Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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AN INTERLEUK1N-5 ANTAGONIST
The present invention relates to modified and variant forms of Interleukin-5
molecules
capable of antagonizing or reducing the activity of IL-5 and their use in
ameliorating,
abating or otherwise reducing the aberrant effects caused by native or mutant
forms of IL-5.
Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid
sequences
referred to in the specification are defined following the description.
Throughout this specification, unless the context requires otherwise, the word
"comprise",
or variations such as "comprises" or "comprising", will be understood to imply
the inclusion
of a stated element or integer or group of elements or integers but not the
exclusion of any
other element or integer or group of elements or integers.
The rapidly increasing sophistication of recombinant DNA technology is greatly
facilitating
research and development in the medical and allied health fields. This is
particularly
important in the area of haemopoietic growth factor research where a number of
disease
conditions are predicated on the aberrant effects of native or mutant forms of
growth
factors.
One particularly important haemopoietic growth factor is IL-5. This molecule
is a
lymphokine secreted by T-cells and mast cells and is a disulfide-linked
homodimeric
glycoprotein. The human form of this molecule comprises 114 amino acids per
monomer.
IL-5 consists of a bundle of four a-helices in an up-up, down-down array. The
phenomenon
of D-helix swapping whereby one bundle is built up of three helices coming
from one
monomer and a fourth helix which is contributed by the second monomer is
unique to IL-5.
The IL-5 molecule also contains two short anti-parallel ~3-strands located
between helices A
and B and helices C and D.
Human and murine IL-5 receptors comprise two different chains, the a and ~3-
subunits.
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Human IL-5 hinds to the a-subunit but the binding affinity is increased upon
association
with the ~i-chain. The ~3-chain is shared by other cytokines such as GM-CSF
and IL-3.
IL-5 is a haemopoietic growth factor with selectivity for production and
activation of human
eosinophils. There is a need, therefore, to develop antagonists of IL-5 to act
as therapeutic
agents for chronic asthma or other disease states with demonstrated
eosinophilia or other
conditions associated with IL-5. It is also important for the IL-5 antagonist
not to interfere
with the activities of other cytokines, such as GM-CSF or IL-3.
Accordingly, one aspect of the present invention contemplates a modified IL-S
comprising
a sequence of amino acids within a first a-helix wherein one or more exposed
amino acids
in said first a-helix having acidic or acidic-like properties are substituted
with a basic amino
acid residue or a non-acidic amino acid residue.
The IL-5 which is subject to modification is generally of mammalian origin
such as from
humans, primates, livestock animals (eg. sheep, cows, pigs, horses),
laboratory test animals
(eg. mice, rats, guinea pigs, rabbits), companion animals (eg. dogs, cats} and
captive wild
animals (eg. kangaroos, foxes, deer). Most preferably, the IL-5 is of human
origin. The
modified IL-5 of the present invention may be glycosylated or unglycosylated
and does not
interfere with GM-CSF or IL-3 activity.
Even more particularly, the present invention is directed to a modified human
IL-5 molecule
comprising a sequence of amino acids wherein Glu at amino acid position 13 (or
its
equivalent position) in a first a-helix is replaced by Arg or Lys or a
chemical equivalent or
derivative thereof. An alternative substitution may also be made using non-
acidic amino
acid residues such as but not limited to Gln and Asn or their chemical
equivalent or
derivatives. A "derivative" may be a naturally occurring or synthetic amino
acid residue.
In accordance with the present invention, it is proposed that the modified IL-
5 molecules
defined above act as antagonists of the native form of IL-5. The term
"modified" is
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considered herein synonymous with terms such as "variant", "derivative" and
"mutant" , The
present invention is particularly directed to a modified IL-5 which exhibits
specific
antagonism of II,-S mitogenic effects such as observed in vitro. The modified
IL-5
molecules may be glycosylated or unglycosylated and do not interfere with GM-
CSF or IL-
3 activity.
Accordingly, another aspect of the present invention is directed to an IL-5
antagonist said
antagonist comprising an IL-5 molecule having an amino acid sequence in its
first a-helix
wherein one or more exposed amino acids in said first a-helix having acidic or
acidic-like
properties are substituted with a basic amino acid residue or a non-acidic
amino acid
residue.
More particularly, the present invention provides an antagonist of IL-S said
antagonist
comprising an IL-5 molecule with Gln at position 13 (or its equivalent
position) in a first a-
helix substituted by Arg or Lys or a chemical equivalent or derivative
thereof. An
alternatively substitution may also be made using non-acidic amino acid
residues such as but
not limited to Gln and Asn or their chemical equivalents or derivatives.
The modified IL-5 molecule of the present invention is preferably in
recombinant or
synthetic form and, with the exception of the amino acid substitutions) in the
first a-helix,
the amino acid sequence of the IL-5 may be the same as the naturally occurring
molecule
(i.e. native molecule) or may carry single or multiple amino acid
substitutions, deletions
and/or additions to the native amino acid sequence. It is then referred to as
a "mutant" IL-S.
The structure of the first a-helix of IL-5 has been determined at 2.4 angstrom
resolution by
X-ray crystallography and comprises amino acid residues 7 to 27 or their
equivalents (see
Milburn et al. Nature 363: 172-176, 1993). The modified IL-5 of the present
invention
may or may not comprise a leader sequence.
The nucleotide and corresponding amino acid sequence for the modified IL-5
having Arg in
position 13 is shown in SEQ ID NOs: 1 and 2 and Figure 1. The leader sequence
is shown
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as amino acids 1 to 6 (Met His Tyr His His His [SEQ ID N0:3]). Consequently,
amino
acids 7 to 27 are shown as amino acids 13-33 in SEQ ID NOs: 1 and 2 and in
Figure 1.
Reference to amino acids 7 to 27 is taken as amino acid residue numbers in a
molecule
without a leader sequence. The amino acid sequence for amino acids 7 to 27 is
shown as
SEQ ID N0:4 except that amino acid 13 is represented as Xaa. In accordance
with the
present invention Xaa is preferably a basic amino acid residue or a non-acidic
amino acid
residue.
Reference to "unglycosylated form" herein means that the molecule is
completely
unglycosylated such as when expressed in recombinant form in a prokaryotic
organism (e.g.
E. coli). Alternatively, a glycosylation-deficient mammalian cell may be used
or complete
deglycosylation may occur in vitro using appropriate enzymes. Different
glycosylation
patterns are encompassed by the present invention such as when recombinant
molecules are
produced in different mammalian cells.
An "exposed" amino acid is taken herein to refer to an amino acid on a solvent-
exposed or
outer portion of an a-helix compared to those amino acids orientated towards
the inside of
the molecule.
An acidic amino acid includes, for example, Glu and Asp. Preferred basic amino
acids are
Arg and Lys. Preferred non-acidic amino acids are Gln and Asn.
According to another aspect of the present invention there is provided a
modified IL,-5
characterised by:
(i) comprising a sequence of amino acids within a first a-helix;
(ii) having one or more exposed amino acids in said a-helix which have acidic
or
acidic-like properties being substituted by a basic amino acid residue or a
non-acidic amino acid residue;
(iii) being in recombinant or synthetic form; and
(iv) being capable of antagonising IL-5 mitogenic activity in vitro.
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In a related embodiment, the present invention provides an IL-5 antagonist
characterised by:
(i) comprising a sequence of amino acids within a first a-helix;
(ii) having one or more exposed amino acids in said a-helix which have acidic
or
acidic-like properties being substituted by a basic amino acid residue or a
non-acidic amino acid residue;
(iii) being in recombinant or synthetic form; and
(iv) being capable of antagonising IL-5 mitogenic activity in vitro.
The IL-5 mutant may be in glycosylated or unglycosylated form.
This aspect of the present invention is predicated in part on the finding that
a mutation in
amino acid 13 (Glu) of human (h) IL-5 to Arg results in the hIL-5 variant
being capable of
antagonising IL-5 mitogenic effects in vitro. This variant is referred to
herein as "IL-5
Argl3" or "E13 R". Such a variant would be unable to bind to high affinity
receptors but
would still able to fully bind the low affinity a chain of the IL-5 receptor.
Importantly,
the IL-5 Arg'3 mutant acts as an antagonist, preventing the stimulatory effect
of native IL-
5. A particularly important use of the IL-5 Argl3 antagonist is in reducing or
otherwise
antagonising IL-5-mediated stimulation and activation of eosinophils in vivo
or in vitro.
The antagonist may also be able to antagonise effects caused by a mutated,
endogenous
IL-5. The present invention extends to a range of other IL-5 mutants such as
IL-5 Lys'3,
IL-5 G1n13 and IL-5 Asnl3 as well as functionally equivalent mutants. The
nucleotide and
corresponding amino acid sequence for IL-5 Arg'3 (E13R) are shown in SEQ ID
NOs: 1
and 2. The present invention extends, in a particularly preferred embodiment,
to an
isolated polypeptide having an amino acid sequence substantially as set forth
in SEQ ID
N0:2 or a genetic sequence encoding same having a nucleotide sequence
substantially as
set forth in SEQ ID NO:1.
By way of a shorthand notation, both single and three letter abbreviations are
used for
amino acid residues in the subject specification and these are defined in
Table 1.
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Where a specific amino residue is referred to by its position in IL-5, a
single or three
letter amino acid abbreviation is used with the residue number given in
superscript (eg.
Xaa", wherein Xaa is the amino acid residue) and "n" is the residue number in
the
molecule.
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Table 1
Amino acid Three-letter Corresponding
abbreviation single-letter
abbreviation
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamine Gln Q
Glutamic acid Glu E
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
The present invention is exemplified using IL-5 Arg'3. This is done, however,
with the
understanding that the present invention extends to all other IL-5 variants
having
antagonistic properties to IL-5 in vitro or against eosinophils in vitro or in
vivo.
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According to another aspect of the present invention there is provided an IL-5
variant
comprising an amino acid sequence in the first a-helix of said IL-5:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu
Ile Ala
S [SEQ ID No. 4];
wherein Xaa is a basic or non-acidic amino acid residue preferably selected
from the group
consisting of Arg, Lys, Gln and Asn and wherein said variant IL-S acts as an
antagonist for
at least one property of the corresponding native IL-5. The amino acid
sequence defined by
SEQ ID N0:4 corresponds to amino acid residues 7 to 27 of human IL-5.
Preferably, Xaa is
Xaa° wherein n is amino acid position 13 of human IL-5. Preferably Xaa"
is Arg'3 or its
equivalent.
In a related embodiment, the present invention contemplates an IL-5 antagonist
comprising
a polypeptide or chemical equivalent thereof comprising amino acids 7 to 27 of
the first a-
helix of human IL-5 with the proviso that one or more exposed amino acids in
said first a-
helix having acidic or acidic-like properties are substituted by a basic amino
acid residue or
a non-acidic amino acid residue.
Preferably, the acidic or acidic-like amino acid residue if Glu or Asp and is
replaced by Arg,
Lys, Gln or Asn.
More preferably, the acidic or acidic-like amino acid residue is replaced by
Arg.
Most preferably, the amino acid sequence of the modified IL,-5 is as set forth
in SEQ ID
N0:2.
In a particularly preferred embodiment, the present invention is directed to
an IL-5
antagonist comprising a modified IL-5 molecule having the following amino acid
sequence
in its first a-helix:
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Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu
Ile Ala
[SEQ ID N0:4]
wherein Xaa is Arg;
or an IL-5 molecule having a single or multiple mutation in its first a-helix
giving
functionally similar antagonistic properties to the mutation wherein Xaa is
Arg.
In respect of the latter embodiment, the mutation in the IL-5 molecule may be
a single or
multiple amino acid substitution, deletion and/or addition or may be an
altered glycosylation
pattern amongst other mutations. Preferably, the IL-5 antagonist comprises an
amino acid
sequence as set forth in SEQ ID N0:2.
The IL-5 antagonists of the present invention and in particular IL-5 Arg'3 are
useful inter
alia in the treatment of allergy, some myeloid leukemias (such as eosinophilic
myeloid
leukaemia), idiopathic eosinophilic syndrome, allergic inflammations such as
asthma,
rhinitis and skin allergies by preventing or reducing IL,-5-mediated
activation of eosinophils.
These and other conditions are considered herein to result from or be
facilitated by the
aberrant effects of an endogenous native IL-5 or an endogenous naturally
mutated IL-5.
The present invention, therefore, contemplates a method of treatment
comprising the
administration to a mammal of an effective amount of a modified IL-5 as
hereinbefore
defined and in particular IL-5 Arg'3 for a time and under conditions
sufficient for effecting
said treatment.
Preferably, the treatment is in respect of the treatment of allergy, some
myeloid leukemias
(such as eosinophilic myeloid leukaemia), idiopathic eosinophilic syndrome,
allergic
inflammations such as asthma, rhinitis and skin allergies.
Generally, the mammal to be treated is a human, primate, livestock animal,
companion
animal or laboratory test animal. Most preferably, the mammal is a human.
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A single modified IL-S may be administered or a combination of variants of the
same IL-5.
For example, a range of IL-5 variants could be used such as a combination
selected from
two or more of IL.-5 Arg'3, IL-5 Lys'3, IL-5 Gln'3 and IL-5 Asn'3. The IL-5
variants of the
present invention are particularly useful in treating eosinophilia and
conditions resulting
S therefrom such as asthma. Administration is preferably by intravenous
administration but a
range of other forms of administration are contemplated by the present
invention including
via an implant device or other form allowing sustained release of the IL-5
variant, in a
nebuliser form or nasal spray. Modified forms of IL-5 permit entry following
topical
application are also encompassed by the present invention.
In addition to the modifications to IL-5 contemplated above, the present
invention further
provides a range of other derivatives of IL-5.
Such derivatives include fragments, parts, portions, mutants, homologues and
analogues of
the IL-S polypeptide and corresponding genetic sequence. Derivatives also
include single or
multiple amino acid substitutions, deletions and/or additions to IL,-S or
single or multiple
nucleotide substitutions, deletions and/or additions to the genetic sequence
encoding IL-5.
Derivatives also contemplated modifications to resident nucleotides.
Alteration of the
nucleotides may result in a corresponding altered amino acid sequence or
altered
glycosylation patterns amongst other effects. "Additions" to amino acid
sequences or
nucleotide sequences include fusions with other peptides, polypeptides or
proteins or fusions
to nucleotide sequences. Reference herein to "IL,-5" includes reference to all
derivatives
thereof including functional derivatives or IL-5 immunologically interactive
derivatives.
All such derivatives would be in addition to the modifications to the first a-
helix
contemplated above. Accordingly, such derivatives would be to IL-5 Arg'3, IL-5
Lys'3, IL-
5 Gln'3 or IL-5 Asn'3.
Analogues of IL-5 contemplated herein include, but are not limited to,
modification to side
chains, incorporating of unnatural amino acids and/or their derivatives during
peptide,
polypeptide or protein synthesis and the use of crosslinkers and other methods
which impose
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conformational constraints on the proteinaceous molecule or their analogues.
The present invention further contemplates chemical analogues of IL-5 capable
of acting as
antagonists or agonists of IL-5 or which can act as functional analogues of IL-
5. Chemical
analogues may not necessarily be derived from IL-5 but may share certain
conformational
similarities. Alternatively, chemical analogues may be specifically designed
to mimic
certain physiochemical properties of IL-S. Chemical analogues may be
chemically
synthesised or may be detected following, for example, natural product
screening.
Other derivatives contemplated by the present invention include a range of
glycosylation
variants from a completely unglycosylated molecule to a fully glycosylated
molecule and
from a naturally glycosylated molecule to molecules with an altered
glycosylation pattern.
Altered glycosylation patterns may result from expression of recombinant
molecules in
different host cells.
All these types of modifications may be important to stabilise the modified IL-
5 molecules
if administered to an individual or when used as a diagnostic reagent. The
modifications
may also add, complement or otherwise facilitate the antagonistic properties
of the modified
IL-5 molecules.
Reference herein to a "modified" IL-5, therefore, includes reference to an IL-
5 with an
altered amino acid sequence in the first a-helix as well as, where
appropriate, a range of
glycosylation variants, amino acid variations in other parts of the molecule,
chemical
modifications to the molecule as well as fusion molecules.
The present invention also provides a pharmaceutical composition comprising
the modified
IL-5 molecules as hereinbefore defined or combinations thereof.
Accordingly, the present invention contemplates a pharmaceutical composition
comprising a
a modified IL-5, said modified IL-5 comprising a sequence of amino acids with
a first a-
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helix wherein one or more exposed amino acids in said first a-helix having
acidic or acidic-
like properties are substituted with a basic amino acid residue or non-acidic
amino acid
residue, said composition further comprising one or more pharmaceutically
acceptable
carriers and/or diluents.
In a related embodiment, the present invention provides a pharmaceutical
composition
comprising a modif=ied human IL-5 or a mammalian homologue thereof said
modified IL-5
comprising a sequence of amino acids in a first a-helix of:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu
Ile Ala
[SEQ ID N0:4]
wherein Xaa is a basic non-acidic amino acid residue preferably selected from
Arg, Lys, Gln
and Asn, said composition further comprising one or more pharmaceutically
acceptable
carriers and/or diluents. Preferably, Xaa is Xaa" where n is amino acid
position 13.
Preferably, Xaa is Arg.
In another related embodiment, the present invention contemplates a
pharmaceutical
composition capable of antagonising IL-5, said composition comprising a
modified IL-5
having an amino acid sequence in its first a-helix:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu
Ile Ala
[SEQ ID N0:4]
wherein Xaa is selected from Arg, Lys, Gln and Asn.
Preferably, Xaa is Arg.
The pharmaceutical compositions may also contain other pharmaceutically active
molecules
including other IL-5 variants. The modified IL-5 molecule and other components
in a
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pharmaceutical composition are referred to below as "active agents" or "active
compounds".
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions
(where water soluble) or dispersions and sterile powders for the
extemporaneous preparation
of sterile injectable solutions or dispersions. It must be stable under the
conditions of
manufacture and storage and must be preserved against the contaminating action
of
microorganisms such as bacteria and fungi. The carrier can be a solvent or
dispersion
medium containing, for example, water, mannitol glycine or suitable mixtures
thereof. The
preventions of the action of microorganisms can be brought about by various
antibacterial
and antifungal agents, for example, parabens, chiorobutanol, phenol, sorbic
acid,
thirmerosal and the like. In many cases, it will be preferable to include
isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the injectable
compositions
can be brought about by the use in the compositions of agents delaying
absorption.
Sterile injectable solutions are prepared by incorporating the active
compounds in the
required amount in the appropriate solvent with various of the other
ingredients enumerated
above, as required, followed by filtered sterilization. Generally, dispersions
are prepared by
incorporating the various sterilized active ingredient into a sterile vehicle
which contains the
basic dispersion medium and the required other ingredients from those
enumerated above.
In the case of sterile powders for the preparation of sterile inj ectable
solutions, the preferred
methods of preparation are vacuum drying and the freeze-drying technique which
yield a
powder of the active ingredient plus any additional desired ingredient from
previously
sterile-filtered solution thereof. Mannitol glycine is a particularly useful
formulation
especially when the modified IL-5 molecules are given as an intravenous drip.
The present invention also extends to forms suitable for inhaling such as a
nasal spray as
well as in nebulizer form. Alternatively, sustained release compositions may
be formulated
as well as a range of implant devices. When suitably modified, the molecules
may also be
given as a cream, lotion or gel. A suitable carrier for a cream, lotion or gel
includes a
polyol such as but not limited to glycerol, propylene glycol, liquid
polyethylene glycol and
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the like.
Pharmaceutically acceptable carriers and/or diluents include any and all
solvents, dispersion
media, and antibacterial and antifungal agents. The use of such media and
agents for
pharmaceutical active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active ingredient, use
thereof in the
therapeutic compositions is contemplated by the present invention.
Supplementary active
ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage
unit form for
ease of administration and uniformity of dosage. Dosage unit form as used
herein refers to
physically discrete units suited as unitary dosages for the mammalian subjects
to be treated;
each unit containing a predetermined quantity of active material calculated to
produce the
desired therapeutic effect in association with the required pharmaceutical
carrier. The
specification for the novel dosage unit forms of the invention are dictated by
and directly
dependent on (a) the unique characteristics of the active material and the
particular
therapeutic effect to be achieved, and (b) the limitations inherent in the art
of compounding
such an active material for the treatment of disease in living subjects having
a diseased
condition involving or facilitated by aberration of IL-S molecules or levels
of IL-5.
The principal active ingredient is compounded for convenient and effective
administration
in effective amounts with a suitable pharmaceutically acceptable carrier in
dosage unit form
as hereinbefore disclosed. A unit dosage form can, for example, contain the
principal active
compound in amounts ranging from 0.5 ug to about 2000 mg. Expressed in
proportions, the
active compound is generally present in from about 0.5 gg to about 2000 mg/ml
of carrier.
In the case of compositions containing supplementary active ingredients, the
dosages are
determined by reference to the usual dose and manner of administration of the
said
ingredients.
The pharmaceutical composition may also comprise genetic molecules such as a
vector
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capable of transfecting target cells where the vector carries a nucleic acid
molecule capable
of modulating modified IL-5 expression or modified IL-5 activity. The vector
may, for
example, be a viral vector.
Still another aspect of the present invention is directed to antibodies to the
modified IL-5
molecules and their derivatives. Such antibodies may be monoclonal or
polyclonal and may
be specifically raised to modified IL-5 or derivatives thereof. In the case of
the latter, a
modified IL-5 or its derivatives may first need to be associated with a
carrier molecule. The
antibodies and/or recombinant modified IL-5 or its derivatives of the present
invention are
particularly useful as therapeutic or diagnostic agents.
For example, a modified IL-5 and its derivatives can be used to screen for
naturally
occurring antibodies to IL-5. These may occur, for example in some autoimmune
diseases.
Alternatively, specific antibodies can be used to screen for a modified or
mutant IL-5.
I 5 Techniques for such assays are well known in the art and include, for
example, sandwich
assays and ELISA. Knowledge of IL-5 levels may be important for diagnosis of
certain
cancers or a predisposition to cancers or for monitoring certain therapeutic
protocols when
modified IL-5 molecules are employed.
Antibodies to modified IL-5 of the present invention may be monoclonal or
polyclonal.
Alternatively, fragments of antibodies may be used such as Fab fragments.
Furthermore,
the present invention extends to recombinant and synthetic antibodies and to
antibody
hybrids. A "synthetic antibody" is considered herein to include fragments and
hybrids of
antibodies. The antibodies of this aspect of the present invention are
particularly useful for
immunotherapy and may also be used as a diagnostic tool for assessing
apoptosis or
monitoring the program of a therapeutic regimen.
For example, specific antibodies can be used to screen for modified IL-5
proteins. The
latter would be important, for example, as a means for screening for levels of
modified IL-5
in a cell extract or other biological fluid or purifying modified IL-5 made by
recombinant
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means from culture supernatant fluid. Techniques for the assays contemplated
herein are
known in the art and include, for example, sandwich assays and ELISA.
It is within the scope of this invention to include any second antibodies
(monoclonal,
polyclonal or fragments of antibodies or synthetic antibodies) directed to the
first mentioned
antibodies discussed above. Both the first and second antibodies may be used
in detection
assays or a first antibody may be used with a commercially available anti-
immunoglobulin
antibody. An antibody as contemplated herein includes any antibody specific to
any region
of modified IL-5.
Both polyclonal and monoclonal antibodies are obtainable by immunization with
the
enzyme or protein and either type is utilizable for immunoassays. The methods
of obtaining
both types of sera are well known in the art. Polyclonal sera are less
preferred but are
relatively easily prepared by injection of a suitable laboratory animal with
an effective
amount of modified IL-5 or antigenic parts thereof, collecting serum from the
animal, and
isolating specific sera by any of the known immunoadsorbent techniques.
Although
antibodies produced by this method are utilizable in virtually any type of
immunoassay, they
are generally less favoured because of the potential heterogeneity of the
product.
The use of monoclonal antibodies in an immunoassay is particularly preferred
because of
the ability to produce them in large quantities and the homogeneity of the
product. The
preparation of hybridoma cell lines for monoclonal antibody production derived
by fusing
an immortal cell line and lymphocytes sensitized against the immunogenic
preparation can
be done by techniques which are well known to those who are skilled in the
art.
Another aspect of the present invention contemplates a method for detecting
modified IL-5
in a biological sample from a subject said method comprising contacting said
biological
sample with an antibody specific for modified IL-5 or its derivatives or
homologues for a
time and under conditions sufficient for an antibody-modified IL-5 complex to
form, and
then detecting said complex.
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The present invention also contemplates genetic assays such as involving PCR
analysis to
detect a modified IL-5 gene or its derivatives. Alternative methods include
direct nucleotide
sequencing or mutation scanning such as single stranded conformation
polymorphism
analysis (SSCP} as well as specific oligonucleotide hybridisation.
The present invention extends to nucleic acid molecules encoding a modified IL-
5 of the
present invention. Such nucleic acid molecules may be DNA or RNA. When the
nucleic
acid molecule is in DNA form, it may be genomic DNA or cDNA. RNA forms of the
nucleic acid molecules of the present invention are generally mRNA.
Although the nucleic acid molecules of the present invention are generally in
isolated form,
they may be integrated into or ligated to or otherwise fused or associated
with other genetic
molecules such as vector molecules and in particular expression vector
molecules. Vectors
and expression vectors are generally capable of replication and, if
applicable, expression in
one or both of a prokaryotic cell or a eukaryotic cell. Preferably,
prokaryotic cells include
E. coli, Bacillus sp and Pseudomonas .sp. Preferred eukaryotic cells include
yeast, fungal,
mammalian and insect cells.
Accordingly, another aspect of the present invention contemplates a genetic
construct
comprising a vector portion and a mammalian and more particularly a human
modified IL-5
gene portion, which modified IL-5 gene portion is capable of encoding an
modified IL-5
polypeptide or a functional or immunologically interactive derivative thereof.
Preferably, the modified IL-5 gene portion of the genetic construct is
operably linked to a
promoter on the vector such that said promoter is capable of directing
expression of said
modified IL-5 gene portion in an appropriate cell.
In addition, the modified IL-5 gene portion of the genetic construct may
comprise all or part
of the gene fused to another genetic sequence such as a nucleotide sequence
encoding
glutathione-S-transferase or part thereof.
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Accordingly, another aspect of the present invention contemplates an isolated
nucleic acid
molecule comprising a sequence of nucleotides encoding or complementary to a
sequence
encoding a modified IL-5, said modified IL-5 comprising a sequence of amino
acids with a
first a-helix wherein one or more exposed amino acids in said first a-helix
having acidic or
acidic-like properties are substituted with a basic amino acid residue or a
non-acidic amino
acid residue.
Preferably the sequence of nucleotides encodes a human modified IL-5 or a
mammalian
homologue which comprises a sequence of amino acids in a first a-helix of:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu
Ile Ala
[SEQ ID N0:4]
wherein Xaa is a basic or non-acidic amino acid residue preferably selected
from Arg, Lys,
Gln and Asn and wherein said modified IL,-S exhibits antagonism of IL-5
induced mitogenic
effects. Preferably, Xaa is Xaa" where n is amino acid position 13.
Preferably, Xaa is Arg.
The present invention extends to such genetic constructs and to prokaryotic or
eukaryotic
cells comprising same.
The present invention further contemplates the use of a modified IL,-5 as
hereinbefore
defined in the manufacture of a medicament in the treatment of allergy, some
myeloid
leukemias (such as eosinophilic myeloid leukaemia), idiopathic eosinophilic
syndrome,
allergic inflammations such as asthma, rhinitis and skin allergies.
The present invention is further described by reference to the following non-
limiting
Examples and/or Figures.
In the Figures:
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Figure 1 is (a) a diagrammatic representation of the IL-5 E13R bacterial
expression plasmid
pPP31 and (b) DNA/amino acid sequence of MHYH3-IL-5 (E13R). The E13R mutation
is
circled.
S Figure 2 is a graphical representation showing the titration E13R for its
ability to
antagonise IL-5-mediated proliferation of TF 1.8 cells.
f IL-5 WT
IL-5 E 13 R
-f- WT@ 3ng/ml+E13R
Figure 3A shows the titration of E13R for its ability to antagonise IL-5-
mediated
proliferation of TF1-8 cells. This figure also shows that E13R exhibits no
detectable agonist
activity.
"'~ IL-5 WT
"~ IL-5 E13R
IL-5 + 11,-5 E 13 R
Figure 3B shows the failure of E13R to antagonise IL-3-mediated proliferation
of TF1.8
cells.
-~- IL-5
-~- IL-S E13R
-~- IL-3 + IL-5 E13R
Figure 3C 5hOW5 the failure of E13R to antagonise GM-CSF-mediated
proliferation of
TF 1.8 cells.
3 0 -~- GM
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IL-S E13R
-~- GM + IL-5 E 13 R
Figure 4 is a graphical representation showing the titration of IL-3 for its
mitogenic effects
S on TF1.8 cells and the failure of high levels of E13R to interfere with this
activity.
iL-5 WT
.~ IL-3
E 13R@ l 00ng/ml+IL3
Figure 5 is a graphical representation showing TF 1.8 proliferation assay for
IL-5.
-~-- IL-5 WT
-~- IL-5 E 13R
-f- WT ILS@3ng/ml+E13R
-~-- WT ILS (HI)
WT IL-5 (HI) was prepared in inclusion bodies in bacteria, dimerized in 2M
urea, purified
by reverse phase HPLC, concentrated in buffer exchange in PBS. It was never
dried down.
IL-5 WT was prepared in the same way as WT IL-5 (HI) but after concentration,
the
preparation was dried down and dissolved in 25mM glycine and 1.25 mg/ml of
mannitol.
Figure 6 is a graphical representation showing the titration of GM-CSF for its
mitogenic
effects on TF1.8 cells and the failure of high levels of E13R to interfere
with this activity.
t IL-5 WT
-o-- GM
~-- E13R@1000ng/ml+GM
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Figure 7 is a graphical representation showing that E13R inhibits IL-5 induced
eosinophil
antibody-dependent cell-mediated cytotoxicity.
- E 13 R+IL- 5
-~- E13R+GM-CSF
In the absence of GM-CSF and IL-~, the %''Cr-release was 4%.
Figure 8 is a graphical representation showing that E 13R inhibits IL-5
induced eosinophil
colony formation.
"~ E 13 R+IL-5
~- E 13 R+GM-C SF
No eosinophil colonies were detected absent of GM-CSF and IL-5.
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EXAMPLE 1
Production of a charge-reversal mutant of interleukin 5(IL-5) with
IL-5-antagonistic properties
The inventors expressed and purified a charge-reversal mutant of IL-5 in which
the
glutamate residue at position 13 (E 13) is replaced by an arginine residue (R)
[E I3Rj. This
mutant, E13R, shows specific antagonism of IL-5 mitogenic effects in vitro. In
order to
maximise expression, this protein is presently being expressed as a fusion
protein with the
leader sequence MHYH3 (MHYHHH) [SEQ ID N0:3]. The leader sequence comprises
amino acids 1 to 6 in SEQ ID NOs: 1 and 2. However, there is no reason to
believe that
removal of this leader sequence (or its replacement by another leader
sequence) will affect
the antagonistic effects of E I 3R. A diagrammatic representation of MHYH3 and
the
sequence of E 13R is shown in Figure 1. The nucleotide and corresponding amino
acid
sequence of E13R is represented in SEQ ID NOs: 1 and 2.
1 S EXAMPLE 2
Production of a bacterial expression plasmid encoding MHYH3IL-5 (E13R)
Human IL-5 E13R mutant coding sequence was derived from IL-5 wild-type
sequence, by
site-specific mutagenesis.
Codon 13 was changed to an Arg codon
CAG ~ CGT
The additional codon changes 1 fisted here were based on bacterial codon
preferences
to promote translational efficiency in E. coli, and did not lead to amino acid
changes.
codon 82: CAA ~ CAG
codon 83: AAA ~ AAG
codon 84: AAA ~ AAG
codon 90: AGA ~ CGT
codon 91: CGG ~ CGT
codon 92: AGA ~ CGT
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codon 97: CTA ~ CTG
codon 110: GAG ~ GAA
codon 112: ATA ~ ATC
codon 113 ATA ~ ATC
codon 115 AGT ~ TCC.
The mutant DNA fragment was modified by polymerase chain reaction using
primers to
generate a sequence encoding the leader MHYH~ at the 5' end, and a stop codon
at the 3'
end. The amplified fragment was subcloned into the plasmid pEC611 to generate
the
expression plasmid pPP3l, containing the trc promoter.
EXAMPLE 3
Production, isolation and refolding of MHYH3 IL-5 (E13R)
Fermentation is by the Fed-Batch method. Escherichia coli MM294/pACYCLacIq
transformed with the E13R-expressing plasmid pPP31 is grown in minimal medium
at
37 °C. The pH is held constant at pH 7.0 during bacterial growth by the
addition of
NH40H. Expression of IL-5 (E13R) is induced at high cell density (OD~> 20)
either by
addition of IPTG or by feeding with lactose. Induction continues for S hours
(not
including lag phase) with the protein being expressed as insoluble inclusion
bodies (IBs).
Cells are lysed by high pressure homogenization. A primary separation of IBs
from
cellular debris is done by differential centrifugation, after which the IBs
are washed and
homogenized one more time. A final centrifugation step isolates IBs of
sufficient
cleanliness for refolding.
The conditions for fermentation, induction, isolation and refolding are as
follows:
1. FERMENTATION
A. Inoculation
1. Escherichia coli MM294/pACYCIacIq carrying pPP31 was streaked out from a
80°C glycerol stock onto a minimal medium plate containing ampicillin
(100~g/ml) and
kanamycin (30~,g/ml) and grown overnight at 37°C.
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2. Multiple colonies were assessed for IL-5 (E13R) expression levels by
microscopic
examination of 20m1 shake flask cultures containing inducer, or by colony size
on agar
plates with or without inducer, in this instance with IPTG.
3. The selected single colony from an agar plate was transferred into 20m1 of
nitrogen-rich minimal medium (2) containing amplicillin ( 100 icg/ml) +
kanamycin (30
~cg/ml). The culture was grown at 37°C overnight with agitation.
4. An amount of lOL of C2 was sterilised in a 22 L fermenter and inoculated to
a
very low density with overnight culture. Growth was at 37°C and a pH of
7.0 was
maintained by the addition of NH40H or HzS04.
5. Agitation was manually controlled and aeration automatically controlled
with
oxygen saturation levels remaining above 10 % v/v p02. Following depletion of
glucose at
16-20 hours the cell mass was fed with a concentrated glucose solution
containing
additional salts. Nutrient feed flow rate was determined by pH or oxygen
saturated levels.
B. Induction
1. At an optical density of Aboo = >20 the recombinant expression of E13R
antagonist was induced, in this instance by changing to a lactose based
nutrient feed.
Induction continued for 5 hours at 37 ° C, pH 7Ø Samples were removed
at immediately
preceding induction and each hour post-induction and examined by microscopy
for the
presence of IB, their size being determined by disc centrifugation.
2. The culture was stored overnight at 4°C.
2. PRIMARY ISOLATION OF IL-5 (E13R) INCLUSION BODIES
1. Homogenization - Step 1
The culture was passed five times through a Gaulin 30CD homogenizer at 13,500
psi,
with homogenate being cooled between passes. Homogenate was then diluted with
an
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equal volume of RO H20.
The IB size was redetermined by disc centrifugation.
2. Centrifugation - Step 1
The homogenate was centrifuged in a Westfalia SB-7 separator with a constant
speed of
9,210 rpm at a flow rate determined by IB size.
The concentrate collected from this first step was diluted to s2.5 % w/v.
3. Homogenization - Step 2
The IB suspension was passed once through a Gaulin 30CD homogenizer at 13,500
psi.
The IB size was again determined by disc centrifugation.
4. Centrifugation - Step 2
The homogenate was centrifuged in a Westfalia SA-1 separator with a constant
speed of
9,700 rpm at a flow rate determined by IB size.
For refolding, IBs are initially dissolved in 6M Guanidine-HCI) containing 5-
lOmM
dithiothretiol and buffered to pH 9.5. Reduced IL-5 monomers of MHYH3IL-
5(E13R) are
then purified from non-reduced protein by Size Exclusion Chromatography (eg.
Superose
12 [Pharmacia]) in 6M Guanidine-HCI, containing 5mM dithiothreitol and
buffered to pH
9.5. The purified reduced monomer is then refolded into dimers by dilution
into 2M urea
buffered to pH 9.5 at a final protein concentration of O.OI-0.1 mg/ml.
Purification of
correctly folded dimers is by reversed phase chromatography on a High
Performance
Liquid Chromatograph (HPLC) using a suitable column (eg. Brownlee butyl-silica
employing 0.1 % v/v trifluoroacetic acid (TFA) in water as Buffer A and 0.1 %
v/v TFA in
acetonitrile as Buffer B). Purified, correctly folded protein can be recovered
in biological
buffers after lyophilization from HPLC buffers in the presence of mannitol and
glycine,
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added as bulking excipient agents. The identity of the purified protein can be
confirmed
using mass spectrometry and N-terminal sequencing.
EXAMPLE 4
Assay for II-5-antagonistic activity
The biological assay for IL-5 antagonism by E13R uses incorporation of
radiolabelled
thymidine to detect IL-5-induced cellular proliferation. The cell line used,
TF1.8, is a
subline of TF 1, a human erythroleukemic cell line which expresses the
receptors for IL-3
and GM-CSF. TF 1. 8 differs from TF 1 in that it has been selected for
expression of the
IL-5 receptor. Stimulation of TF1.8 proliferation by IL-5 is inhibited by 50%
in the
presence of a 30-fold excess of E13R, and abolished in the presence of a 300-
fold excess
(Figure 2). However, stimulation of TF1.8 proliferation by IL3 or GM-CSF is
unaffected
by the presence of E13R. These results demonstrate that E13R is a specific and
potent
antagonist of IL-5. The 3H-thymidine assay was conducted as follows:
Wash cells 24hrs, prior to assay (1500rpm/Smin/RT) and resuspend them in
cytokine free medium.
Dilute standards and samples to concentrations required, in culture medium
mentioned above (range between 1000ng/ml-lpg/ml as example). Dilutions were
done in triplicate.
Aliquot dilutions of appropriate standards (GM-CSF, IL-3, IL-5, etc.) and
samples
in 100u1 final volumes, in sterile 96 well, flat bottom microtitre plates.
Check viability of washed cells and resuspend cells to a concentration of 5 x
104
cells/well.
Each cell contains 100u1 of diluted standard/sample + 100u1 resuspended cells.
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Plates are incubated @ 37 ° C for 72 hrs, in a humidified COZ incubator
(48 hrs, for
TF-1 & TF I . 8 cells).
Pulse cells with 3H-Thymidine (I/20 dilution of stock, then add IOuI/well)
O.SuCI/well.
Incubate plates @ 37°C for 5 hrs in a humidified COZ incubator.
Harvest contents of each well onto filterpaper using cell harvester and
determine
the radioactivity incorporated into the DNA, by liquid scintillation counting.
The results are shown in Figures 2 to 6. The results show that E13R
specifically
antagonises IL-5 activity but does not interfere with GM-CSF or IL-3 activity.
In
particular, the Figures show that E13R does not antagonise TF1.8 cell
proliferation
1 S induced by IL-3 nor does it antagonise TFI .8 cell proliferation induced
by GM-CSF.
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EXAMPLE 5
hIL-5 Lys"
Thr Ser Ala Leu Val Lys Lys Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu
Ile Ala
[SEQ ID NO. 5]
EXAMPLE 6
hIL-5 Arg'3
Thr Ser Ala Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu
Ile Ala
[SEQ ID NO. 6]
EXAMPLE 7
hIL-5 Gtn"
Thr Ser Ala Leu Val Lys Gln Thr Leu Ala Leu La Ser Thr His Arg Thr Leu Leu Ile
Ala
[SEQ ID NO. 7]
EXAMPLE 8
hIL-5 Asn"
Thr Ser Ala La Val Lys Asn Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu Ile
Ala
[SEQ ID NO. 8]
EXAMPLE 9
Antagonistic properties of E13R on eosinophils
An antibody dependent cell-mediated cytotoxicity (ADCC) assay was used to
determine the
effect of E13R on eosinophil function. This assay measures the ability of
eosinophils to kill
target cells radiolabelled with chromium and expressing a certain antibody
recognized by
the eosinophils (Vadas et al. J Immunol 130: 795, 1983). Blood was collected
from a
healthy donor and eosinophils were prepared by metrizamide gradient separation
of
leukocytes after removal of red cells with dextran (Vadas et al. J Immunol
122: 1228, 1979).
The eosinophil purity exceeded 95%. The eosinophils were then incubated with
the target
cells at a ratio of 100 eosinophils per target cell, and a fixed dose of
either GM-CSF (10
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ng/ml) or IL-5 (10 ng/ml) and a titration of E13R. Figure 7 shows that E13R
dose-
dependently antagonised IL-5-induced ADCC, but had no effect on GM-CSF-induced
ADCC. Values are mean and sem of triplicates.
EXAMPLE 10
Effect of E13R on proliferation and differentiation of eosinophilic
progenitors
Bone marrow was collected from a healthy donor and subjected to density
centrifugation
(Lymphoprep.). The mononuclear cells were plated in agar supplemented with a
fixed dose
of either GM-CSF ( 10 ng/ml) or IL-5 ( 10 ng/ml) and a titration of E 13R. The
cells were
allowed to grow at 37°C for 2 weeks. The agar plates were then fixed in
gluteraldehyde
before the plates were stained with Luxol Fast Blue to detect eosinophilic
colonies. Figure 8
shows that E13R dose-dependently antagonized the colony-promoting effects of
IL-5, while
E13R had no effect on GM-CSF stimulation of eosinophilic colony formation.
Values are
mean and sem of triplicates.
Those skilled in the art will appreciate that the invention described herein
is susceptible to
variations and modifications other than those specifically described. It is to
be understood
that the invention includes all such variations and modifications. The
invention also
includes all of the steps, features) compositions and compounds referred to or
indicated in
this specification, individually or collectively, and any and all combinations
of any two or
more of said steps or features.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: BRESAGEN LIMITED and MEDVET SCIENCE PTY. LTD.
(ii) TITLE OF INVENTION: AN INTERLEUKIN-5 ANTAGONIST
(iii) NUMBER OF SEQUENCES: 8
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: FETHERSTONHAUGH & CO.
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,26,368
2 0 (B) FILING DATE: 23-MAY-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: AU PO 0054
(B) FILING DATE: 24-MAY-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FETHERSTONHAUGH & CO.
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 23199-221
(ix) TELECOMMUNICATION INFORMATION:
3 0 (A) TELEPHONE: (613)-235-4373
(B) TELEFAX: (613)-232-8440
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
23199-221
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- 30a -
(A) LENGTH: 377 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 4..366
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CAT ATG CAC TAT CAC CAT CAC ATC CCC ACA GAA ATT CCC ACA AGT GCA 48
Met His Tyr His His His Ile Pro Thr Glu Ile Pro Thr Ser Ala
1 5 10 15
TTG GTG AAA CGT ACC TTG GCA CTG CTT TCT ACT CAT CGA ACT CTG CTG 96
Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu
25 30
ATA GCC AAT GAG ACT CTG AGG ATT CCT GTT CCT GTA CAT AAA AAT CAC 144
2 0 Ile Ala Asn Glu Thr Leu Arg Ile Pro Val Pro Val His Lys Asn His
35 40 45
CAA CTG TGC ACT GAA GAA ATC TTT CAG GGA ATA GGC ACA CTG GAG AGT 192
Gln Leu Cys Thr Glu Glu Ile Phe Gln Gly Ile Gly Thr Leu Glu Ser
50 55 60
CAA ACT GTG CAA GGG GGT ACT GTG GAA AGA CTA TTC AAA AAC TTG TCC 240
Gln Thr Val Gln Gly Gly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser
65 70 75
TTA ATA AAG AAA TAC ATT GAC GGC CAG AAG AAG AAG TGT GGA GAA GAA 288
Leu Ile Lys Lys Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu Glu
80 85 90 95
CGT CGT CGT GTA AAC CAA TTC CTG GAC TAC CTG CAA GAG TTT CTT GGT 336
Arg Arg Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly
100 105 110
GTA ATG AAC ACC GAA TGG ATC ATC GAA TCC TGATGAAGCT T 377
4 0 Val Met Asn Thr Glu Trp Ile Ile Glu Ser
115 120
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 121 amino acids
(B) TYPE: amino acid
50 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
23199-221
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- 30b -
Met His Tyr His His His Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu
1 5 10 15
Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu Ile
20 25 30
Ala Asn Glu Thr Leu Arg Ile Pro Val Pro Val His Lys Asn His Gln
35 40 45
Leu Cys Thr Glu Glu Ile Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln
50 55 60
Thr Val Gln Gly Gly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser Leu
65 70 75 80
Ile Lys Lye Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg
85 90 95
Arg Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val
100 105 110
Met Asn Thr Glu Trp Ile Ile Glu Ser
115 120
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
3 0 (A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met His Tyr His His His
1 5
(2) INFORMATION FOR SEQ ID N0. 4:
4 0 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0. 4:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu
5 10 15
23199-221
Leu Ile Ala
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(2) INFORMATION FOR SEQ ID N0. 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
10 (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0. 5:
Thr Ser Ala Leu Val Lys Lys Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu
1 5 10 15
Leu Ile Ala
20 20
(2) INFORMATION FOR SEQ ID NO. 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: amino acid
(C) STRANDEDNESS: single
3 0 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 6:
Thr Ser Ala Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu
1 5 10 15
Leu Ile Ala
40
(2) INFORMATION FOR SEQ ID NO. 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: amino acid
(C) STRANDEDNESS: single
23199-221
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20
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0. 7:
Thr Ser Ala Leu Val Lys Gln Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu
1 5 10 15
Ile Ala
(2) INFORMATION FOR SEQ ID N0. 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 8:
Thr Ser Ala Leu Val Lys Asn Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu
1 5 10 15
Leu Ile Ala
23199-221
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-31 -
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 377 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 4..366
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CAT ATG CAC TAT CAC CAT CAC ATC CCC ACA GAA ATT CCC ACA AGT GCA 48
Met His Tyr His His His Ile Pro Thr Glu Ile Pro Thr Ser Ala
1 5 10 15
TTG GTG AAA CGT ACC TTG GCA CTG CTT TCT ACT CAT CGA ACT CTG CTG 96
Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu
20 25 30
ATA GCC AAT GAG ACT CTG AGG ATT CCT GTT CCT GTA CAT AAA AAT CAC 144
Ile Ala Asn Glu Thr Leu Arg Ile Pro Val Pro Val His Lys Asn His
35 40 45
CAA CTG TGC ACT GAA GAA ATC TTT CAG GGA A7.'A GGC ACA CTG GAG AGT 192
Gln Leu Cys Thr Glu Glu Ile Phe Gln Gly Il.e Gly Thr Leu Glu Ser
50 55 60
CAA ACT GTG CAA GGG GGT ACT GTG GAA AGA CTA TTC AAA AAC TTG TCC 240
Gln Thr Val Gln Gly Gly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser
65 70 75
TTA ATA AAG AAA TAC ATT GAC GGC CAG AAG AAG AAG TGT GGA GAA GAA 288
Leu Ile Lys Lys Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu Glu
80 85 90 95
CGT CGT CGT GTA AAC CAA TTC CTG GAC TAC CTG CAA GAG TTT CTT GGT 336
Arg Arg Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly
100 105 110
GTA ATG AAC ACC GAA TGG ATC ATC GAA TCC TGATGAAGCT T 377
Val Met Asn Thr Glu Trp Ile Ile Glu Ser
115 120
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(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 121 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met His Tyr His His His Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu
1 S 10 15
Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu Ile
20 25 30
Ala Asn Glu Thr Leu Arg Ile Pro Val Pro Val His Lys Asn His Gln
35 40 45
Leu Cys Thr Glu Glu Ile Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln
50 55 60
Thr Val Gln Gly Gly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser Leu
65 70 75 80
Ile Lys Lys Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg
85 90 95
Arg Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val
100 105 110
Met Asn Thr Glu Trp Ile Ile Glu Ser
115 120
(2} INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid ,
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met His Tyr His His His
(2) INFORMATION FOR SEQ ID NO. 4:
CA 02256368 1998-11-23
WO 97/45448 PCT/AU97100322
- 33 -
(i) SEQUENCE CHARACTERISTIC:>
(A) LENGTH: 21
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 4:
Thr Ser Ala Leu Val Lys Xaa Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu
10 15
Leu Ile Ala
(2) INFORMATION FOR SEQ ID NO. 5:
(i) SEQUENCE CHARACTERISTIC:>:
(A) LENGTH: 21
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 5:
Thr Ser Ala Leu Val Lys Lys Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu
5 10 15
Leu Ile Ala
(2) INFORMATION FOR SEQ ID NO. 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypept:ide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 6:
Thr Ser Ala Leu Val Lys Arg Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu
5 10 15
Leu Ile Ala
(2) INFORMATION FOR SEQ ID NO. 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: amino acid
i I
CA 02256368 1998-11-23
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-34-
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 7:
Thr Ser Ala Leu Val Lys Gln Thr Leu Ala Leu La Ser Thr His Arg Thr Leu Leu
10 15
Ile Ala
(2) INFORMATION FOR SEQ ID NO. 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 8:
Thr Ser Ala La Val Lys Asn Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu Leu
5 10 15
Ile Ala
