (e.g., creams, ointments, and sprays). It should be
understood that local administration can result in systemic levels
of the composition or formulation following administration (e.g.,
an inhaled composition may result in systemic levels of the
composition).
[0064] As used herein, the term "aerosol" refers to any gaseous
suspension of fine solid or liquid particles. As such, the term
"aerosolized" refers to being in the form of microscopic solid or
liquid particles dispersed or suspended in air or gas. A typical
microscopic solid will have a mass median aerodynamic diameter
.ltoreq.20 .mu.m. As used herein, the term "nebulize" refers to the
act of converting (a liquid) to a fine spray or atomizing.
Accordingly, the term "dry powder aerosol" refers to any
microscopic solid suspended in gas, typically air. It is also
possible for the compositions of the invention to be formulated as
a slow-release preparation. A short-term therapy or a continuous
therapy is possible in the case of all the therapy forms.
[0065] Therapeutic formulations of the IL-4 antagonist are prepared
for administration and/or storage by mixing the IL-4 antagonist,
after achieving the desired degree of purity, with pharmaceutically
and/or physiologically acceptable carriers, auxiliary substances or
stabilizers (Remington's Pharmaceutical Sciences, loc. cit.) in the
form of a lyophilisate or aqueous solutions. The term
"pharmaceutically acceptable" or "physiologically acceptable," when
used in reference to a carrier, is meant that the carrier, diluent
or excipient must be compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
[0066] In general, the pharmaceutical compositions are prepared by
uniformly and intimately bringing the active ingredient into
association with a liquid carrier or a finely divided solid carrier
or both, and then, if necessary, shaping the product into the
desired formulation. Acceptable carriers, auxiliary substances or
stabilizers are not toxic for the recipient at the dosages and
concentrations employed; they include buffers such as phosphate,
citrate, tris or sodium acetate and other organic acids;
antioxidants such as ascorbic acid; low molecular weight
polypeptides (less than approximately 10 residues), proteins such
as serum albumin, gelatin or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, arginine, leucine or lysine;
monosaccharides, disaccharides and other carbohydrates, for example
glucose, sucrose, mannose, lactose, citrate, trehalose,
maltodextrin or dextrin; chelating agents such as EDTA; sugar
alcohols such as mannitol or sorbitol; salt-forming counter-ions
such as sodium, and/or non-ionic surface-active substances such as
Tween, Pluronics or polyethylene glycol (PEG).
[0067] Such pharmaceutical compositions may further contain one or
more diluents, fillers, binders, and other excipients, depending on
the administration mode and dosage form contemplated. Examples of
therapeutically inert inorganic or organic carriers known to those
skilled in the art include, but are not limited to, lactose, corn
starch or derivatives thereof, talc, vegetable oils, waxes, fats,
polyols such as polyethylene glycol, water, saccharose, alcohols,
glycerin and the like. Various preservatives, emulsifiers,
dispersants, flavorants, wetting agents, antioxidants, sweeteners,
colorants, stabilizers, salts, buffers and the like can also be
added, as required to assist in the stabilization of the
formulation or to assist in increasing bioavailability of the
active ingredient(s) or to yield a formulation of acceptable flavor
or odor in the case of oral dosing. The muteins of the instant
invention can be administered alone, or in various combinations,
and in combination with other therapeutic compositions.
[0068] The IL-4 antagonist of the invention is normally stored in
lyophilized form or in solution. The IL-4 antagonists of the
invention are typically water soluble and available as a dry solid
to be administered as a dry powder, or reconstituted in water or
saline. Respirable powders for pulmonary delivery can be produced
by a variety of conventional techniques, such as jet milling, spray
drying, solvent precipitation, supercritical fluid condensation,
and the like.
[0069] Pulmonary delivery represents a nonparenteral mode of
administration of the drug to the circulation. The lower airway
epithelia are highly permeable to a wide range of proteins of
molecular sizes up to about 20 kDa. Micron-sized dry powders
containing the medicament in a suitable carrier such as mannitol,
sucrose or lactose may be delivered to the distal alveolar surface
using dry powder inhalers (DPI) such as those of Nektar.TM.,
Vectura (Gyrohaler.TM.), and GSK (Discus.TM.), or Astra
(Turbohaler.TM.) propellant based metered dose inhalers. Solution
formulations with or without liposomes may be delivered using
ultrasonic nebulizers such as those of PARI (LC Plus.TM.) and
Aerogen (Aeroneb Pro.TM.).
[0070] The compositions of the invention can also have formulations
whereby the modified human IL-4 receptor antagonists are in a
delayed-released format. Suitable examples of preparations having a
delayed release are, for example, semi-permeable matrices
consisting of solid hydrophobic polymers which contain the protein;
these matrices are shaped articles, for example film tablets or
microcapsules. Examples of matrices having a delayed release are
polyesters, hydrogels [e.g. poly(2-hydroxyethyl
methacrylate)--described by Langer et al., J. Biomed. Mater. Res.,
15:167-277 [1981] and Langer, Chem. Tech., 12:98-105 [1982]--or
poly(vinyl alcohol)], polyactides (U.S. Pat. No. 3,773,919, EP
58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate
(Sidman et al., Biopolymers, 22:547-556 [1983]), non-degradable
ethylene/vinyl acetate (Langer et al., loc. sit.), degradable
lactic acid/glycolic acid copolymers such as Lupron Depot.TM.
(injectable microspheres consisting of lactic acid/glycolic acid
copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric
acid (EP 133,988). While polymers such as ethylene/vinyl acetate
and lactic acid/glycolic acid enable the molecules to be released
for periods of greater than 100 days, the proteins are released
over relatively short periods of time in the case of some
hydrogels. If encapsulated proteins remain in the body over
relatively long periods of time, they can then be denatured or
aggregated by moisture at 37.degree. C., resulting in a loss of
biological activity and possible changes in immunogenicity.
Meaningful strategies for stabilizing the proteins can be
developed, depending on the mechanism involved. If it is found, for
example, that the mechanism which leads to the aggregation is based
on intermolecular S--S bridge formation as a result of
thiodisulphide exchange, stabilization can be achieved by modifying
the sulphydryl radicals, lyophilizing from acid solutions,
controlling the moisture content, using suitable additives and
developing special polymer/matrix compositions.
[0071] The formulations of the invention exhibiting delayed release
also include modified human IL-4 receptor antagonists which are
enclosed in liposomes. IL-4 antagonist-containing liposomes are
prepared by methods which are known per se: DE 3,218,121; Epstein
et al., Proc. Natl. Acad. Sci. USA, 82; 3688-3692 (1985); Hwang et
al., Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52,322; EP
36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent
Application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and
also EP 102,324. As a rule, the liposomes are of the small
(approximately 200-800 Angstrom) unilamellar type having a lipid
content of greater than approximately 30 mol % cholesterol, with
the proportion in each case being adjusted for the optimum IL-4
antagonists. Liposomes exhibiting an extended circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0072] Other formulations of the invention include albumin
microspheres, microemulsions, nanoparticles, nanocapsules and
macroemulsions. Such techniques are mentioned in Remington's
Pharmaceutical Sciences, 16th edition, Osol, A., Ed. (1980), which
is incorporated herein by reference.
[0073] The following examples are intended to illustrate but not
limit the invention.
Example 1
Fermentation of E. coli
[0074] Cells containing genes for the production of muteins were
grown in LB medium (10 g Bacto tryptone, 5 g Yeast extract, 10 g
NaCl per liter, pH 7.5) until OD600 reached 0.8-1.0. Expression was
induced by addition of IPTG to a final concentration of 0.5 mM and
incubation continued for 5 hours. Cells were harvested by
centrifugation. E. coli transformants expressing hIL4 mutant
proteins were cultured as described in U.S. Pat. No. 6,130,318.
Briefly, E. coli were fermented in LB nutrient solution of the
following composition: Bacto tryptone 10 g/l, Bacto yeast extract 5
g/l, and sodium chloride 10 g/l. The constituents were dissolved in
deionized water, which was sterilized at 121.degree. C. for 20 min.
Prior to inoculation, an antibiotic which was suitable for
selecting the transformants (e.g., 100 mg/I Na ampicillin or 50
mg/l kanamycin sulphate depending on the selection marker used in
the vector) was added to the nutrient solution under sterile
conditions. Strain stocks of all the E. coli transformants were
laid down by taking 2 ml aliquots of a preliminary culture and
storing them in liquid nitrogen. The preliminary culture
fermentations were carried out in 1 1 tr. shaking flasks which
contained 200 ml of LB nutrient solution. The nutrient solution was
inoculated with a strain stock or with a single colony from an LB
agar plate. The cultures were incubated at 30.degree. C. for 12-18
hrs. while being shaken continuously.
[0075] The main culture fermentations were carried out in LB
nutrient solution using 10 liter stirred tank fermenters. The
nutrient solution was inoculated with 1-5% by vol. of a preliminary
culture, with the biomass being centrifuged out of the preliminary
culture and resuspended in fresh LB medium prior to the
inoculation. The fermentation conditions for the 10 liter main
culture were as follows: 37.degree. C., stirrer revolution rate 500
rpm, aeration rate 0.5 vvma.
[0076] In order to monitor the growth of the biomass, sterile
samples were removed from the culture broth at intervals of approx.
1 hr., and their optical density was determined at 600 nm (OD600).
The cultures were induced when an OD600 of 0.8-1.2 had been
reached. Induction took place as follows, IPTG induction: Sterile
addition of isopropyI-.beta.-D-thio-galactopyranoside (IPTG) to a
concentration of 0.4 mM. The induction time was typically 4-8
hrs.
[0077] After the fermentation had finished (6-14 hrs.), the
contents of the fermenter were cooled down to 10-15.degree. C., and
the bacterial cells were harvested using standard centrifugation
techniques (e.g., bucket centrifuge). The cell mass which was
obtained after centrifugation was temporarily stored, where
appropriate, in the frozen state. The product was worked up from
the biomass which had been obtained in this way.
Example 2
Expression of Interleukin 4 Mutant Proteins in E. coli Using
Inducible Promoters
[0078] Surprisingly, a custom made vector system and a E.
coli-codon optimized gene of the IL-4 mutein has demonstrated that
bacteria transformed with said plasmid according to U.S. Pat. No.
6,506,590 gives expression rates, plasmid and expression stability
values many times higher than those observed after transforming the
identical hosts with plasmids known in the art.
[0079] The E. coli phage T5 promoter together with two lac operator
sequences is derived from the pQE30 plasmid (Qiagen) belonging to
the pDS family of plasmids (Bujard et al., Methods Enzymol. 155,
416-433, 1987; and Stuber et al., Immunological Methods, I.
Lefkovits and B. Pernis, eds., Academic Press, Inc., Vol. IV,
121-152, 1990).
[0080] The ribosomal binding site (rbs) is derived from the region
upstream from gene 10 of the phage T7 (T7 g 10 leader). Gene 10 of
phage T7 codes for the coat protein, which is the major protein
expressed after T7 infection. The T7 g10 rbs was obtained from the
vector pET-9a (Studier et al., Methods Enzymol. 185, 60-89, 1990).
The T7 g10 leader spans a region of about 100 bp (Olins et al.,
Gene 227-235, 1988). In the final expression construct the region
upstream of the XbaI site is deleted. The T7 g10 leader sequence
now spans 42 bp and harbors one base exchange from G to A in
position 3638 of the preferred plasmid.
[0081] As an effective measure of synonymous codon usage bias, the
codon adaptation index (CAI) can be useful for predicting the level
of expression of a given gene (Sharp et al., Nucleic Acids Res. 15,
1281-1295, 1987; and Apeler et al., Eur. J. Biochem. 247, 890-895,
1997). The CAI is calculated as the geometric mean of relative
synonymous codon usage (RSCU) values corresponding to each of the
codons used in a gene, divided by the maximum possible CAI for a
gene of the same amino acid composition. RSCU values for each codon
are calculated from very highly expressed genes of a particular
organism, e.g., E. coli, and represent the observed frequency of a
codon divided by the frequency expected under the assumption of
equal usage of the synonymous codons for an amino acid. Highly
expressed genes, e.g., genes encoding ribosomal proteins, have
generally high CAI values.gtoreq.0.46. Poorly expressed genes like
lad and trpR in E. coli have low CAI values.ltoreq.0.3. The
calculated E. coli CAI value for the natural IL-4 sequence is
0.733. This means that the natural gene should be well-suited for
high level expression in E. coli. Nevertheless, a synthetic gene
with optimal E. coli codon usage (CAI value=1) has the potential to
further increase the expression level. Therefore synthetic IL-4 and
IL-4 mutein genes were designed and cloned.
[0082] A T7 DNA fragment containing the transcription terminator
T.phi. is derived from the vector pET-9a (Studier et al., Methods
Enzymol. 185, 60-89, 1990). Transcriptional terminators determine
the points where the mRNA-RNA polymerase-DNA complex dissociates,
thereby ending transcription. The presence of a transcriptional
terminator at the end of a highly expressed gene has several
advantages: they minimize sequestering of RNA polymerase that might
be engaged in unnecessary transcription, they restrict the mRNA
length to the minimal, thus limiting energy expense, as strong
transcription may interfere with the origin of replication, a
transcriptional terminator increases plasmid stability due to copy
number maintenance (Balbas and Bolivar, Methods Enzymol. 185,
14-37, 1990).
[0083] The kan resistance gene is derived from the vector pET-9a
(Studier et al., Methods Enzymol. 185, 60-89, 1990). Originally,
this is the kan gene of Tn903 from the vector pUC4KISS (Barany,
Gene 37, 111-123, 1985). In the preferred plasmid the kan gene and
the IL-4 and IL-4 mutein gene have opposite orientations, so there
should not be an increase in kan gene product after induction due
to read-through transcription from the T5 promoter. Kanamycin was
chosen as selective marker because it is the preferred antibiotic
for GMP-purposes. In addition, kan gene based vectors are more
stable than ampicillin resistant (bla) plasmids. Ampicillin
selection tends to be lost in cultures as the drug is degraded by
the secreted .beta.-lactamase enzyme. The mode of bacterial
resistance to kanamycin relies upon an aminogly-coside
phosphotransferase that inactivates the antibiotic.
[0084] Controlled gene expression is absolutely necessary for the
set-up of a stable plasmid system, particularly if the protein of
interest is deleterious to the host cell. The preferred plasmid
uses a lac-based inducible system consisting of a lac repressor
gene (lad) and two synthetic lac operator sequences fused
downstream to the E. coli phage T5 promoter. The lacI.sup.q
promoter and the lacI structural gene were isolated from the vector
pTrc99A (Amann et al., Gene 69, 301-315, 1988). I.sup.q is a
promoter mutation which leads to overproduction of the lad
repressor. The wild-type lac repressor is a tetrameric molecule
comprising four identical subunits of 360 amino acids each. The lac
repressor tetramer is a dimer of two functional dimers. The four
subunits are held together by a four-helix bundle formed from
residues 340-360. Due to the isolation of the lad gene from the
vector pTrc99A by a NarI cut the residues beyond amino acid 331 are
deleted and 10 amino acids not normally encoded in the lad gene are
added. It is known that mutations or deletions that occur in the
C-terminal part of lacI, beyond amino acid 329, result in
functional dimers that appear phenotypically similar to the
wild-type repressor (Pace et al., TIBS 22, 334-339, 1997).
[0085] The origin of replication (ori) of the preferred plasmid is
derived from the vector pET-9a, the ori of which originates from
pBR322. The preferred plasmid therefore carries the pMBI (ColE1)
replicon. Plasmids with this replicon are multicopy plasmids that
replicate in a `relaxed` fashion. A minimum of 15-20 copies of
plasmid are maintained in each bacterial cell under normal growth
conditions. The actual number for the preferred plasmid is within
this range. Replication of the ColE1-type ori is initiated by a
555-nucleotide RNA transcript, RNA II, which forms a persistent
hybrid with its template DNA near the ori. The RNA II-DNA hybrid is
then cleaved by RNase H at the ori to yield a free 3'0H that serves
as a primer for DNA polymerase I. This priming of DNA synthesis is
negatively regulated by RNA I, a 108-nucleotide RNA molecule
complementary to the 5' end of RNA II. Interaction of the antisense
RNA I with RNA II causes a conformational change in RNA II that
inhibits binding of RNA II to the template DNA and consequently
prevents the initiation of plasmid DNA synthesis. The binding
between RNAs I and II is enhanced by a small protein of 63 amino
acids (the Rop protein, Repressor of primer), which is encoded by a
gene located 400 nucleotides downstream from the origin of
replication (Sambrook et al., Molecular Cloning, Cold Spring
Harbor, 1989). Deletion of the rop gene leads to an increase in
copy number and due to a gene dosage effect to enhanced expression
levels of the plasmid encoded heterologous gene. This observation
was also made for the IL-4 expression vectors tested. But it turned
out that rop-plasmids are instable and lost very rapidly during
fermentation under non-selective conditions. Therefore, the
replicon of the preferred plasmid contains the rop gene to ensure
high plasmid stability. The preferred plasmid lacks the mob gene
that is required for mobilization and is therefore incapable of
directing its own conjugal transfer from one bacterium to
another.
Example 3
Preparation of an IL-4 Mutant Protein
[0086] Cell disruption and isolation of the inclusion bodies: 25 g
of E. coli moist mass from Example 1 were taken up in 200 ml of
buffer (0.1 M phosphate buffer, pH 7.3, 0.1% Triton, 1 mM EDTA, 1
.mu.g/ml pepstatin) and disrupted by sonication (Branson B 15
sonifier). The inclusion bodies, which contain the product, were
isolated by centrifugation (35,000.times.g, 20 min) and washed in
disruption buffer which additionally contained 4M urea.
[0087] The washed inclusion bodies were solubilized in 125 ml of
buffer (0.2 M Tris, pH 8.1, 8M guanidine hydrochloride). 4 g of
sodium sulphite and 2 g of potassium tetrathionate were added and
the reaction mixture was stirred for 2 h. Undissolved constituents
were removed by centrifugation (35,000.times.g, 20 min) after the
reaction had finished. The supernatant was loaded onto a gel
filtration column (Sephacryl S-300 HR, Pharmacia, 10.times.90 cm)
and subjected to gel filtration in PBS buffer containing 6M
guanidine hydrochloride at a flow rate of 280 ml/h.
Product-containing fractions were identified by means of SDS-PAGE
and combined.
[0088] .beta.-Mercaptoethanol (final concentration 15 mM) was added
in order to reduce the molecules. Following a two-hour incubation
at room temperature, the mixture was diluted 5 times with water and
dialyzed against buffer (3 mM NaH.sub.2PO.sub.4, 7 mM
Na.sub.2HPO.sub.4, 2 mM KCl, 120 mM NaCl) for 3-4 days. The
dialyzed material was adjusted to pH 5.0 with acetic acid and its
conductivity was decreased to .ltoreq.10 mS/cm by adding water. 50
ml of CM Sepharose-FF (Pharmacia), which was equilibrated with 25
mM ammonium acetate, pH 5.0, were added to the mixture while
stirring. Unbound material was filtered off and the gel was used to
fill a column. The product was eluted with a linear gradient from 0
to 1 M NaCl in 25 mM ammonium acetate, pH 5.0, at a flow rate of
300 ml/h. Product containing fractions were identified by SDS-PAGE
or by analytical RP chromatography.
[0089] The pool of CM sepharose was loaded on to a Vydac C-4 column
(1.times.25 cm, 10 .mu.m) which was equilibrated with 0.1% TFA and
eluted with an increasing gradient of acetonitrile. Fractions which
contained the pure product were combined and lyophilized.
Example 4
IL-4 Mutant Protein Decreases Pre-Existing Asthma in a Therapeutic
Primate Model
[0090] The therapeutic effect of IL-4RA subcutaneous treatment on
allergen-induced airway inflammation and airway hyperresponsiveness
(an animal model of asthma) was evaluated in Cynomolgus monkeys
naturally allergic to Ascaris suum antigen as shown in (FIG. 1).
For these experiments, the study period was extended to 24 days
during which animals received inhaled antigen challenges on Days 3,
5, 7, 12, 14, 19 and 21. Airway responsiveness to inhaled
methacholine and airway cellular composition (BAL) were examined on
Days 0, 10, 17 and 24. The first treatment with IL-4RA (0.5 mg/kg,
s.c.) occurred following assessment of airway responsiveness and
airway inflammation on Day 10 and continued twice daily on each
consecutive day through to Day 23 (total of 14 days treatment).
Thus, this study was designed to assess the efficacy of IL-4RA in
reversing existing airway inflammation and airway
hyperresponsiveness.
[0091] In contrast to vehicle treatment control studies,
administration of IL-4RA (0.5 mg/kg/bid, s.c.) prevented any
further increase in airway hyperresponsiveness at study Day 17, and
reversed airway hyperresponsiveness by approximately -79% at Day 24
(p=0.018, FIG. 2a). An effect of IL-4RA treatment on airway
inflammation was also observed. Net eosinophil influx was
significantly reduced (p=0.05) at Day 17 and appeared still reduced
on Day 24 (FIG. 2b, Values are means.+-.SD, n=6).
[0092] These studies demonstrate that IL-4RA, administered
subcutaneously, can effectively reverse airway hyper-responsiveness
in the presence of continued antigen challenge suggesting that this
compound may have therapeutic utility in clinical disease.
Example 5
Study of IL-4 Mutant Protein Delivered Subcutaneously to Human
Asthmatics
[0093] The effect of subcutaneous (s.c.) IL-4RA on allergen-induced
changes in lung function, airway hyperresponsiveness and other
signs and symptoms associated with asthma was assessed in 24
asthmatics in a single centre, randomized, double blind,
placebo-controlled, parallel group study. Subjects were treated
daily with 25 mg IL-4RA s.c. (n=12) or placebo (sterile saline,
n=12) for 28 days and late asthmatic response (LAR) to allergen
challenge (as measured by the forced expiratory volume in 1 sec
(FEV1)) was evaluated as the primary endpoint. Additionally, the
effect on inhaled methacholine airway responsiveness was evaluated.
Since patients were maintained on their current therapy during the
trial, medication use was monitored along with patient reporting of
symptoms.
[0094] Relative to placebo, the IL-4RA treated group showed a 46%
improvement in LAR (p=0.05) and a 26% improvement in the maximum
fall in forced expiratory volume in 1 second (FEV1). FIG. 3 shows
the improvement in the treated group from before drug (screening
visit 2) to after the last treatment on Day 28. There was also a
trend toward improvement in methacholine airway responsiveness in
patients that received IL-4RA. In addition to positive effects on
the subjects' lung function, those who received IL-4RA reported 57%
fewer and milder asthma-related adverse events (6 events in 4
subjects with 3 requiring .beta.-agonists) than subjects who
received placebo (14 events in 6 subjects with 11 requiring
.beta.-agonists). The difference between placebo and IL-4RA treated
patients needing .beta.-agonist therapy was significant (p=0.03,
FIG. 4).
Example 6
Aerosol Characterization of an IL-4 Mutant Protein in Aeroneb
Pro.RTM. for Use in Nonhuman Primate Studies
[0095] Prior to studies in nonhuman primate the effect of
aerosolization on the stability of IL-4RA has been determined.
Solutions of IL-4RA (5 mL), citrate or lactate
formulations.+-.0.01% tween, were placed in an Aerogen Aeroneb
Pro.TM. nebulizer and the nebulizer was run to dryness. IL-4RA
samples were collected pre and post nebulization and assayed for
concentration, aggregation and activity. Protein concentration was
assessed using several methods. No differences in protein
concentration were determined via a Bradford type method or RP-HPLC
in pre and post nebulized samples. SEC measurements also
demonstrated 95-101% protein recovery in the post nebulized
samples. Additionally, SEC-HPLC demonstrated that no soluble
aggregates were present in the samples. SDS-PAGE analysis was
performed to determine if nebulization of the solutions resulted in
degradation of IL-4RA. No evidence of degradation products such as
aggregates or fragments were observed in the top of the gel,
indicative of large insoluble aggregates, or in the gel itself.
Activity of IL-4RA in the pre- and post-nebulized samples was
assessed in a. TF-1/IL-4 proliferation assay. The ability of IL-4RA
to inhibit IL-4-induced cell proliferation (EC.sub.50 approximately
0.2-0.3 nM) was not reduced following nebulization as demonstrated
by a comparison of EC.sub.50 values determined from pre and post
nebulization samples (FIG. 5). The binding activity of IL-4RA to
IL-4-receptor alpha chain in nebulized samples was measured using a
Biacore assay. The results indicated no difference between pre- and
post-nebulized samples with a Kd of approximately 0.1 nM obtained.
The data demonstrate that protein content, activity and integrity
of IL-4RA were maintained following nebulization.
Example 7
Inhalation Study of IL-4 Mutant Protein in Monkeys
[0096] The effect of aerosolized IL-4RA on allergen-induced airway
inflammation and airway hyperresponsiveness (an animal model of
asthma) was evaluated in Cynomolgus monkeys naturally allergic to
Ascaris suum antigen. The studies were performed using a 7 day
primate asthma model. Airway responsiveness to inhaled methacholine
and airway cellular composition by bronchoalveolar lavage (BAL)
were determined 2 days before (Day 0) and 2 days after (Day 7)
three consecutive-day (Days 3, 4, 5) inhalations of Ascaris suum
extract. Treatment studies were bracketed by control studies to
assure that no changes in sensitivity to antigen occurred over
time. All animals were rested 4 to 6 weeks between control and
treatment studies to allow airway responsiveness and inflammation
to return to baseline (pre-antigen) levels.
[0097] In this twice daily treatment study, IL-4RA was administered
on the afternoon of day 2, 1 hr prior and 5 hrs after antigen
challenge on days 3, 4, and 5, and in the morning and afternoon of
day 6. Inhaled IL-4RA was evaluated at nominal doses in the
nebulizer device of 0.5, 1.0, and 3.0 mg (in a volume of 3 ml).
Inhalation studies were performed using an Aerogen Aeroneb Pro
nebulizer system, coupled to a Bird Mark 7A respirator. Using this
system, IL-4RA has been shown to retain activity and integrity
following nebulization (Example 6). During aerosol delivery,
animals were ventilated via an endotracheal tube using 5
breaths/min (20 cmH.sub.20 inspiratory pressure cut-off) with a 5
sec breath hold. The change in log methacholine provocative
concentration (PC100, mg/ml) and change in BAL total cell number
and eosinophil number from Day 0 to Day 7 were determined for the
two bracketing control studies and averaged for comparison to the
treatment study.
[0098] The effects of twice daily inhaled IL-4RA on antigen-induced
airway hyperresponsiveness are shown in FIG. 6 (n=6-10,
mean.+-.SEM). Inhaled IL-4RA effectively prevented the induction of
antigen-induced airway hyperresponsiveness in a dose-dependent
manner, reaching a maximum inhibitory effect of 64% (p<0.001) at
a nominal dose of 3 mg BID.
[0099] An effect of inhaled IL-4RA on airway inflammation was also
observed. Inhaled IL-4RA delivered twice daily (BID) on
antigen-induced airway (BAL) eosinophilia (n=6-10, mean.+-.SEM), as
a marker of airway inflammation was studied. A significant
inhibitory effect of inhaled IL-4RA on allergen-induced BAL
eosinophilia (60% inhibition, p=0.003) was observed at a nominal
dose of 3 mg BID (FIG. 7). Bioavailability of IL-4RA is
approximately 6-30%.
Example 8
Aerosol Characterization of an IL-4 Mutant Protein in Pari LC
Plus.RTM. for Use in Human Asthma Studies
[0100] Prior to studies in asthmatic humans, the effect of
aerosolization on IL-4RA using the Pari LC Plus nebulizer and Pari
Pro Neb Ultra Compressor was also determined. Solutions of IL-4RA
(3 mL, lactate formulation), were placed in a Pari LC Plus.TM.
nebulizer and the nebulizer was run to dryness. IL-4RA samples were
collected pre and post nebulization and assayed for concentration,
aggregation and activity. TF-1/IL-4 proliferation assay was used to
evaluate activity of the post nebulized IL-4RA samples.
Spectrophotometry (A280) and RP-HPLC assays were used to establish
the concentration of the post nebulized sample. Integrity of IL-4RA
post nebulization was confirmed by SDS-PAGE and RP-HPLC. TF-1
proliferation assay demonstrated a pre nebulization IC50 of 0.4594
nM, a post nebulization IC50 of 0.4826 nM, indicating the activity
of IL-4RA was maintained following nebulization (FIGS. 8A and 8B).
The integrity of IL-4RA post-nebulization was also confirmed by
SDS-PAGE and RP-HPLC. In a separate study, using the same nebulizer
and compressor, the mass mean aerodynamic particle size was
determined using an Anderson Cascade impactor and found to be 4
.mu.m. The fine particle fraction (% of particle below 4.7 .mu.m
and thus of respirable size) was 57%. Using a breath simulator and
an adult breathing pattern, it was estimated that approximately 38%
of the dose was delivered to the lung.
Example 9
Inhalation Study of IL-4 Mutant Protein in Human Asthmatics
[0101] The effect of aerosolized IL-4RA on allergen-induced changes
in lung function, airway responsiveness and other signs and
symptoms associated with asthma was evaluated in 30 asthmatic
subjects. Equal numbers of subjects were randomized to receive
either IL-4RA (60 mg) or a matched volume of placebo. Treatments
were administered by nebulization from a PARI LC Plus nebulizer
which has been shown to leave IL-4RA intact and fully active
(Example 8). Subjects received twice daily administration of IL-4RA
or placebo for 27 days and a single morning dose on Day 28. Prior
to and during the study, symptoms, vital signs, ECG, exhaled nitric
oxide and lung function were periodically measured. The late
asthmatic response (LAR) to allergen challenge (as measured FEV1)
was evaluated as the primary endpoint. Additionally, blood samples
were obtained to measure IL-4RA, Anti-IL-4RA antibodies, IgE,
sIL-13R.alpha.2, IFN gamma and genetic markers of response (Single
Nucleotide Polymorphisms), as well as, standard hematology and
clinical chemistry parameters. On Day 27, the patients were
challenged with an inhaled antigen to which they had previously
demonstrated hypersensitivity, and on Day 28, airway
hyperresponsiveness was examined during inhalation challenge with
adenosine monophosphate (AMP).
[0102] On Day 27, compared to placebo, there was 72% reduction in
the late asthmatic FEV1 response after inhalation challenge
(p<0.01) to an antigen to which they had previously demonstrated
hypersensitivity. FIG. 9 shows the improvement in the treated group
from before drug (screening) to after the last treatment on Day 27.
Similarly, there was also a reduction in airway responsiveness to
AMP, compared to placebo, on Day 28. Evaluation of expired nitric
oxide, a biomarker of the severity of asthma inflammation,
indicated that with 27 days of treatment with twice daily inhaled
IL-4RA, expired nitric oxide was decreased (FIG. 10).
[0103] IL-4RA protects subjects against the development of antigen
induced decreases in lung function and decreases non specific
airway responsiveness. Both monkeys and asthmatic humans show
improvement in lung function after inhalation challenge to an
antigen to which they have a demonstrated hypersensitivity
regardless of route of exposure. The monkey data also indicate that
lung inflammation, as measured by lung lavage eosinophils, is
reduced with IL-4RA treatment, while reduced expired nitric oxide,
symptoms and .beta.-agonist use suggest a similar response occurs
in asthmatics. Evaluation of the pharmacokinetics of IL-4RA in
humans and monkeys after subcutaneous or inhalation treatment
indicates that after inhalation, the systemic dose is roughly 10
times lower than the systemic exposure after subcutaneous IL-4RA
treatment (FIG. 11, human; FIG. 12, monkey). This higher pulmonary,
but lower systemic dose, is associated with a better or similar
outcome in humans and monkeys after inhalation treatment. Taking
both the monkey and the human data together, the data suggest that
the effect of IL-4RA is directly on the lung and lung-associated
tissue (vasculature and lymph nodes) rather than primarily mediated
by other systemic organs and tissues.
[0104] Although the invention has been described with reference to
the above example, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
41129PRTHomo sapiens 1His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile
Lys Thr Leu Asn Ser1 5 10 15Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu
Leu Thr Val Thr Asp Ile 20 25 30Phe Ala Ala Ser Lys Asn Thr Thr Glu
Lys Glu Thr Phe Cys Arg Ala 35 40 45Ala Thr Val Leu Arg Gln Phe Tyr
Ser His His Glu Lys Asp Thr Arg 50 55 60Cys Leu Gly Ala Thr Ala Gln
Gln Phe His Arg His Lys Gln Leu Ile65 70 75 80Arg Phe Leu Lys Arg
Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 85 90 95Asn Ser Cys Pro
Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe 100 105 110Leu Glu
Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser 115 120
125Ser2387DNAHomo sapiens 2cacaagtgcg atatcacctt acaggagatc
atcaaaactt tgaacagcct cacagagcag 60aagactctgt gcaccgagtt gaccgtaaca
gacatctttg ctgcctccaa gaacacaact 120gagaaggaaa ccttctgcag
ggctgcgact gtgctccggc agttctacag ccaccatgag 180aaggacactc
gctgcctggg tgcgactgca cagcagttcc acaggcacaa gcagctgatc
240cgattcctga aacggctcga caggaacctc tggggcctgg cgggcttgaa
ttcctgtcct 300gtgaaggaag ccaaccagag tacgttggaa aacttcttgg
aaaggctaaa gacgatcatg 360agagagaaat attcaaagtg ttcgagc
3873130PRTArtificial sequenceSynthetic construct 3Met His Lys Cys
Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn1 5 10 15Ser Leu Thr
Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp 20 25 30Ile Phe
Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg 35 40 45Ala
Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr 50 55
60Arg Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu65
70 75 80Ile Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala
Gly 85 90 95Leu Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu
Glu Asn 100 105 110Phe Leu Glu Arg Leu Lys Thr Ile Met Asp Glu Lys
Asp Ser Lys Cys 115 120 125Ser Ser 1304396DNAArtificial
sequenceSynthetic construct 4atgcacaaat gcgatatcac cctgcaggaa
atcatcaaaa ccctgaattc tctgaccgaa 60cagaaaaccc tgtgcaccga actgaccgtt
accgacatct tcgctgcttc gaaaaacacc 120accgaaaaag aaaccttctg
ccgtgctgct accgttctgc gtcagttcta ctctcaccac 180gaaaaagaca
cccgttgcct gggtgctacc gctcagcagt tccaccgtca caaacagctg
240atccgtttcc tgaaacgtct ggaccgtaac ctgtggggtc tggctggtct
gaacagctgc 300ccggttaaag aagctaacca gtctaccctg gaaaacttcc
tggaacgtct gaaaaccatc 360atggacgaaa aagactctaa atgctcttct taataa
396
* * * * *