Note: Descriptions are shown in the official language in which they were submitted.
WO 03/020762 CA 02459348 2004-03-03 PCT/AU02/01204
1
Antibodies to non-functional P2X7 receptor, diagnosis and treatment of cancers
and other conditions
TECHNICAL FIELD
This invention concerns diagnosis and treatment of diseases, including
cancers.
The types of diseases with which this invention is concerned include cancers
derived from epithelial cells and malignant lymphoma. The invention also
concerns
other conditions, such as preneoplastic states, irritable bowel syndrome and
viral
and other infections. It is quite possible that the invention is also
applicable to other
diseases and conditions.
BACKGROUND
Adenosine triphosphate (ATP) can activate ligand-gated purinergic receptors
known as P2X receptors. Receptor subtypes P2X1 to P2X7 have been identified.
It
is known that different P2X receptor subtypes are present in many cells,
including
epithelial cells and leukocytes, including lymphocytes, thymocytes,
macrophages
and dendritic cells.
P2X receptors are permeable to calcium ions as well as some other cations,
such as
potassium and sodium. An influx of calcium ions into a cell via a P2X receptor
can
be associated with cell death.
It is believed that the P2X7 subtype is involved in apoptosis, or programmed
cell
death, in many cell types. In the presence of ATP, the P2X7 receptor expressed
on
the surface of a cell is capable, within a second, of opening calcium channels
through the cell membrane. Continued exposure to ATP can lead to the formation
of large pores, within a few seconds to tens of seconds, that enable the cell
to be
flooded with excess calcium, inducing apoptosis.
The amino acid sequences of the human and rat P2X7 receptors are known, for
example, from US patent No. 6,133,434 (Buell et al). Refer also to Figure 1
herein.
CA 02459348 2004-03-03
WO 03/020762 PCT/AU02/01204
2
Exposure to ATP does not generally result in apoptosis in the case of
epithelial
cancer cells, for example. It has been found that such cells express P2X7
receptors
that are unable to form pores. These are regarded as non-functional receptors.
In human cancer cell lines, such as prostate PC3 and breast MCF7, as well as
in
animal cell lines including rodent hybridomas, the P2X7 receptor is found on
the
cell surface in a non-functional conformation.
The B-cells of patients with malignant lymphoma express non-functional P2X7
receptors. Lymphoma develops from malignant clones that escape cytolytic
destruction. This process leads to the progressive accumulation of malignant B-
lymphocytes and thus lymphadenopathy and/or splenomegaly.
SUMMARY OF THE INVENTION
In a first aspect, this invention provides a probe for detection of a disease
or
condition, the probe being adapted to distinguish between functional P2X7
receptors and non-functional P2X7 receptors. Preferably, the probe
distinguishes
between functional and non-functional P2X7 receptors by detecting change in
relation to binding of adenosine triphosphate (ATP) to the receptors or by
detecting
change in binding of one or more proteins necessary for pore formation in P2X7
=
receptors. In an alternate embodiment, the probe detects one or more parts of
the
P2X7 receptor exposed in the absence of bound ATP. Such receptor part may
include a P2X7 monomer.
The invention also provides a method for detecting a disease or condition, the
method including the steps of using the probe of the invention to distinguish
between functional P2X7 receptors and non-functional P2X7 receptors, providing
a
receptor expression profile, and comparing the receptor expression profile
with that
of a normal profile. The change may be detected, for example, as indicated
above
in connection with the probe itself.
WO 03/020762 CA 02459348 2004-03-03 PCT/AU02/01204
3
The probe may be natural or artificial. Preferably, the probe is an antibody,
which
may be poIyclonal, monoclonal, recombinant, a humanised antibody, a human
antibody or an appropriate fragment thereof. The antibody is preferably
directed
against an epitope located in an extracellular domain adjacent to a site for
binding
ATP. In the case of human P2X7 receptors, the probe is preferably adapted to
distinguish between functional receptors having a sequence in which proline at
amino acid 210 is in the trans conformation and non-functional receptors
having a
sequence in which the proline at amino acid 210 is in the cis conformation
that acts
to impart a significant alteration in the local protein structure.
The probe may be prepared using any suitable technique, as will be readily
apparent to one skilled in the art.
It is within the scope of the invention that the probe may distinguish between
functional and non-functional receptors through detection of other
conformational
changes occurring at a site for binding ATP. For example, the change detected
may
be in an amino acid other than the proline referred to above. An example of
such an
amino acid is Pro199 which, when in the cis conformation, significantly alters
the
local protein structure. As another example, the change detected may be in
some
other respect.
The probe may also be adapted to detect other regions of the P2X7 receptor
unchanged by functional state. The conformation of the monomeric subunits
lacking bound ATP may be detectable using the probe, as the epitope chosen may
specifically detect the shape of a region of the surface of the receptor
accessible
only when ATP is not bound. The probe may detect change in binding of one or
more proteins, such as accessory or other proteins, necessary for pore
formation.
Non-limiting examples of such proteins are laminin, integrin, beta-actin,
alpha-
actinin and supervillin.
In the present invention, a P2X7 subtype-specific antibody can be used to
specifically detect or bind to non-functional P2X7 receptors expressed in or
on cells
WO 03/020762 CA 02459348 2004-03-03
PCT/AU02/01204
4
forming part of preneoplastic tissue, very early neoplastic tissue, advanced
neoplastic tissue and on any neoplastic cell expressing non-functional P2X7
receptors. Thus, the P2X7 receptor is detected or bound only when in the close-
gated or non-functional conformation, even though it may be normally expressed
in the cell membranes and may otherwise be partially able to function as a
channel.
Further, the conformation of the monomeric subunits lacking bound ATP is also
detectable with the antibody, because the epitope chosen specifically detects
the
shape of a region of the surface accessible only when ATP is not bound.
In the present invention, the non-functional P2X7 receptors can be detected or
bound by using an antibody directed against an epitope that undergoes a
conformational change from the structure present in functional receptors. It
has
been found that the amino acid sequence of the non-functional receptors can be
identical to the amino acid sequence of functional receptors, so that the
cause of the
conformational change in the receptors relates to the interaction of the
receptors
with ATP. As set out above, the ATP molecules act as receptor agonists, so
that
when ATP is bound to the receptors, they are able to open a channel through
the
cell membrane for the inflow of calcium ions. Non-functionality is therefore
. caused by a lack of appropriate binding of the ATP agonists to the
receptors, for
reasons that may include a deficit in the local availability of ATP through
production deficit or increase in the rate of degradation. If ATP binding to
the
receptors is disrupted, the receptor conformation is altered. This can be
detected by
using an antibody specially designed to bind to the region of the protein
affected by
the binding of the ATP.
In the case of human P2X7 receptors, the specific sequence involved in the
conformational change may include Pro210, which undergoes a change in
conformation from the trans form to the cis form in the absence of bound ATP.
Thus, in the case of human receptors, an appropriate epitope sequence against
which an antibody must be raised may include Pro210, and may extend either
side
WO 03/020762 CA 02459348 2004-03-03 PCT/AU02/01204
5
of this residue, to an appropriate extent necessary to induce an antibody
response.
By way of non-limiting example, this may include a segment extending from
Gly200 to Cys216. Further, a homologous segment from other mammals, such as
rat, may be used where this cross-reacts with human tissue. As an example, the
same segment G1y200 to Cys216 in rat may be used, although there are two amino
acid substitutions in the rat sequence compared with the human sequence (refer
US
patent No. 6,133,434, for example).
In the case of non-human receptors, the specific sequence may be ascertained
by
suitable experiment.
The detection of non-functional P2X7 receptors according to the invention may
show a distribution pattern in which functional receptors (and hence normal
cells)
may remain essentially unlabelled. However, non-functional conformations of
P2X7 receptors may be detected, initially in the nuclei and cytoplasm of
cells, at a
very early stage in preneoplasia. For example, in the case of epithelial cell
cancer,
using the method of the invention it may be possible to detect preneoplasia
several
years prior to the normal pathological appearance of cancer as detected by
haematoxylin and eosin ("H & E") stained slides of biopsied tissues. Thus,
cancers
such as prostate, skin and breast may be detected far earlier than is
currently the -
case, with the advantages of introduction of early therapy.
The full scope of the diseases and conditions which may be detected by the
probe
and method of the invention has not yet been ascertained. However, it is
believed
that these include epithelial cell cancers, such as prostate, breast, skin,
lung, cervix,
uterus, stomach, oesophagus, bladder, colon and vaginal cancers, as well as
blood
cancers including malignant lymphoma, irritable bowel syndrome and infection
by
viruses such as HIV or other pathological organisms, such as Mycobacterium
tuberculosis. Infection may cause non-functional receptors to be expressed
either
directly through inhibition of co-factors required for functionality, or
through the
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
6
up-regulation of co-factors acting to inhibit P2X7 function on epithelial or
other
cells, so rendering the infected cell less amenable to destruction by,
apopto.sis.
Unless otherwise indicated, the term "disease or condition" as used herein is
intended to include all those specific diseases and conditions set out in the
preceding paragraph.
In the specific case of irritable bowel syndromes ("IBS"), it has now been
found
that, in patients with this condition, the gut mucosa, that normally expresses
P2X7
receptors in the widely distributed lymphocytes present in the stoma beneath
the
epithelium, becomes up-regulated. In affected patients, this increased
expression
can be observed from duodenum to rectal mucosa. The increased expression may
be found in isolated regions, or to be generally increased over the entire
length of
the intestinal tract in more extreme cases.
In the least affected cases, total P2X7 receptors are up-regulated, but these
are all
functional and they do not penetrate into the epithelium. In more severe
cases, total
P2X7 receptor expression is even higher, and the most affected areas of the
gut
exhibit receptors that are non-functional. These may be localised to caecal
mucosa,
for example, and may penetrate into the epithelium. The most severe cases are
*those in which total P2X7 receptor expression is further increased and most
of the
receptors are non-functional with increased epithelial cell penetration.
As already discussed, non-functionality of P2X7 receptors is caused by lack of
appropriate binding of the ATP agonist to the receptors. The reasons for this
may
include a deficit in the local availability of ATP through production deficit
or
increase in rate of degradation through ecto-ATPase enzymatic degradation of
ATP. If ATP binding to the receptors is disrupted, the receptor conformation
is
altered as already discussed, and this can be detected using the probe of the
invention. However, the detection of total P2X7 receptor distribution is best
achieved using an epitope to other regions of the extracellular domain of the
P2X7
receptor that is not affected by ATP binding. The probe may be capable of
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
7
detecting regions of the P2X7 receptor unchanged by functional state, by
detecting
an epitope common to both functional and non-functional conformations, such as
Va165-Lys81.
It is within the scope of this invention to use one or two P2X7 subtype-
specific
antibodies to specifically distinguish between total P2X7 distribution and the
proportion of receptors that are non-functional and expressed in gut mucosa.
Thus
the two antibodies used together can detect both total receptor count and
those
receptor channels. present only in a close-gated or non-functional
conformation.
The first antibody is adapted to detect total P2X7 receptor expression. The
probe
comprising or attached to the antibody of the invention can provide the second
antibody for detection of IBS, not only distinguishing between functional and
non-
functional nx, receptors, but also allowing for detection of other regions in
which
the receptor is unchanged by functional state. The antibodies may be used
separately or together. Preferably, they are used in combination.
The detection of all P2X7 receptors, separately from non-functional P2X7
reeeptors,
determines the severity of the condition. Expression of non-functional P2X7
receptors in the gastrointestinal mucosa occurs in a pattern in which normal
cells
remain essentially unlabelled. Thereafter, the non-functional conformation of
P2X7
is first detected in the stroma underneath the epithelium ranging from
isolated
patches in mild cases of the syndrome to extensive expression throughout the
length of the gastrointestinal tract with isolated patches of infiltration of
non-
functional receptors into the epithelium.
The invention also provides a method of diagnosing irritable bowel syndrome,
comprising detecting the P2X7 expression profile of cells and/or tissue and
comparing the profile with a predetermined expression profile of normal cells
and/or tissue. Preferably, the detection of the P2X7 expression profile
includes use
of one or more antibodies. Further, it is preferred that such antibody or
antibodies
are different from the probe of the invention in that they do not detect
change in
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
8
relation to binding of ATP to the P2X7 receptors. The preparation of such
antibodies will be readily apparent to one skilled in the art.
The invention also includes use of one or more antibodies to diagnose
irritable
bowel syndrome.
Therapeutic treatment for this condition is discussed below, in connection
with the
third aspect of this invention.
The diagnostic can be used in standard microscopy employing standard
irnmunohistochernical techniques. The diagnostic may also be used in vivo.
Diagnosis using the probe and method of the invention may be carried out using
in
situ imagine techniques to detect distribution in body tissues, in addition,
standard
microscopy, confocal microscopy and fluorescence activated cell sorting may be
used. Normal immunohistochemical techniques for testing lymph, prostate,
breast,
skin, lung, uterus, bladder, cervix, stomach, oesophagus and similar biopsies,
also
fine needle aspirates of breast and other tissue and cell smears such as those
taken
for the detection of cervical cancer, may be used.
For in vivo diagnosis, it is preferred that the probe is a human antibody or
domain,
manufactured with no animal components. The antibody is preferably labelled
with a short-lifetime radiolabel, detectable by means of scanning technology
such
as positron emission tomography (PET scanner). Such imaging can detect the
aggregation of labelled antibody anywhere in the body, thus signalling the
presence
of non-functional receptors, associated with the presence of any tumour.
Ideally,
such a test should be conducted only after detection of primary cancer and for
the
purpose of checking for secondary cancer, or after a general screen by means
of a
blood test (refer below) has detected the likelihood of the presence of one of
more
tumours.
The probe and method of the invention may be employed to provide a blood test
for detecting non-functional P2X7 receptors and hence cancer or pre-cancerous
WO 03/020762 CA 02459348 2004-03-03
PCT/AU02/01204
9
conditions. By way of example, the probe in the form of a fluorescent labelled
antibody (monoclonal or polyclonal) can be used in flow cytometry against
blood
cell fractions of the patient in order to detect binding to non-functional
receptors on
various gated leukocytes, including T lymphocytes, B lymphocytes or
macrophages.
In another form of blood test, the probe preferably takes the form of a
labelled
antibody attached to a matrix provided in a kit, enabling detection by the
presence
of a colour reaction to the binding of the fixed antibody to positive white
blood ,
cells. Such a kit may be suitable for use by medical practitioners.
In a similar blood test, the antibody probe of the invention may be used as a
diagnostic tool for screening patients who may not have cancer but in whom the
normal cell killing pathways are inhibited through lack of function in P2X1 on
one
or more leukocytes. Such patients may express non-functional receptors on
macrophages, indicating inhibition of the ability of those macrophages to kill
infected cells, such as those infected by organisms like Mycobacterium
tuberculosis, or other infectious agents including malaria and HIV. Such
organisms
preferentially proliferate in patients for whom the normal cell killing
pathways are
inhibited through lack of function in P2X7 on one or more leukocytes.
Other techniques may be used with the probe and method of the invention.
This invention provides an antibody for treating a disease or condition, the
antibody being adapted to distinguish between functional P2X7 receptors and
non-
functional P2X7 receptors and being adapted to bind only to non-functional
receptors. Preferably, the antibody distinguishes between the functional and
non-
functional receptors by detecting change in relation to binding of adenosine
triphosphate (ATP) to the receptors, or by detecting change in binding of one
or
more proteins necessary for pore formation in P2X7 receptors and being adapted
to
bind only to non-functional receptors. In another embodiment, the antibody
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
10
distinguishes between the functional and non-functional receptors by detecting
parts of the receptor exposed in the absence of bound ATP.
The antibody for treating diseases and conditions may be the same as the
antibody
which may be used as the probe for diagnosing diseases and conditions. Such an
antibody could be used to treat skin cancers topically, for example. For
systemic
treatment of cancer, the antibody or its active fragments should be human or a
human domain, in order to minimise undesirable immune response side effects.
The antibody of the invention may be used to treat diseases or conditions in
mammals, including humans. Examples of the diseases or conditions have been
set
out above in connection with the probe of the invention.
The invention also provides an epitope capable of causing the generation of
the
antibody of the second aspect of the invention. The epitope preferably
includes
Pro21 0 and encompasses the segment G1y200 to Cys216 (in the P2X7 sequence of
the human receptor). The epitope should preferably have attached to the C-
terminal
end a Cys residue (Cys216) that is cross-linked to diphtheria toxin via the
chemical
cross-linker rnaleimidocaproyl-N-hydroxysuccinimide (MCS), so that the
conformation adoptcci by the attached epitope peptide occupies a stable cis
proline
configuration.
This specific peptide conformation is intended to be presented to humans or
animals with one or more diseases or conditions, especially epithelial cell
cancers,
such as prostate, breast, skin, lung, cervix, uterus, stomach, oesophagus,
bladder,
colon and vaginal cancers, as well as malignant lymphoma, irritable bowel
syndrome and infection by viruses such as HIV or other pathological Organisms,
such as Mycobacterium tuberculosis. The patient will preferably mount an
immune
response to the applied conjugated epitope and so generate antibodies
recognising
the non-functional P2X7 receptors present on the surface of the affected
cells, thus
binding to them and alerting the appropriate immune cell to destroy the
complexed
cells. Other cells primed for cell death may also be affected.
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
11 =
It is to be understood that the sequence referred to above is not limiting on
the
scope of the invention, which includes alternate sequences and carriers and
cross-
linkers that similarly produce a specific immune response, preferably against
only
non-functional P2X7 receptors, preferably ignoring all functional receptors
expressed on cell surfaces, and so avoiding side effects.
The invention, in this second aspect, also provides for the use of the
antibody of the
invention as a therapeutic vehicle for treatment of a disease or condition in
a
patient to regulate programmed cell death by targeting aberrant or non-
functional
P2X7 receptors expressed on the surface of cells, while leaving all cells
expressing
normal (functional) receptors untouched. The invention also covers the use of
the
epitope of the invention to cause the generation of the antibody, as above.
The invention also provides a pharmaceutical composition for treatment or
prevention of a disease or condition in a patient, the composition including a
pharmaceutically effective amount of an antibody, or an epitope to cause the
generation of such an amount, capable of reg-ulating programmed cell death of
cells
having expressed on their surface aberrant or non-functional P2X7 receptors.
The phannaceutically effective amount of the antibody or epitope will vary
= = according to the patient and the nature of the disease or condition.
These variables
can be ascertained by one skilled in the art.
The pharmaceutical composition of the invention may be administered in
conjunction with a pharmaceutically acceptable carrier, which may be any of
those
known in the art or devised hereafter and suitable for the intended use. As
well as
carriers, the pharmaceutical compositions of the invention may include other
ingredients, including dyes, preservatives, buffers and antioxidants, for
example.
The pharmaceutical composition of the invention may take any desired form and
may be administered, for example, in the form of an ointment, cream, solution,
suspension, powder, tablet, capsule, suppository or pessary.
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
12
The pharmaceutical composition of the invention may be administered in any
suitable way, which may include oral, parenteral, intravenous, intramuscular,
subcutaneous or topical administration.
The invention also provides a method of treating or preventing a disease or
condition in a patient, the method including administering to the patient a
pharmaceutical composition according to the invention.
The invention also provides the use of the pharmaceutical composition of the
invention, in the treatment or prevention of a disease or condition, in a
patient.
It will be apparent to one skilled in the art that the pattern of use of the
pharmaceutical composition of the invention may need to be altered for optimum
effect it may be necessary to take into account the nature of the disease or
condition as well as its severity.
The third aspect of the invention focuses on the expression of ATPases
(enzymes)
that control the supply of ATP to P2X7 receptors, for example in the B-cells
of a
patient having malignant lymphoma. Channel opening of P2X7 receptors on
leukocytes is terminated through the rapid hydrolysis of ATP agonist by ecto-
- ATPases and ecto-ATPdiphosphohydrolases (ecto-ATPDases). These enzymes
regulate numerous physiological processes that are dependent on ATP. Substrate
specificity of ATPase and ATPDase activity on lymphocytes indicates the
presence
on the lymphocytes of more than one type on the cell surface, including CD39.
Proliferation of one or more of these ATPases or ATPDases could limit the
supply
of ATP needed to control P2X7 pore formation and the subsequent programmed
cell death needed to regulate B-cell numbers.
Similarly, it is believed that, in the case of IBS, proliferation of ATPases
may
contribute to lack of appropriate binding of the agonist ATP to the P2X7
receptors.
Accordingly, in this third aspect, the invention provides a preparation for
treatment
or prevention of a disease or condition in a patient, the preparation
including one or
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
13
more substances adapted to regulate the expression of ATPases that control the
supply of ATP to P2X7 receptors in the patient's cells or tissues. The
invention also
provides a method of treating or preventing a disease or condition in a
patient, the
method including the step of administering to the patient a preparation
including
one or more substances adapted to regulate the expression of ATPases that
control
the supply of ATP to P2X7 receptors in the cells or tissue of the patient.
Examples of such ATPases may be CD39 or CD73.
Such a substance may take the form of an ATP analogue, preferably non-
hydrolysable, and specific for P2X7, or another substance that inhibits the
action of
local ATPases depleting the availability of ATP for the P2X7 binding site. The
preparation may be in the form of a human antibody directed specifically
against
non-functional P2X7 receptors.
A substance such as an ATP analogue may bind to the P2X7 and hold it in open
pore configuration, thus forcing the pore to assume a functional state, in
which it is
able to take up both large and small cation permeants. In this way the use of
such a
synthetic agonist may act to restore receptor function, at the same time as
controlling the growth advantage that P2X7 provides cells in its role as a
calcium
channel.
The disease or condition is preferably malignant lymphoma or IBS but the
invention may also extend to other diseases or conditions, including other
epithelial
cell or blood cancers or viral and other pathological infections.
In the case of malignant lymphoma, the ATPases control the local supply of ATP
to the P2X7 receptors so as to reduce the concentration of ATP available for
binding to the P2X7 receptors and so deactivate them leading to a significant
reduction in programmed B-cell death. These ATPases may be specifically
expressed on the surface of the B-cells and appear to be up-regulated in
malignant
lymphoma. Preferably, application of a specific ATPase inhibitor may be used
to
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
14
regulate the availability of ATP on the P2X7 receptors, so regulating
programmed
B-cell death.
For treatment of malignant lymphoma, the substance may include a synthetic
agonist capable of blocking ATPases or ATPDases, of the form of non-
hydrolysable P2X7 agonist.
In relation to irritable bowel syndrome, administration of the preparation of
the
invention is intended to restore receptor function that may be depleted
through
overactivity of the muscle underlying the affected region of mucosa. The
preparation of the invention may act on the mucosa directly to remove these
non-
functional receptors and thereby restore local normal gastrointestinal
secretory
mechanisms. Therapeutic treatment is aimed at restoring the local supply of
ATP to
the non-functional receptors, so that normal receptor function is restored.
The
consequences of control of receptor function include restoration of normal
control
of gastrointestinal secretions and peristalsis. This may be achieved by
application
of enteral or systemic supply of synthetic P2X7-specific agonist, preferably
non-
hydrolysable by ATPases, by systemic application of an antibody directed
against
non-functional P2X, receptors, preferably a small human specific antibody to
remove the non-functional receptors, leaving only functional receptors.
If abnormalities of peristalsis in the underlying smooth muscle are
responsible for
depleting the local availability of ATP for binding to the normal P2X7
receptors,
treatment may involve restoration of this natural supply of agonist by means
of a
limit on the uptake or use of ATP by the smooth muscle through application of
a
treatment to temporarily limit gut motility.
The invention also provides a pharmaceutical composition for treatment of a
disease or condition, the composition including a pharmaceutically effective
amount of one or more substances adapted to regulate the expression of ATPases
(enzymes) that control the supply of ATP to P2X7 receptors.
WO 03/020762 CA 02459348 2004-03-03 PCT/AU02/01204
15
The invention in all its aspects extends to such similar applications that
could be
made in other medical conditions in which aberrant P2X7 receptOrs are involved
as
a result of viral infection where the virus is protected in the infected cell
by up-
regulating non-functional P2X7 receptor or where such receptors are up-
regulated
from the normal cell condition.
The invention also provides a method of treating irritable bowel syndrome,
comprising administering to a patient a pharmaceutical composition as defined
above.
The invention also provides the use of such a pharmaceutical composition in
the
treatment of irritable bowel syndrome.
The pattern of use of one or more of the above pharmaceutically effective
agents
may need to be altered for optimum effect.
Expressed another way, the invention provides a method of treating irritable
bowel
syndrome, the method including administering a composition adapted to restore
P2X7 receptor function. The receptor function may have been depleted through =
overactivity of the muscle underlying the affected region of mucosa. The
composition may be the same as that set out above for the substance included
in the
preparation of the invention.
In a further aspect, the invention provides a method for distinguishing
between
different conformations of proteins by using an epitope capable of causing the
generation of an antibody, or the antibody itself, to effect specific
pharmaceutical
outcomes (active as well as passive immunisation) from binding to all members
of
the proteins with a selected conformation. An example of this would be prion
proteins in the conformation that leads to the condition v013. The abnormal
form
of the protein could be targeted by a specific antibody or epitope causing the
generation of the antibody, preferably human and reduced in size for optimum
pharmacological effect.
WO 03/020762 CA 02459348 2004-03-03 PCT/AU02/01204
16
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 shows the amino acid sequence of the human P2X7 receptor (prior art).
Sequences 65 to 81 and 200 to 216 are highlighted and are referred to below.
DETAILED DESCRIPTION OF THE INVENTION
To raise the antibody specifically to non-functional P2X7,the epitope used was
the
sequence 200 to 216 in Figure 1, containing a Cys at 216.
To raise the antibody to non-discriminatory P2X7,the epitope used was the
sequence 65 to 81 in Figure 1, to which was added an N-terminal Cys. This
antibody could not detect whether the receptor was non-functional but was
designed to detect all receptor so that the proportion of receptor that was
functional
could be determined by comparing the staining obtained by using the two
antibodies separately. =
The Cys residues on the epitopes were coupled via a maleimidocaproyl-N-
hydroxysuccinimide (MCS) cross linker to diphtheria toxin (DT) carrier with
ten
peptide epitopes attached to each DT carrier, to maintain conformational
stability
end provide a larger antiacnic structure These conjugated epitopes were used
as
the antigens for injection into several animal species (sheep, rabbit and
mouse) to
raise antibodies specific to the epitopes, in the usual manner.
The procedure for raising antibodies is well documented in the prior art by
use of
antigen/adjuvant mixtures injected into animals at particular times. Specific
examples for raising the antibodies are set out below:
Example 1
Sheep anti-P2X7 antibodies
500 Rg of conjugate (approximately 100 xg of P2X7 epitope) was diluted in
phosphate-buffered saline (PBS) to 0.8 mL and was emulsified with 1.2 mL of
Freund's Complete adjuvant. Sheep were injected at multiple sites both
CA 02459348 2010-05-31
17
subcutaneously and intramuscularly with the antigen/adjuvant emulsion. Eight
weeks later the sheep were again injected with the same amount of conjugate
emulsified with Freund's Incomplete adjuvant at multiple sites. This was
repeated
4 weeks later and the animals were bled from the jugular vein. The serum
collected was tested for antibody specificity. The sheep were then routinely
injected and bled at eight week intervals to provide a pool of serum
containing the
specific antibodies.
Other sheep were injected with the same dose of conjugated antigen similar to
the
schedule above but a different adjuvant was used. In these animals, 0.7 raL of
the
diluted antigen was mixed with 0.1 mL of a Quill A / DEAE*Dextran solution
(2.5
mg Quill A +25 mg DEAE Dextran per mL of PBS) and 1.2 mL of ISA 50V
Montanide. The emulsion was injected at multiple sites both subcutaneously and
intramuscularly. The antibodies produced using this adjuvant produced the same
specificities as those produced using Freund's adjuvant.
.x_ample 2
Rabbit anti-P2X7 antibodies
Antibodies were raised in rabbits using the same two adjuvants as with the
sheep
and the same injection schedules, the only difference being that 300 ug
amounts of
the conjugate were used for the injection. The antibodies raised had the same
specificities as those produced in the sheep and could readily discriminate
between
the epitopes against which they were raised.
Example 3
Mice anti-P2X7 antibodies
Antibodies were raised in mice against the conjugated epitopes and also
against the
unconjugated epitope of the non-functional P2X7 epitope (which is able to
discriminate receptors that cannot from pores and thus fail to be apoptotic).
* Trade-mark
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
18
In these experiments, the adjuvant used was the QAIGEN Pty Ltd product,
ImmunEasyTm which contains the irrununo-stimulatory product CpG DNA
(trademark of Coley Pharmaceutical Group Inc.)
pz of epitope or conjugated epitope was diluted in 70 ILL of PBS and 30 ILL of
IrnmunEasyTM adjuvant. Mice were injected at multiple sites subcutaneously and
intramuscularly. This regime was repeated two weeks later and again at a
further
two weeks. Mice were bled eight days after the third injection. Antibodies
raised
in mice by this method were again able to discriminate between the different
P2X7 .
epitopes and the antibodies against the P2X7 non-functional epitope gave the
same
results as those raised in sheep and rabbits.
As the above Examples illustrate, antibodies to various epitopes of the P2X7
receptor in different species and using different adjuvants may be raised
consistently. In particular, antibodies to an epitope of the P2X7 receptor
which
identifies the receptor in the non-functional state, in which it cannot form a
pore
and carry out its apoptotic function under normal physiological conditions,
may be
raised rountinely.
Example 4
The antibody detecting non-functional P2X7 was tested by binding the antibody
to
cells expressing P2X7 (human) with known function as revealed through the
ability
of the P2X7 to take up ethidium or rubidium. These P2X7 protein channels may
have been mutated at base pair 1513, such that the channels would not form
apoptotic pores. These and similar non-functional P2X7 receptors expressed on
malignant B lymphocytes also bound the antibody in flow cytornetry and in
standard immunohistochemistry while cells expressing normal functional P2X7
(capable of taking up calcium, ethidium and rubidium with large fluxes) were
unable to bind the antibody, because the epitope chosen to detect the non-
functional receptors was unavailable in functional receptors. The Pro210
adopted a
cis conformation in the non-functional receptors and it was specifically this
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
19
conformation that was stabilised in the conjugated epitope used to raise the
antibody. The Pro210 was in the trans conformation in the receptors that were
shown to be functional. This was a result of the binding of ATP (adenosine
triphosphate) to the P2X7 receptor. When ATP was bound, the Pro210 on a
segment immediately adjacent to the ATP binding site adopted a trans
configuration.
This was verified using site directed mutagenesis to change the Pro210 to an
Ala
that was fixed in the trans configuration and this mutant protein was found to
be
fully functional and unable to bind the antibody raised to detect the non-
functional
receptor.
Eliainple 5
Further verification of the specificity of the antibody to detect the non-
functional
receptor came in experiments that labelled macrophages expressing P2X7. The
macrophages bound antibody to the P2X7 receptors using the P2X7 universal
antibody but did not bind the antibody to non-functional P2X7 until they had
been
exposed to cancer cells such as mouse hybridoma cells. Contact between the
macrophages and the hybriclorna cells induced the expression on the
rnacropliac,,,es
of non-functional P2X7 that was detected by the antibody to non-functional
P2X7
as well as the universal P2X7 antibody.
The macrophages and B-cell lymphocytes extracted from patients with malignant
lymphoma were tested ana all these cells bound the antibody to universal P2X7
as
well as the antibody to the non-functional P2X7 receptors, verifying that P2X7
was
non-functional in all the cancer cells detected, with the apoptotic pore
formed by
functional P2X7 unable to form and thus induce apoptosis in cancer cells.
All such cancer cells from all epithelial cell cancers in humans such as
prostate,
breast, bowel, skin, stomach, cervix and others as well as malignant lymphoma,
chronic lymphocytic leukaemia and brain tumours, as well as the same tumours
in
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
20
other mammals that were tested, including breast and prostate in dog and skin
in
cat as well as all mouse hybridoma eel's and mouse fibrosarcoma cells, all
express
the same non-functional P2X7. Sequence similarity between human, rat, cat, dog
and mouse at the chosen epitopes is sufficient for positive identification to
be made
in all the above cases. This shows that the mechanism of cancer in these
mammals
is identical in that all cancer cells express non-functional P2X7 receptors
unable to
form apoptotic pores that would normally kill the cell when activated. In this
way
the cancer cells become immortal, with apoptosis being switched off.
Example 6
As further verification that the cancer cells such as affected B-cell
lymphocytes are
unable to induce apoptosis through P2X7 function, B cells from leukaemia
patients
containing non-functional P2X7 receptors were incubated with 5 mM ATP for 2
hours in culture. The results were that all the non-functional receptors were
forced
by the excess ATP to open and induce apoptosis that killed the affected cells.
Example 7
As further verification that the antibody selectively binds cancer cells, skin
from
patients with basal cell carcinomas (BCC) were treated with the antibody to
the
non-fiinctional P2X7 receptors, suspended in an inert cream base and applied
to the
lesion and surrounding skin (refer Example 10, below). Within 1 week of daily
application of the topical antibody, all trace of the BCCs had disappeared
with no
effect on surrounding skin since normal skin was devoid of the receptors.
DIAGNOSTIC APPLICATIONS
Descriptions are provided here by way of example, using the specific non-
functional P2X7 antibody in animals and demonstrating the universal
application of
the probe and method of the invention to the diagnosis of most cancers in
humans
and other mammals.
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
21
In prostate tissue from humans and mammals, such as cats and dogs, when the
antibody of the invention is used for diagnosis, no labelling is obtained in
the
absence of cancer or pre-cancerous lesions. However, the diagnostic method of
the
invention reveals first signs of neoplastic change while there is still no
accompanying morphological changes detectable by H&E stain.
At this stage, it is necessary to stain for the receptor units first appearing
in the
nuclei of epithelial cells. These migrate to the cytoplasm in later stages of
the
disease, acting as a field effect throughout the prostate, so that less tissue
need be
biopsied to be certain of the existence of a tumour. In later stages of the
disease,
the staining becomes more confined to the apical epithelium.
Similarly, other epithelial cell cancers, like breast, lung, colon and skin in
humans
and in other mammals, such as cats and dogs, can be detected with margins as
there
is no longer a clear field effect in these other tissues.
The same stage development is seen in these other tissues, like breast and
cervix,
with nuclear stain preceding cytoplasmic stain, while normal tissue is
unstained.
Affected ducts and lobules in breast tissue are readily detected due to the
local field
2ffected duct system in the breast even where normal
morphology suggests there is no cancer. Adjacent unaffected ducts appear
unstained. Similarly, affected lymph nodes, directly draining tissue
containing a
tumour, show signs of the tumour through the field effect of affected
lymphocytes.
Thus, sentinel nodes can be detected without there being any metastatic
cellular
spread to the node.
Skin cancers, such as basal cell carcinoma, squamous cell carcinoma and
dysplastic
naevi as well as malignant melanomas show positive staining for non-functional
receptors and channel components (monomers) in keratinocyte and melanocyte
layers with clear margins beyond which normal skin is unlabelled on both
epidermis and deep within the dermis.
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
22
All tested mammalian cancer cell lines such as human prostate (PC3) and breast
(MCF7) and rodent hybridomas are positive for the non-functional receptors on
the
cell surface so that apoptosis is inhibited in these cancer cells. The general
application of this diagnostic is seen by way of the same label on mouse
hybridoma
cells showing the ubiquitous nature of the receptor in other animal types
besides
human. Normal human B-cell lymphocytes show that functional P2X7 receptors are
expressed on the cell surface, so enabling apoptosis when necessary, while
human
B-cell lymphocytes from patients with malignant lymphoma show that non-
functional P2X7 receptors are expressed on the cell surface, so curtailing
apoptosis.
THERAPEUTIC APPLICATIONS
Targeting this apparently ubiqitous P2X7 non-functional conformer expressed on
the cell surface of cancer cells attempting to undergo apoptosis may be used
to
treat most cancers in humans and other mammals. Examples are set out below:
Example 8
Mouse hybridoma cells were grown on a macrophage base both in the presence and
absence of affinity purified antibody to non-functional P2X7. Cell counts
revealed
that over 4 days while cells coincubated with purified normal IgG grew from 1
x
104 to 7 x 104, coincubation with non-functional P2X7. antibody kept the cell
count
to only 1.5 x 104.
Example 9
This example shows that antibodies raised against the non-functional epitope
of the
P2X7 receptor can inhibit tumour formation in vivo.
As shown above, antibodies raised in sheep against the non-functional P2X7
epitope identified this non-functional P2X7 apoptotic receptor on the surface
of
mouse hybridoma cells. Addition of this antibody to hybridoma cell cultures
CA 02459348 2004-03-03
WO 03/020762 PCT/AU02/01204
23
retarded the growth of the cells. Mouse hybridoma cells when injected into
prepared inbreed mouse strains will cause tumour formation.
In this experiment, three groups of 10 Balb-c female mice each received the
following treatments:
Group 1: 10 mice each injected intraperitoneally (IP) with 1 x 106
hybridoma cells in 0.5 niL of cell culture medium on Day 1.
On Days 2 and 3, they received an intraperitoneal injection of
0.5 niL of cell culture medium.
Group 2: 10 mice each injected intraperitoneally (TIP) with 1 x 106
hybridoma cells in 0.5 mL of cell culture medium containing 1
mg of purified sheep IgG on Day 1. On Days 2 and 3, they
were injected with 0.5 rnL of cell culture medium containing 1
mg of purified sheep IgG.
Group 3: 10 mice each injected intraperitoneally (IP) with 1 x 106
hybridoma cells in 0.5 rnL of cell culture medium containing 1
mg of purified sheep anti-P2X7 non-functional epitope IgG on
Day 1. On Days 2 and 3, they, received a further injection of
0.5 ml of cell culture medium containing 1 mg of purified =
sheep anti-P2X7 IgG.
Mice from all the groups were killed on Day 11 and examined for the presence
of
tumour. The tumours were excised and weighed.
The results were as follows:
Groups Observations Mean Tumour
Weight per mice
( SD) (g)
1: Control 1 9 out of 10 mice had tumours. 3.98 1.1
WO 03/020762 CA 02459348 2004-03-03 PCT/AU02/01204
24
2: Control 2 10 out of 10 mice had tumours 2.93 0.9
3: Experimental 9 out of 10 mice had tumours 1.13 0.4
An analysis of variance showed a significant difference in tumour weight
between
the groups (probability P <0.01). The experimental group treated with the anti-
P2X7 non-functional antibodies was significantly different (P <0.01) from the
two
control groups. That is, treatment with antibodies against the P2X7 non-
functional
epitope significantly reduced the amount of tumour in the experimental
animals.
Example 10
Specific affinity purified antibody (to zreatly improve specificity) was
applied to 3
human basal cell carcinomas ("BCC") either as a liquid held in place for 7
days or
suspended in a dimethicone cream base. No trace of the BCC lesions was
detectable after treatment, while control skin was entirely unaffected due to
the
absence of the protein target.
Example 11
Skin lesions of the form of basal cell carcinoma (BCC) and squamous cell
carcinoma (SCC) (both primary tumours and secondary nunours), including
relapsed tumours and dysplastic naevi, were treated in a further trial using
purified
antibody, IgG either affinity purified or not, mixed in dimethicone cream base
or a
penetrating cream base. Since there were no non-functional receptors present
in the
normal skin there were no side effects detected in normal skin of any kind.
The
cancers of all types all responded to the presence of the antibody by
disappearing
within a period from thirty six hours to one week with twice daily
applications. No
relapse has occurred in periods of up to twelve months. The size of the
tumours
treated ranged from 3mm diameter with no raised border to 5crn diameter and up
to
4mm thick. A total of thirty four histologically confirmed tumours have been
successfully eliminated within one week treatment periods.
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
25
It is believed that application to patients in general would involve
production of a
human monoclonal antibody (such as herceptin) so that internal cancers could
be
treated with the same efficacy as is revealed with topical application. All
normal
functional P2X7 expressed on the cell surfaces of cells such as lymphocytes
would
need to remain unaffected by the presence of the antibody to avoid side
effects.
The antibody should therefore only bind to proteins expressed on the cell
surface of
cells attempting to but unable to initiate apoptosis. Thus all cells targeted
would be
only those attempting to kill themselves through programmed cell death,
including
cancer cells. The P2X7 receptors on these cells, particularly cancer cells,
would be
in a non-functional or ATP-depleted state.
ACTIVE IMMUNISATION
Active immunisation may also be used for therapeutic purposes. In this case
the
humans or other mammals need to be immunised against a specific epitope or
epitopes that are in a conformation that mimics the conformation adopted only
by
the receptors in their non-functional (ATP-depleted) shape on the cell
surface,
Conformational flexibility that includes partial exposure of an epitope shape
that is
present in functional receptors should be avoided. The cis configuration of
the
epitope Gly200-Cys216 as an example should be fixed before use by appropriate
means. As added proof that this concept is sound is the observation that
numerous
animals including mice, rabbits and sheep used to raise the antibodies have
not
been immuno-compromised. None of these many animals have ever developed any
tumours
A specific example illustrates this:
Example 12
Protocol: The experiment was conducted on the basis of a mouse tumour model.
Forty
ten-week old female inbred Balb C mice were used, and divided into two groups
of
twenty, Group 1 being experimental and Group 2 being the control group.
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
26
Day 1: The twenty experimental animals in Group 1 were injected with 0.1 mg of
the peptide epitope (hP2X7 sequence 200-216) conjugated to diphtheria toxin
via -
the MCS crosslinker. This contained approximately 0.02 mg of the peptide
epitope.
The Peptide conjugate was emulsified with a QUILL A/DEAE Dextran/Montanide
ISA 50V adjuvant mix and injected in a volume of 0.1 m1., at multiple
subcutaneous and intramuscular sites.
The twenty mice in the control group, Group 2, were injected with 0.1 mL of
the
adjuvant mix without peptide conjugate at multiple subcutaneous and
intramuscular sites.
Day 8: The twenty Group 1 mice were injected with 0.01 mg of the peptide
epitope
(hP2X7 sequence 200-216) conjugated to diphtheria toxin via the MCS
crosslinker
(containing approximately 0.002 mg of the peptide epitope). The peptide was
contained in a phosphate buffered saline solution and mixed according to the
protocol with the commercially available CpG DNA adjuvant ImmunEasy (from
Qiagen). A volume of 0.1 mL of peptide conjugate/adjuvant solution was
injected
at multiple subcutaneous and intramuscular sites in each mouse.
The twenty (Jroup 2 mice were injected with the comparable phosphate buffered
saline/ CpG DNA adjuvant mix. This was injected in a volume of 0.1 mL in each
mouse at multiple subcutaneous and intramuscular sites.
Day 26: The twenty Group I mice were injected with 0.025 mg of the peptide
epitope (hP2X7 sequence 200-216) conjugated to diphtheria toxin via the MCS
crosslinker (containing approximately 0.005 mg of the peptide epitope). This
was
contained in a phosphate buffered saline solution and mixed with the Qiagen
CpG
DNA adjuvant ImmunEasy. Again 0.1 mL of the mix was injected in each mouse
at multiple subcutaneous and intramuscular sites. The control group was
injected as
before on Day 8.
WO 03/020762 CA 02459348 2004-03-03 PCT/AU02/01204
27
Day 29: All mice received an injection of tumour cells at a single
subcutaneous site
located at the back of the neck in 0.1 tnL of tissue culture media. The tumour
cells
used were a mouse fibrosarcoma cell line developed by the Walter and Eliza
Hall
Institute in Melbourne Australia designated cell line WEHI 164.
The cells were injected at two concentrations into both the experimental and
control groups of mice. Each group was subdivided into two. Ten mice from each
of the experimental and control groups received 160,000 cells per mouse and
ten
mice from each group received 320,000 cells per mouse.
The cells from this cell line had previously been tested for the presence of
the non- ,
functional P2X7 epitope on their cell surface. This was done using an antibody
raised in sheep which specifically recognises the non-functional form of the
receptor.
Day 38: All mice were killed and blood collected for analysis of antibodies to
the
non-functional P2X7 epitope. All mice were weighed and the tumours were
excised
and weighed.
Results
Group Control Experimental Control Experimental
160,000 cells 160,000 cells 320,000 cells 320,000 cells
n 10 10 10 10
Mean tumour 599 270 1147 750
wt (mg)
SD 307 108 633 363
SEM 97 34 200 115
Analysis of variance of the results showed a statistically significant
difference
between control and treatment groups and between low and high dose groups
WO 03/020762 CA 02459348 2004-03-03PCT/AU02/01204
28
(P-31.0003). The lower dose group showed a larger difference due to the lower
tumour load having less effect on the ability of the mice immune systems to
cope.
ATP ANALOGUE
The efficacy of use of a synthetic agonist to effectively bind to ATP binding
sites
on the P2X7 pore, to force the pore to enter the functional state, thereby
acting to
restore receptor function as well as controlling the growth advantage that
P2X7
provides cells, is shown in the following experiment in culture. Tumour B-
cells
collected from a patient with CLL, when mixed with a similar number of like
cells
from a normal patient were treated with ATP at 2.5 mM for four hours. No
tumour
cells remained, only normal cells. The use of ATP or the more selective P2X7
agonist benzoyl, benzoyl ATP is not appropriate in vivo. Thus, a selective ATP
analogue able to selectively bind to P2X7 at much higher affinity than either
ATP
or BzATP may be designed to reinstate the process of apoptosis in a range of
affected tumour cell types.
INDUSTRIAL APPLICABILITY
The invention in all its aspects has application to the fields of human and
veterinary medicine and health, with the potential to enable early and
accurate
diagnosis of diseases and effective treatment, which in many cases is far less
invasive or traumatic than those available in the prior art.
CA 02459348 2004-07-05
28a
SEQUENCE LISTING
<110> Intreat Pty Limited
<120> Antibodies to non-functional P2X7Receptor Diagnosis and
Treatment of Cancers and Other Conditions
<130> 15696-70CA
<140> 2,459,348
<141> 2002-09-03
<150> AU PR 7430
<151> 2001-09-03
<150> AU PR 7431
<151> 2001-09-03
<150> AU PCT/AU02/00061
<151> 2002-01-17
<160> 1
<170> PatentIn version 3.2
<210> 1
<211> 595
<212> PRT
<213> Homo sapien P2X7 receptor
<400> 1
Met Pro Ala Cys Cys Ser Cys Ser Asp Val Phe Gin Tyr Glu Thr Asn
1 5 10 15
Lys Val Thr Arg Ile Gin Ser Met Asn Tyr Gly Thr Ile Lys Trp Phe
20 25 30
Phe His Val Ile Ile Phe Ser Tyr Val Cys Phe Ala Leu Val Ser Asp
35 40 45
Lys Leu Tyr Gin Arg Lys Glu Pro Val Ile Ser Ser Val His Thr Lys
50 55 60
Val Lys Gly Ile Ala Glu Val Lys Glu Glu Ile Val Glu Asn Gly Val
65 70 75 80
Lys Lys Leu Val His Ser Val Phe Asp Thr Ala Asp Tyr Thr Phe Pro
85 90 95
Leu Gin Gly Asn Ser Phe Phe Val Met Thr Asn Phe Leu Lys Thr Glu
100 105 110
Gly Gin Glu Gin Arg Leu Cys Pro Glu Tyr Pro Thr Arg Arg Thr Leu
115 120 125
CA 02459348 2004-07-05
28b
Cys Ser Ser Asp Arg Gly Cys Lys Lys Gly Trp Met Asp Pro Gin Ser
130 135 140
Lys Gly Ile Gin Thr Gly Arg Cys Val Val His Glu Gly Asn Gin Lys
145 150 155 160
Thr Cys Glu Val Ser Ala Trp Cys Pro Ile Glu Ala Val Glu Glu Ala
165 170 175
Pro Arg Pro Ala Leu Leu Asn Ser Ala Glu Asn Phe Thr Val Leu Ile
180 185 190
Lys Asn Asn Ile Asp Phe Pro Gly His Asn Tyr Thr Thr Arg Asn Ile
195 200 205
Leu Pro Gly Leu Asn Ile Thr Cys Thr Phe His Lys Thr Gin Asn Pro
210 215 220
Gin Cys Pro Ile Phe Arg Leu Gly Asp Ile Phe Arg Glu Thr Gly Asp
225 230 235 240
Asn Phe Ser Asp Val Ala Ile Gin Gly Gly Ile Met Gly Ile Glu Ile
245 250 255
Tyr Trp Asp Cys Asn Leu Asp Arg Trp Phe His His Cys His Pro Lys
260 265 270
Tyr Ser Phe Arg Arg Leu Asp Asp Lys Thr Thr Asn Val Ser Leu Tyr
275 280 285
Pro Gly Tyr Asn Phe Arg Tyr Ala Lys Tyr Tyr Lys Glu Asn Asn Val
290 295 300
Glu Lys Arg Thr Leu Ile Lys Val Phe Gly Ile Arg Phe Asp Ile Leu
305 310 315 320
Val Phe Gly Thr Gly Gly Lys Phe Asp Ile Ile Gin Leu Val Val Tyr
325 330 335
Ile Gly Ser Thr Leu Ser Tyr Phe Gly Leu Ala Ala Val Phe Ile Asp
340 345 350
Phe Leu Ile Asp Thr Tyr Ser Ser Asn Cys Cys Arg His His Ile Tyr
355 360 365
Pro Trp Cys Lys Cys Cys Gin Pro Cys Val Val Asn Glu Tyr Tyr Tyr
370 375 380
Arg Lys Lys Cys Glu Ser Ile Val Glu Pro Lys Pro Thr Leu Lys Tyr
385 390 395 400
Val Ser Phe Val Asp Glu Ser His Ile Arg Met Val Asn Gin Gin Leu
405 410 415
Leu Gly Arg Ser Leu Gin Asp Val Lys Gly Gin Glu Val Pro Arg Pro
420 425 430
CA 02459348 2004-07-05
28c
Ala Met Asp Phe Thr Asp Leu Ser Arg Leu Pro Leu Ala Leu His Asp
435 440 445
Thr Pro Pro Ile Pro Gly Gin Pro Glu Glu Ile Gin Leu Leu Arg Lys
450 455 460
Glu Ala Thr Pro Arg Ser Arg Asp Ser Pro Val Trp Cys Gin Cys Gly
465 470 475 480
Ser Cys Leu Pro Ser Gin Leu Pro Glu Ser His Arg Cys Leu Glu Glu
485 490 495
Leu Cys Cys Arg Lys Lys Pro Gly Ala Cys Ile Thr Thr Ser Glu Leu
500 505 510
Phe Arg Lys Leu Val Leu Ser Arg His Val Leu Gin Phe Leu Leu Leu
515 520 525
Tyr Gin Glu Pro Leu Leu Ala Leu Asp Val Asp Ser Thr Asn Ser Arg
530 535 540
Leu Arg His Cys Ala Tyr Arg Cys Tyr Ala Thr Trp Arg Phe Gly Ser
545 550 555 560
Gin Asp Met Ala Asp Phe Ala Ile Leu Pro Ser Cys Cys Arg Trp Arg
565 570 575
Ile Arg Lys Glu Phe Pro Lys Ser Glu Gly Gin Tyr Ser Gly Phe Lys
580 585 590
Ser Pro Tyr
595
