U.S. patent application number 12/079894 was filed with the patent office on 2008-12-25 for scd fingerprints.
This patent application is currently assigned to Cambridge University Technical Services Limited. Invention is credited to Charles Nicholas Hales, Margaret Hales, Cesar Milstein, Celia Prilleltensky Milstein, Adrian Woolfson.
Application Number | 20080318836 12/079894 |
Document ID | / |
Family ID | 27791909 |
Filed Date | 2008-12-25 |
United States Patent
Application |
20080318836 |
Kind Code |
A1 |
Woolfson; Adrian ; et
al. |
December 25, 2008 |
sCD Fingerprints
Abstract
The present invention relates to the use of cluster of
differentiation (CD) molecules in detecting the presence and
progression of one or more disease states in an individual. In
particular it relates to the use of profiles of shed CD (sCD)
molecules in detecting an assessing the progress of one or more
disease states in an individual. Further uses of sCD profiles
according to the present invention are also described.
Inventors: |
Woolfson; Adrian; (Primrose
Hill, GB) ; Hales; Charles Nicholas; (Cambridge,
GB) ; Hales; Margaret; (Great Wilbraham, GB) ;
Milstein; Cesar; (Cambridge, GB) ; Prilleltensky
Milstein; Celia; (Cambridge, GB) |
Correspondence
Address: |
Edwards Angell Palmer & Dodge LLP
P.O. Box 55874
Boston
MA
02205
US
|
Assignee: |
Cambridge University Technical
Services Limited
London
GB
Medical Researach Council
Addenbrookes NHS Trust
|
Family ID: |
27791909 |
Appl. No.: |
12/079894 |
Filed: |
March 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10506906 |
Jun 27, 2006 |
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PCT/GB03/00974 |
Mar 7, 2003 |
|
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12079894 |
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Current U.S.
Class: |
514/1.1 ;
530/300; 702/19 |
Current CPC
Class: |
G01N 33/6845 20130101;
A61P 35/00 20180101; G16H 40/63 20180101; G01N 33/68 20130101; G01N
33/6803 20130101; G16H 70/60 20180101; G01N 33/54306 20130101; Y02A
90/10 20180101 |
Class at
Publication: |
514/2 ; 702/19;
530/300 |
International
Class: |
A61K 38/02 20060101
A61K038/02; G06F 19/00 20060101 G06F019/00; C07K 2/00 20060101
C07K002/00; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 7, 2002 |
GB |
0205394.0 |
Apr 3, 2002 |
GB |
0207746.9 |
Dec 3, 2002 |
GB |
0228195.4 |
Claims
1. A shed CD (sCD) fingerprint of one or more disease states.
2. A method of generating a shed CD (sCD) fingerprint of one or
more disease state/s comprising the step of measuring the levels in
parallel of more than one shed CDs from one or more individuals and
collating the data.
3. A sCD fingerprint according to claim 1 or a method according to
claim 2 wherein the disease state is any one or more selected form
the group consisting of: infectious, neoplastic, autoimmune,
metabolic, immunological, degenerative, psychological, psychiatric,
iatrogenic, inflammatory, drug or toxin related, vascular,
traumatic and endocrine diseases.
4. A sCD fingerprint or a method according any preceding claim
wherein the disease state is any one or more selected from the
group consisting of the following: infection, Bence Jones
Proteinuria, Chronic Myeloid Leukemia, Colorectal cancer, chronic
renal failure, Crohn's Disease, Diabetic Nephropathy, Cardiac
pathology, Infection, Liver damage, Lymphoma, macrocytic anaemia,
Prostate Cancer, Oligoclonal Banding and Pulmonary Embolism/Deep
Vein Thrombosis and appendicitis.
5. A sCD fingerprint according to claim 1 or claim 3 or claim 4 or
a method according to claim 2, claim 3 or claim 4 wherein the sCDs
referred to comprise two or more selected from the group
consisting: CD 14, CD25, CD31, CD44, CD50, CD54, CD62E, CD62L,
CD86, CD95, CD106, CD116, CD124, CD138, CD141, CD40L, CD8, CD23,
CD30, CD40 and their homologues present in other mammalian or
non-mammalian species.
6. A method according to any of claims 2 to 5 wherein the sCD
levels are measured in samples of one or more body fluids from an
individual.
7. A method according to claim 6 wherein the body fluid is
serum.
8. A method according to any of claims 2 to 7 wherein sCD levels
are measured using one or more methods selected from the group
consisting of: immunoassay and flow cytometry.
9. A method according to claim 8 wherein sCD levels are measured
using any one or more method selected from the group consisting of
the following: multiplexed particle flow cytometry, chip based
monoclonal antibody technology, chips comprising engineered
antibodies, non protein agents which bind to one or more sCDs.
10. A method for predicting the presence of one or more disease
states in an individual comprising the step of comparing one or
more sCD fingerprints generated from that individual with one or
more reference sCD fingerprint/s.
11. A method for detecting the presence of one or more disease
states in an individual comprising the step of comparing one or
more sCD fingerprint/s generated from that individual with one or
more reference sCD fingerprint/s.
12. A method for detecting the extent of one or more disease states
in an individual comprising the step of comparing one or more sCD
fingerprint/s generated from that individual with one or more
reference sCD fingerprint/s.
13. A method for assessing the progression of a disease state in an
individual comprising the step of comparing the sCD fingerprint of
an individual at two or more periods during the life-span of the
disease.
14. A method for assessing the effect of one or more agent/s on one
or more disease states in an individual comprising the step of
comparing a sCD fingerprint of an individual at two or more
different time periods.
15. The use of a sCD fingerprint to assess the effect of one or
more agent/s on an individual.
16. A method for sub-categorising a sCD fingerprint profile
comprising the steps of identifying within one disease category one
or more group/s of sCDs wherein each group of sCDs exhibits common
characteristics distinguishing it from any other group within that
disease category.
17. A sCD database comprising pathological and/or normal sCD
fingerprint patterns.
18. A method for treating one or more diseases comprising the step
of inhibiting the production of one or more sCDs within an
individual.
19. A method according to claim 18 wherein the one or more sCDs are
any one or more of those selected from the group consisting of the
following: CD14, CD25, CD31, CD44, CD50, CD54, CD62E, CD62L, CD86,
CD95, CD106, CD116, CD124, CD138, CD141, CD40L, CD8, CD23, CD30,
CD40.
20. A method according to claim 19 wherein at least one sCD is
sCD1.
21. A method according to claim 18 or claim 19 wherein the
production of one or more sCDs is inhibited by the use of one or
more CD specific alternative splicing inhibitors.
22. A method according to any of claims 18 to 21 wherein the
disease is any one or more of those selected from the group
consisting of the following: tumourigenesis, infection, vascular
disease, endocrine disease.
23. The use of an inhibitor of the production of one or more sCDs
in the preparation of a medicament for the treatment of
disease.
24. The use according to claim 23, wherein that use exhibits any
one or more of the features of claims 18 to 22.
Description
[0001] This application is a continuation of Ser. No. 10/506,906,
filed Jun. 27, 2006, which is a 371 national phase application of
PCTGB03/00974 filed Mar. 7, 2003, which claims the benefit of
GB0205394.0 filed Mar. 7, 2002; GB0207746.9 filed Apr. 3, 2002; and
GB0228195.4, filed Dec. 3, 2002. Each of these applications in
their entirety is incorporated by reference herein.
[0002] The present invention relates to the use of cluster of
differentiation (CD) molecules in detecting the presence and/or
assessing the progression and/or assessing the response to
therapeutic intervention of one or more disease states in an
individual. In particular I relates to the use of
profile/fingerprints of shed CD (sCD) molecules in body fluids in
detecting and/or assessing the progression of one or more disease
states in an individual. Further uses of sCD profiles according to
the present invention are also described.
BACKGROUND TO THE INVENTION
[0003] Rapid and accurate diagnosis is essential in medicine as in
many cases early diagnosis and successful treatment correlates with
a better outcome and reduced hospitalisation. Currently, the
clinical diagnosis and staging of many diseases of global
significance involve different invasive procedures such as
histopathological analysis of biopsy samples which are usually
obtained when the disease process is at a relatively advanced
stage. In many cases, a classic histopathological approach may not
be sufficient to produce accurate diagnosis and any delay in
confirming the diagnosis would have financial and morbidity
repercussions for the healthcare institution and most importantly
for the individual. Disease states and disease staging are also
determined by different imaging techniques such as X-rays, nuclear
magnetic resonance (NMR), CT analysis and others, however, these
are expensive and impractical when dealing with large numbers of
individuals, or when it is necessary to monitor disease progression
closely, or in health institutes or clinical situations where such
equipment is unavailable. Furthermore such investigations are
impractical for individuals because it would result in such
individuals obtaining high radiation doses. For this reason such
tests cannot be carried out serially and are thus of little use in
monitoring drug responses and monitoring disease progression.
[0004] A variety of diseases or the predisposition to such a
disease can be characterised by changes in the overall patterns
and/or expression levels of various genes and their proteins. For
example, some cancers are associated with changes in the expression
of oncogenes or tumour suppressor genes. Furthermore, disease
conditions or disorders associated with disregulated cell cycle and
development can be attributed to changes in transcriptional
regulation of specific genes.
[0005] Although there are several genetic assays available to
assess gene mutations, the identification of specific genetic
changes may not always be a direct indicator of a disease or
disorder and thus cannot be relied upon as an accurate prognostic
indicator.
[0006] Certain genetic changes are exhibited by alterations in cell
surface antigens. Again, however, prior attempts to develop a
diagnostic assay for complex disease conditions or disorders based
on the identification of single antigen or very small numbers of
antigens have not been uniformly successful.
[0007] In addition, or alternatively, biochemical analysis of a
patient may be used to diagnose a disease state. For example, the
presence of Bence Jones proteins in urine is an indicator that an
individual has multiple myeloma. However, classical biochemical
methods are limited, for example an elevated cholesterol in serum
indicates hypercholesterolaemia but does not definitively indicate
atherosclerosis. A further disadvantage of biochemical methods of
diagnosis is that they generally permit the measurement of only one
or two indicator/s of disease in any one test. Consequently, they
provide an incomplete picture of the disease state of an
individual. Moreover, if several tests are performed in an attempt
to provide a more complete picture, this inevitably increases the
number of variables which complicates interpretation. Furthermore,
for many diseases there are no reliable biochemical markers,
especially for diseases of global importance such as breast cancer,
colorectal cancer and lung cancer. In the case of solid tumours
such as colorectal cancer, a number of carcinoembryonic antigen
(CEA) markers have been identified, however they have poor
sensitivity and very low specificity. The situation is similar with
disease conditions requiring surgical intervention. There is still,
for example, no marker for acute appendicitis and consequently, a
great many patients undergo unnecessary invasive surgery. It has
been estimated that more than 40,000 unnecessary appendicitis
operations occur each year due to misdiagnosis with associated
costs of $700 million. In a recent larger retrospective study,
Flume and colleagues show that misdiagnosis occurs in 15% of
instances.
[0008] Therefore, there is a pressing need in the art to provide a
simple and complete picture of the disease state or condition of an
individual. Such a `picture` would be of use in predicting and/or
detecting the presence of a disease or condition, in assessing the
therapeutic strategies and the potential of various agents and in
monitoring the progression and successful treatment of disease
states or conditions
[0009] Lymphocytes and other leukocytes express a large number of
different antigens associated with their outer plasma membranes
that can be used to identify distinct functional cell subsets. Many
of these antigens were "classically" known to be receptors for
growth factors, cell-cell interactions, viruses eg CD4, CD 112 and
CD 155 are the HIV, poliovirus receptor 2 and poliovirus receptor
respectively), and immunoglobulins; molecules for cell adhesion or
complement stimulation; enzymes and ion channels. A single
systematic nomenclature has been adopted to classify monoclonal
antibodies to human leukocyte cell surface antigens termed cluster
of differentiation (CD) antigens, also referred to as CD
molecules/antigens (Kishimoto et al., 1996 Proceedings of the Sixth
International Workshop and Conference held in Kobe, Japan. 10-14
Garland Publishing Inc, NY, USA). This work originated as the
direct result of the work of one of the inventors (Dr. Cesar
Milstein) of the present application who invented monoclonal
antibody technology with his colleague Georges Kohler (Kohler and
Milstein. Continuous cultures of fused cells secreting antibody of
defined specificity (1975), Nature August 7, 256 (5517), 495-7) and
who identified and raised the first monoclonal antibodies to both
non-human and human CD antigens (McMichael et al. A human thymocyte
antigen defined by a hydrid myeloma monoclonal antibody).
[0010] The data required in order to define a CD has changed over
the years, not surprisingly in view of the advances in modem
technology. Initially, clustering depended absolutely on the
statistical revelation of similarities in reaction pattern of two
or more antibodies, analysed on multiple tissues. It is now
accepted that CD molecules may also be classified by molecular
characteristics. Thus it has become customary to use the CD marker
(for example CD21) to indicate the molecule recognised by each
group of monoclonal antibodies. The current list of CD markers is
constantly updated as new antigens are identified and eventually,
the CD list will encompass all human lymphocyte cell surface
antigens and their homologues in other mammalian and non-mammalian
species (Mason et al., 2001, Immunology, 103, 401-406). It should
be noted that although CD antigens were initially defined in the
basis that they are expressed on the cell surface of leukocytes, a
great many of them are also expressed on numerous other cell types
including brain, liver, kidney, red blood cells, bone marrow,
dendritic antigen presenting cells, epithelial cells, stem cells,
thymocytes, osteoclasts, NK cells, B cells, macrophages, to name
but a few.
[0011] Historically, CD cell surface antigens have been used as
markers in diagnosis. Indeed leukemias are diagnosed on the basis
of cell morphology, expression of specific CD antigens, lymphoid
(LY) and myeloid (MY) antigens, enzyme activities and cytogenetic
abnormalities such as chromosome translocations. The expression of
up to three CD antigens on leukemia cells is determined using
labelled antibodies to particular CD antigens with analysis by flow
cytometry.
[0012] Significantly, however, it has been observed that often (if
not always in normal or disease states) the surface bound CD
immunological specificity molecules (intact CD molecules or
fragments thereof) are found soluble in the serum and in other body
fluids. Subsequent research has shown that indeed CD molecules can
be secreted from cells as a result of "active" processes such as
alternative splicing (Woolfson and Milstein, PNAS, 91 (14)
6683-6687 (1994)) or "passive" processes such as cell surface
shedding. Thus, CD molecules can be found in three forms, membrane
associated CD molecules, shed CD molecules (sCD) produced by
alternative splicing or other mechanisms and intracellular CD
molecules. Each of these can be complete molecules or fragments
thereof.
[0013] It is generally accepted however that the change in levels
of any one sCD is not specific to a given disease state and cannot
therefore usefully be used in the diagnosis of disease states.
[0014] Recent studies (those of WO 00/39580) have described a
system for the diagnosis of haematological maligancies, whereby
immunoglobulins are immobilised on a solid support and are used to
detect cell-surface antigen levels, in particular cell-surface CD
antigen levels in samples of cells. Using this approach, a pattern
of expression of cell-surface bound CD antigens is generated which
the inventors have shown to be indicative of the presence of
various defined leukemias in a patient. However, there are several
disadvantages with this technique. Firstly and importantly, it is a
cell-based technique. Such techniques have many disadvantages
associated with them, for example that of background noise and the
difficulty of measuring antigen levels accurately. Such methods
only allow semiquantitative determination of the relative densities
of sub-populations of cells of distinct immunophemotypes, indeed
absolute quantification using this method may not be possible.
Another problem with this prior art method is that at equilibrium,
the number of cells captured by the immobilised antibody dot
depends not only on the affinities of the interactions, the
concentration of the antibody dot, the level of expression of the
CD antigen on the cell surface and in addition to this the
stereochemical availability and accessibility of the monoclonal
antibody immobilised on the nitrocellulose membrane of the CD
antibody array. Furthermore computerised quantification of the cell
density as indicated by the pixel intensity corresponding to each
dot of arrayed antibody, depends not only on the number of cells in
the test sample, but in addition to the cell size and morphology.
In addition to all of these factors, the absolute requirement for
purification of cells from whole blood and the possible need to
fractionate blood cells still further makes such an approach both
labour intensive and time consuming.
[0015] Therefore, there still exists a need in the art for a simple
method for diagnosis of different diseases and conditions by the
measurement of CD antigens wherein such method produces a complete,
sensitive, specific and accurate picture of disease.
SUMMARY OF THE INVENTION
[0016] The present inventors have surprisingly found that
particular disease states can be characterised by specific patterns
of levels of shed/soluble/secreted (sCD) (as herein defined) CD
molecules derived from the body fluids of an individual. That is,
the profile or `sCD print` of the levels of sCD antigens correlate
with particular diseases or disorders or physiological states such
as those induced by administration of a drug or toxin. This finding
is especially surprising since the levels of sCDs found in the body
fluids of an individual are generally very low, and the sCD
released by cells would only be expected to change in some, and not
all cell types of an individual when affected by one or more
diseases, the change of levels of shed CD levels, as herein defined
detectable in the body fluids of diseased individuals as compared
with non-diseased individuals would be expected to be minimal.
[0017] Thus, in a first aspect, the present invention provides a
shed CD (sCD) fingerprint (sCD print) of one or more disease
states.
[0018] In the context of the present invention, the term `CD`
refers to a different cell surface leukocyte molecule recognised by
a given monoclonal or group of monoclonal antibodies which
specifically `cluster` to the antigen/molecule in question. Many,
if not all of these molecules produce forms which are released from
the cell surface by alternative splicing, proteolytic cleavage,
dissociation or other mechanisms. Thus in the context of the
present invention, the term `shed CD molecule (sCD)` is synonymous
with the term secreted/soluble CD (sCD) and refers to a released
form of a cell surface leukocyte molecule in which at least a
portion of that molecule is recognised by a given monoclonal or
group of monoclonal antibodies as herein described. It should be
noted however, that the antibody used to recognise the CD molecule
may not be monoclonal. It may be engineered, an artificial
construct consisting of an expressed fragment derived from an
antibody molecule with intact recognition, or it may be a
non-protein molecular recognition agent, or a protein recognition
agent which is not an antibody, or is an antibody hydrid, for
example made by introducing antibody binding sites into a different
scaffolding. Advantageously, as herein defined a shed form of sCD
is generated by various mechanisms including but not limited to any
of those selected from the group consisting of the following:
alternative splicing, proteolytic cleavage and dissociation.
[0019] In the context of the present invention it is important to
note that the CD nomenclature is a simple method for representing a
whole range of molecules. For example: CD14 is the
lipopolysaccharide receptor, LP5-R; CD21 is the EBV receptor, CD 25
is the IL-2Ralpha receptor; CD 31 is PECAM-1; CD 44 is H-CAM; CD 50
is ICAM-3; CD 54 ICAM-1; CD 62E is LECAM-2; CD 62L is LECAM-1; CD
86 is B 70; CD 95 is FAS apoptosis antigen; CD 102 is ICAM-2; CD
106 is VCAM-1; CD 116 is GM-CSFR alpha; CD 117 is c-kit stem cell
factor receptor; CD 124 is IL-4R alpha; CD 126 is IL-6Ralpha; CD
130 is gp 130; CD 138 is syndican-1; CD 141 is thrombomodulin; CD
91 is low density lipoprotein receptor-related antigen; CD 132 is
common cytokine receptor gamma), CD 89 is IgA Fc receptor, CD 74 is
class II specific chaperone, CD 95 is apoptosis antigen; CD220 is
the insulin receptor and CD 184 is the chemokine receptor 4.
(CXCR4) CD8 is Lin 2; CD 27 is low affinity IgE-R; CD 30 is
Ki-1.
[0020] The present inventors realised that sCDs act as
representatives/ambassadors for the families of molecules from
which they are shed. Thus sCD 184 stands as an ambassador for all
shed (as defined herein) cell surface chemokine receptors and for
example sCD54 acts as an ambassador for all intercellular adhesion
molecules. Furthermore they realised that cell behaviour can be
interregated on the basis of the patterns of sCD molecules shed by
cells.
[0021] In this regard it should be noted, as mentioned above that
sCDs are ambassadors for a vast range of molecules including but
not limited to the following: integrins, adhesion molecules, Fc
receptors, apoptosis antigens, blood group antigens, viral
receptors, coagulation factors, selectins, chemokine receptors,
macrophage receptors, insulin receptors, prion proteins,
glycophorins, rhesus antigens, T cell receptor zeta chain and
pregnancy specific antigens.
[0022] As herein defined, the term `shed CD fingerprint (sCD)`
describes the pattern or profile of levels of more than one shed
CDs in one or more individuals. A sCD fingerprint as herein defined
may be representative of one or more non-diseased individual/s or
one or more diseased individual/s. Preferably, a shed CD
fingerprint describes the level of five or more shed CD molecules,
more preferably it is 6, 7, 8, 9, 10 or more sCD molecules, more
preferably still a shed CD fingerprint describes the levels of 15
or more sCD molecules. More preferably a shed CD fingerprint
describes the levels of 20 or more sCD molecules. Most preferably,
it comprises the levels of sCDs for the complete set of sCDs for a
given individual.
[0023] sCD levels from normal and/or diseased individuals may be
collated in order to generate one or more reference sCD
fingerprints. A `reference` sCD fingerprint (sCD print) is a
fingerprint which is advantageously generated from sCD measurements
from more than one individual and is representative of the sCD
levels of either a `normal` or diseased individual. Advantageously,
these reference fingerprints are collected together to form a
database so that abnormal fingerprints generated from patient
samples can be distinguished from normal reference fingerprints in
the database. In addition, by comparing one or more patient sample
fingerprints with one or more reference fingerprint/s corresponding
to one or more diseases, then the disease state of an individual
may be established.
[0024] A shed CD (sCD) fingerprint (sCDprint) may be generated from
one individual. Preferably however, each fingerprint is generated
from more than one individual. Advantageously, it is generated from
more than five, ten, fifteen or twenty, 50, 100, 500, 1000, 5000,
10,000, 50,000 or 100,000 individuals. One skilled in the art will
appreciate that the greater the number of individuals used to
generate the reference fingerprint, then the more representative of
any given disease state or of a normal individual the reference sCD
profile/s will be. Fingerprints may be simplified by using the
average values of the data obtained for each sCD for a number of
individuals. For example, the modal value is used for data obtained
from a very small number of samples. Such information may then be
placed within a database as herein described.
[0025] One skilled will appreciate that often more than one disease
state may be present in an individual at a given time. This may
complicate the CD fingerprint obtained, such that the fingerprint
is an aggregate fingerprint of several disease states. The effect
of multiple disease states (composites) in an individual may be
minimised if the reference fingerprint for any given disease state
is generated from several or many individuals. Importantly,
composites may generate their own patterns and be used as reference
in their own right.
[0026] Measuring the levels of sCD molecules is carried out using
methods known to one skilled in the art and described herein.
[0027] sCD levels are measured in samples of body fluids. Suitable
body fluids for measuring sCDs include any one or more selected
from the group consisting of the following: tissue fluid, serum,
blood, cerebrospinal fluid, synovial fluid, urine, plural fluid,
saliva, lymphatic fluid, aspirate, bone marrow aspirate and mucus.
One skilled in the art will appreciate that this list is not
intended to be exhaustive.
[0028] In a further aspect the present invention provides a method
of generating a shed CD (sCD) fingerprint of one or more disease
state/s comprising the step of measuring in parallel the levels of
more than one shed CD in one or more samples from one or more
individuals and collating the data.
[0029] According to the above aspect of the invention, the term
`measuring in paralell` (sCD levels) refers to the process where
sCD levels are measured in one or more samples taken from an
individual at substantially the same time. Those skilled will
appreciate that in the case where sCD levels are measured from more
than one body fluid sample, then it may not be practical to take
more than one sample of body fluid from the same individual at
precisely the same time. Thus according to the above aspect of the
invention, when more than one body fluid sample is to be taken from
an individual for the generation of a sCDprint, then such samples
should be taken from the same individual as close together in time
as possible. Advantageously, the term `close together` (in time)
means within 5 hrs of one another, more advantageously within 4
hrs, 3 hrs, 2 hrs, 1 hr, 30 mins, 20 mins, 10 mins, 5 mins, 1 min
of one another. Those skilled will appreciate that so long as there
is no change in levels of measured sCDs between the first sample
and the last sample being taken, then such a time interval can be
considered `close together` as described herein. Thus so long as
there is no change in sCD levels between the first sample and the
last sample being taken, then such samples can be considered to be
taken `in paralell` and likewise such sCD levels can be considered
to be measured `in paralell` as referred to herein.
[0030] According to the above aspect of the invention, the samples
for testing are used to generate a given sCD
profile/fingerprint/pattern/barcode (sCDprint) and may be from one
body fluid type or more than one body fluid type. Advantageously
the one or more samples for testing and used to generate a sCD
profile are taken from more than one body fluid for any given
disease state. Advantageously, a number of sCD levels are measured
from the same body fluid sample. More advantageously, all of the
sCD levels comprising a fingerprint are measured in parallel from
one body fluid sample.
[0031] As used herein, the term `collating` the data means to put
the data into a form so that one or more pattern/s of the levels of
sCDs within that disease state is apparent. Advantageously, the
data will be entered into a database as described herein. More
advantageously, the database is an integrated database as described
herein, comprising clinical data linked to specific sCD fingerprint
patterns/profiles.
[0032] As the present inventors surprisingly found that a sCD
fingerprint/profile (sCDprint) of a diseased individual is
different from that of a non-diseased individual, it was realised
that by comparing the sCD fingerprint/profile (sCDprint) of a
sample from a diseased patient with that of one or more reference
sCD fingerprints representing one or more defined disease states
then the presence and nature of a disease in that individual could
be ascertained.
[0033] Thus, in a further aspect, the present invention provides a
method for predicting the presence of one or more disease states in
an individual comprising the step of comparing one or more sCD
fingerprint/s (sCDprint/s) generated from that individual with one
or more reference sCD fingerprints.
[0034] In the context of the present invention, the term
`predicting the presence of one or more disease states` refers to
the process of detecting the presence of one or more disease states
before the onset of the clinical signs of the disease are apparent
in the individual. The clinical signs of disease are characteristic
of each disease state or group of disease states, as long as the
disease is present in that individual.
[0035] As referred to herein, the comparing step may refer to
comparing an individuals sCD fingerprint/profile (sCDprint) with
one or more reference sCD fingerprint/s of one or more disease
state/s and/or with a reference sCD fingerprint of a non-diseased
`normal` individual.
[0036] In a further aspect still, the present invention provides a
method for detecting the presence of one or more disease states in
an individual comprising the step of comparing one or more sCD
fingerprint/s generated from that individual with one or more
reference sCD fingerprint/s.
[0037] The term `detecting the presence of one or more disease
states` refers to the detection of the presence of one or more
disease states in an individual once the clinical signs of one or
more disease states are apparent in that individual. In addition,
the term refers to the process of detecting the presence of one or
more disease states in an individual other than the disease state
whose clinical signs are apparent in that individual.
[0038] According to the above two aspects of the invention, the
reference sCD fingerprint/s may be from non-diseased (normal)
individuals and/or from diseased individuals. Alternatively, or in
addition the reference sCD fingerprints/profiles may be derived
from normal subjects who have undergone some form of intervention.
Such interventions include but are not limited to treatment with
chemotherapeutic or other agents, exposure to radiation and
exposure to pathogens. Those skilled in the art will be aware of
other interventions as used herein. According to the above two
aspects of the invention, preferably, the sCD
fingerprint/s/profiles (sCDprint) are from diseased
individuals.
[0039] In a further aspect still, the present invention provides a
method for detecting the extent of one or more disease states in an
individual comprising the step of comparing one or more sCD
fingerprint/s generated from that individual with one or more
reference sCD fingerprint/s.
[0040] As referred to above, the term `detecting the extent of one
or more disease states` includes within its scope detecting the
severity of one or more disease states within an individual. For
example it may allow low grade and high grade forms of the disease
to be distinguished. It allows localised and metastasised forms of
a particular disease to be distinguished. In such cases one or more
sCD fingerprints of an individual are compared with one or more
reference disease sCD fingerprints representative of disease states
at one or more degrees of severity. For example, in the case of
neoplastic disease the presence or absence of metastasis may be
detected using the method of the present invention.
[0041] In a further aspect, the present invention provides a method
for assessing the progression of a disease state in an individual
comprising the step of comparing the sCD fingerprint of an
individual at two or more periods during the occurrence of the
disease.
[0042] In the context of the present invention, the term `assessing
the progression of a disease state` means assessing whether the
disease has increased in severity, decreased in severity or remains
the same severity compared with a different period during the
life-span of the disease. In addition the term `assessing the
progression of a disease state` includes within its scope
monitoring the progression of a disease state.
[0043] The term `period` in the context of the present invention,
generally refers to a time period.
[0044] As defined herein, the term a `disease state` refers to any
impairment of the normal physiological functions affecting an
organism or any disease condition, disorder or the presence of a
particular microbial, viral, parasitic or other pathogenic agent
known to one skilled in the art. Suitable disease states for
analysis as described herein include but are not limited to:
infectious, neoplastic, autoimmune, immunological, metabolic,
degenerative, psychological, psychiatric, iatrogenic, inflammatory,
drug or toxin related, vascular, traumatic and endocrine diseases.
Advantageously, `a disease state` as herein defined refers to any
one or more disease selected from the group which includes but is
not limited to: infections such as bacterial, fimgal, protozoan,
parasitic, prion and viral infections, non-neoplastic disorders;
stroke; heart condition; atherosclerosis; pain; diabetes, obesity;
anorexia; bulimia; asthma; Parkinson's disease; thrombosis; acute
heart failure; hypotension; hypertension; urinary retention;
metabolic bone diseases such as osteoporosis and osteo petrosis;
angina pectoris; hepatitis; myocardial infarction; ulcers; asthma;
allergies; rheumatoid arthritis; inflammatory bowel disease;
irritable bowel syndrome benign prostatic hypertrophy;
pancreatitis; chronic renal failure and psychotic and neurological
disorders, including anxiety, schizophrenia, manic depression,
delirium, dementia, severe mental retardation and dyskinesias, such
as Huntington's disease or Gilles de la Tourett's syndrome and
others. Most preferably it refers to appendicitis; Bence Jones
Proteinuria; Chronic Myoloid Leukemia; Colorectal cancer; chronic
renal failure; Crohn's Disease; Diabetic Nephropathy; Cardiac
pathology; Infection; Liver damage; Lymphoma; macrocytic anaemia;
Prostate Cancer; Oligoclonal Banding and Pulmonary Embolism/Deep
Vein Thrombosis (eg DVT/PE). One skilled in the art will appreciate
that this list is not intended to be exhaustive. Indeed this method
should be suitable for most if not all diseases.
[0045] Examples of shed cluster of differentiation molecules
suitable for measurement to generate a sCD fingerprint for use in
the methods of the present invention include but are not limited to
CD14, CD25, CD31, CD44, CD50, CD54, CD62E, CD62L, CD86, CD95,
CD106, CD116, CD124, CD138, CD141, CD40L, CD8, CD23, CD30, CD40.
Those skilled in the art will be aware of other suitable sCD
molecules for analyses according to the methods of the present
invention. They will also be aware of other members of the family
that each sCD stands as an ambasador for, such as chemokine
receptors, interleukin receptors and inter-cellular adhesion
molecules.
[0046] Measuring CD levels may be carried out using methods known
to those skilled in the art and described herein. Shed CD (sCD)
levels may suitably be measured in samples of tissue fluids which
include, but are not limited to: serum, plasma, lymph fluid,
pleural fluid, synovial fluid, follicular fluid, seminal fluid,
amniotic fluid, milk whole blood, urine, cerebrospinal fluid (CSF),
ascites, saliva, sputum, tears, perspiration, and mucus.
Advantageously, sCD levels are measured from samples of serum using
reagents suitable for detecting shed CDs that include but are not
limited to antibodies raised against those CDs. Preferably
monoclonal antibodies or engineered antibodies, including phage
antibodies raised against shed CDs or their membrane bound forms
are used for their detection. However non-protein agents may also
in principle be used to detect sCD molecules. Similarly the
detecting molecule may contain antibody bonding site fragments
incorporated into the scaffold of another molecules or an
engineered scaffold. Commercially available kits for measuring CD
levels include those from Diaclone 1, Bd A Fleming BP 1985 F-25020
Besancon Cedex-France and Medsystems Diagnostics GmbH, Rennweg 95b,
A-1030 Vienna Austria.
[0047] Suitable techniques for measuring levels of sCDs include but
are not limited to immunoassay including ELISA using commercially
available kits such as those described above, flow cytometry
particularly multiplexed particle flow cytometry as herein
described. Those skilled in the art will be aware of other suitable
techniques for measuring CD levels in samples from an individual
including antibody `chip` array type technologies or chip
technologies utilizing non-classical antibody binding site grafted
molecules. Suitable techniques for measuring levels of sCDs are
described in more detail in the detailed description of the
invention.
[0048] Shed CD levels are also measured in a number of individuals
with one or more disease states as herein defined, such as
appendicitis and the like. Generally one or more shed CDs levels
will be elevated in a given disease state as compared with the
range found in normal individuals. However, some sCD levels
decrease in some disease states compared with the range found in
`normal` individuals and such decreases may also form part of a sCD
fingerprint of the present invention. Thus, by measuring the ranges
of levels of various shed CDs found in a number of individuals with
one or more defined disease state/s a `fingerprint` of shed CD
levels for any defined one or more diseases is generated. Likewise
by measuring the ranges of levels of various shed CDs found in a
number of individuals who have undergone one or more interventions
(such as chemotherapeutic treatment, exposure to pathogens,
exposure to radiation, individuals who have undergone a given
vaccination program etc) then a `fingerprint` (sCDprint) of shed CD
levels for a given intervention may be generated. Those skilled in
the art will appreciate that a sCD fingerprint (sCDprint)
representative of an intervention may be generated form diseased or
non-diseased individuals.
[0049] Preferably, the sCD fingerprint of an individual is
generated from any two or more sCDs selected from the group
consisting of the following: CD 14, CD25, CD31, CD44, CD50, CD54,
CD62E, CD62L, CD86, CD95, CD106, CD116, CD124, CD138, CD141, CD40L,
CD8, CD23, CD30, CD40. One skilled in the art will appreciate that
this list is not intended to be exhaustive and may include CD
homologues of human and other mammalian or non-mammalian species.
One skilled in the art will appreciate though that in general
animal reference sCD fingerprints cannot be used to analyse human
diseases and vice-versa. For instance sCD
patterns/profiles/fingerprints could be defined in the rat or mouse
using rat or mouse equivalent CD monoclonal antibodies as herein
described. This would be an invaluable adjunct for studying these
animal model systems, especially in the area of therapeutics, gene
knockouts and other such proteomic and genomic studies.
[0050] The invention can also be used for testing human and other
mammalian and non-mammalian species using sCD fingerprints from the
appropriate animal.
[0051] One skilled in the art will appreciate that the methods of
the present aspect of the invention can be used to test potential
therapeutic agents suitable for the prophylaxis and/or treatment of
diseases. An agent of therapeutic potential will affect the sCD
profile or `fingerprint` of the disease: If several fingerprints
are taken at various stages of a disease and compared with those
obtained from samples in which an individual has been treated with
a potential therapeutic agent, then the effect on one or more sCD
fingerprints can readily be assessed.
[0052] In addition, the method of the present invention may also be
used to monitor patient compliance with taking a particular drug
(agent), and/or undergoing a particular treatment regime.
[0053] Thus, in a further aspect still, the present invention
provides a method for assessing the effect of one or more agent/s
on one or more disease states in an individual comprising the step
of comparing a sCD fingerprint of an individual at two or more
different time periods.
[0054] According to the above aspect of the invention, preferably
the agent is a potentially therapeutic agent.
[0055] In the context of the present invention the term to `assess
the effect` means to detect any changes in the severity or other
characteristics of any one or more diseases in an individual. Such
changes will be reflected in a change in sCD profile/fingerprint of
an individual.
[0056] Preferably the agent is a potentially therapeutic agent. The
term `potentially therapeutic agent` means any agent that may cause
a beneficial effect on an individual suffering from one or more
diseases. Such beneficial effects may be for example reducing the
clinical signs of the one or more diseases. It is an important
feature of the present invention though, that a change in level of
any one sCD in isolation is not always indicative of a change in
severity of a disease. It will appreciated though that in some
cases, a change in the level of one sCD in isolation will be
indicative of a change in severity of a disease.
[0057] Generally, individual sCD levels will be elevated in a
disease state as compared with a `normal` non-diseased individual.
Occasionally however, the level of an individual sCD will decrease
in a disease state as compared with a normal non-diseased
individual. However, according to the present invention, it is the
changes of the profile of a number of sCDs (that is a fingerprint)
during a disease which provides an accurate measure of the effect
of one or more agents on a disease state in an individual.
[0058] One skilled in the art will appreciate that on occasion a
selection of the complete repertoire of sCDs available for testing
may be measured. The selection chosen may vary according to the
disease state being tested.
[0059] According to this aspect of the invention, sCDs suitable for
generating a sCD fingerprint are as described herein.
[0060] Therapeutic agents may be tested for their effect on any one
or more disease states selected from the group consisting of the
following: infections, autoimmune disease, neoplastic, vascular
endocrinological, metabolic, inflammatory degenerative, psychiatric
psychological, traumatic, drug/toxin-related, bacterial, fungal,
protozoan and viral infections, non-neoplastic disorders; pain;
diabetes, obesity; anorexia; bulimia; asthma; pregnancy; endocrine;
vascular; metabolic; gastro-intestinal; iatrogenic; psychiatric;
psychoclogical; exercise-induced; diet-related; ME; degenerative;
Parkinson's disease; thrombosis; atherosclerosis; acute heart
failure; hypotension; hypertension; erectile dysfunction; urinary
retention; metabolic bone diseases such as osteoporosis; angina
pectoris; hepatitis; myocardial infarction; ulcers; allergies;
rheumatoid arthritis; inflammatory bowel disease; irritable bowel
syndrome benign prostatic hypertrophy; psychosis; psychiatric
disorders; including anxiety; schizophrenia; manic depression;
delirium; dementia; severe mental retardation and dyskinesias, such
as Huntington's disease or Gilles de la Tourett's syndrome; and
preferably tumours which can be benign or malignant cancers; breast
cancer; myeloma; melanoma; bladder cancer; leukaemia; plasmocytoma
and others, but most preferably appendicitis; Bence Jones
Proteinuria; Chronic Myeloid Leukaemia; Colorectal cancer; chronic
renal failure; Crohn's Disease; Diabetic Nephropathy; Cardiac
pathology; Infection; Liver damage; Lymphoma; macrocytic anaemia;
Prostate Cancer; Oligoclonal Banding and PE/DVT
[0061] Suitable agents for assessment according to the method of
the present invention may be naturally occurring or synthetic.
Naturally occurring agents include proteins, peptides or nucleic
acids. They may be agents known to be of therapeutic value or they
may be of unknown therapeutic value.
[0062] In a further aspect, the present invention provides a method
for sub-categorising a sCD fingerprint profile comprising the steps
of identifying within one or more disease states one or more
sub-group/s of sCDs wherein each sub-group of sCDs exhibits common
characteristics distinguishing it from any other sub-group within
that disease category.
[0063] As used herein the term a `sCD sub-category` describes a
sub-group of sCDs which show a defined fingerprint/profile
(sub-fingerprint) of sCD levels within a larger fingerprint of one
or more disease states wherein each sub-group of sCDs exhibits
common characteristics distinguishing it from any other sub-group
within those one or more disease states.
[0064] In a further aspect still, the present invention provides a
sCD reference database comprising pathological and/or normal sCD
fingerprint patterns.
[0065] As herein described the term `a reference database` refers
to a collection of sCD fingerprints from normal `non-diseased`
and/or diseased individuals. Advantageously, the database is
computer generated and/or stored. Advantageously the data from more
than 5 individuals is present in the database. More advantageously
the data from more than 10, 100, or 1000 individuals comprises the
database. More advantageously still the data from more than 10,000
or more than 50,000 individuals comprises the database. Most
advantageously the data from more than 100,000 individuals
comprises the database. Advantageously the database, in addition to
sCD data will also comprise clinical information relating to
various patients and/or disease conditions. Alternatively or in
addition, a database according to the present invention comprises
genomic information such as mRNA expression profiles, and/or sCD
body fluid profiling data, and/or CD cell surface pattern data,
and/or clinical data. Most advantageously, the database will be in
the form of an integrated clinical database comprising accurate
patient details including co-morbidity, age, sex, smoking status
etc.
[0066] Recent studies which have investigated the impaired
expression of NKG2D and T-cell activation by tumour-derived soluble
MHC ligands (Nature, vol 419, 17 Oct. 2002). Studies have shown
that tumours release large amounts of the MHC class I homologue MIC
into the serum. Activation of the NKG2D receptor on natural T cells
is known to stimulate their ability to destroy tumours, but the
high levels of tumour derived MIC seem to downregulate the NKG2D
receptor and block the antitumour effect. (Nature, vol 419, 17 Oct.
2002, p 679, pg 734). These soluble forms of MHC are produced
either by enzymatic cleavage or by alternative splicing (Nature,
vol 419, 17 Oct. 2002, p 679, pg 734).
[0067] It is apparent from the present disclosure that sCDs may be
produced by alternative splicing (Woolfson and Milstein, PNAS Vol
91, pp 6683-6687), enzymatic cleavage or other mechanisms and that
such shed forms are associated with disease (Sugiyama et al.,
Non-invasive detection of bladder cancer by identification of
abnormal CD44 proteins in exfoliated cancer cells in urine, Journal
of Clinical pathology, 48, 3, 142-147; Yoshida, K et al, Abnormal
retention of intron 9 in CD44 transcripts in human gastrointestinal
tumours. Cancer research 55, 4273-4277). The present inventors
consider that sCD molecules may also bind to a ligand/receptor and
thereby block down stream effects. Thus, the present inventors have
realised that the blockage of the production of sCD molecules via
the inhibition of any of the methods of sCD generation described
herein, may be a therapeutically. useful method for the prophylaxis
or treatment of one or more diseases or disorders including but not
limited to any of those in the group consisting of infections,
inflammation, vascular, iatra genic, endocrine, drug-related
disorders, toxin related disorders, and cancer in particular
metastasis and leukemia.
[0068] Thus in a further aspect still, the present invention
provides a method for treating one or more diseases comprising the
step of inhibiting the production of one or more sCDs within an
individual.
[0069] In a further aspect still, the present invention provides
the use of an inhibitor of the production of one or more sCDs in
the preparation of a medicament for the treatment of disease.
[0070] According to the above aspect of the invention, the term `an
inhibitor of the production of one or more sCDs` refers to one or
more agents which inhibit the production of a shed form of sCD as
herein defined. Advantageously, the inhibitor is a specific
inhibitor of those one or more sCDs. Suitable inhibitors include
alternative splicing inhibitors and/or enzymatic cleavage
inhibitors. Advantageously, the inhibitor is an alternative
splicing inhibitor. Such alternative splicing inhibitors include
for example inhibitors of exonic splicing enhancers (Fairbrother et
al, Science, vol 297, 9 Aug. 2002).
[0071] According to the above aspects of the invention, the
production of any one or more sCDs present in the body fluid of an
individual may be inhibited. Advantageously, the one or more sCDs
are any one of those selected from the group consisting of the
following: CD14, CD25, CD31, CD44, CD50, CD54, CD62E, CD62L, CD86,
CD95, CD106, CD116, CD124, CD138, CD141, CD40L, CD8, CD23, CD30,
CD40. More advantageously the sCD is CD1. Advantageously, the sCD
is CD1 and the inhibitory agent is an alternative splicing
inhibitor and/or a gene specific CD1 inhibitor.
[0072] It should be noted that the present inventors consider that
the invention described herein can be used to post hoc re-analyse
clinical trial data, assigning patients to different sub-groups for
analysis on the basis of their sCDprints and in doing so
potentially revealing previously unseen statistical effects and at
the same time identifying responders or non-responders to a
therapeutic intervention, and those who respond adversely to the
intervention in the case in which a therapeutic agent has not been
taken further due to adverse responses in small numbers of
individuals.
DEFINITIONS
[0073] The term `CD` refers to a different cell surface leukocyte
molecule recognised by a given monoclonal or group of monoclonal
antibodies which specifically `cluster` to the antigen/molecule in
question. Many, if not all of these molecules produce forms which
are released from the cell surface by alternative splicing,
proteolytic cleavage, dissociation or other mechanisms.
[0074] Thus in the context of the present invention, the term `shed
CD molecule (sCD)` refers to a released form of a cell surface
leukocyte molecule in which at least a portion of that molecule is
recognised by a given monoclonal or group of monoclonal antibodies
as herein described. Advantageously, as herein defined a shed form
of sCD is generated by various mechanisms including but not limited
to any of those selected from the group consisting of the
following: alternative splicing, proteolytic cleavage and
dissociation.
[0075] As herein defined, the term `shed CD fingerprint/profile
(sCD)` or `sCDprint` describes the pattern or profile of levels of
more than one shed CD measured in one or more body fluids from one
or more individuals. A sCD fingerprint as herein defined may be
representative of one or more non-diseased individual or a one or
more diseased individual/s. Preferably, a shed CD fingerprint
describes the level of five or more shed CD molecules, more
preferably it is 6, 7, 8, 9, 10 or more sCD molecules, more
preferably still a shed CD fingerprint describes the levels of 15
or more sCD molecules. Most preferably a shed CD fingerprint
describes the levels of 20 or more sCD molecules.
[0076] As used herein the term a `sCD sub-category` describes a
sub-group of sCDs which show a defined fingerprint/profile
(sub-fingerprint) of sCD levels within a larger fingerprint of one
or more disease states wherein each sub-group of sCDs exhibits
common characteristics distinguishing it from any other sub-group
within those one or more disease states.
[0077] As defined herein, the term a `disease state` refers to any
impairment of the normal physiological functions affecting an
organism or any disease condition, disorder or the presence of a
particular microbial, viral, parasitic or other pathogenic agent
known to one skilled in the art. Suitable disease states for
analysis as described herein include but are not limited to:
infectious, neoplastic, autoimmune, immunological, metabolic,
degenerative, psychological, psychiatric, iatrogenic, inflammatory,
drug or toxin related, vascular, traumatic and endocrine diseases.
Advantageously, `a disease state` as herein defined refers to any
one or more disease selected from the group which includes but is
not limited to: infections such as bacterial, fungal, protozoan,
parasitic, prion, and viral infections, non-neoplastic disorders;
stroke; heart condition; atherosclerosis; pain; diabetes, obesity;
anorexia; bulimia; asthma; Parinson's disease; thrombosis; acute
heart failure; hypotension; hypertension; urinary retention;
metabolic bone diseases such as osteoporosis and osteo petrosis;
angina pectoris; hepatitis; myocardial infarction; ulcers; asthma
allergies; rheumatoid arthritis; inflammatory bowel disease;
irritable bowel syndrome benign prostatic hypertrophy;
pancreatitis; chronic renal failure and psychotic and neurological
disorders, including anxiety, schizophrenia, manic depression,
delirium, dementia, severe mental retardation and dyskinesias, such
as Huntington's disease or Gilles de la Tourett's syndrome and
others. Most preferably it refers to appendicitis; Bence Jones
Proteinuria; Chronic Myoloid Leukemia; Colorectal cancer; chronic
renal failure; Crohn's Disease; Diabetic Nephropathy; Cardiac
pathology; Infection; Liver damage; Lymphoma; macrocytic anaemia;
Prostate Cancer; Oligoclonal Banding (myaesthenis gravis) and
Pulmonary Embolism/Deep Vein Thrombosis (eg DVT/PE). One skilled in
the art will appreciate that this list is not intended to be
exhaustive.
[0078] Examples of shed cluster of differentiation molecules
suitable for measurement to generate a sCD fingerprint for use in
the methods of the present invention include but are not limited to
CD14, CD25, CD31, CD44, CDSO, CDS4, CD62E, CD62L, CD86, CD95,
CD106, CD116, CD124, CD138, CD141, CD40L, CD8, CD23, CD30,
CD40.
[0079] Those skilled in the art will be aware of other suitable sCD
molecules for analyses according to the methods of the present
invention.
[0080] As defined herein the term `an antibody` includes within its
scope for example IgG, IgM, IgA, IgD or IgE) or fragment (such as a
FAb, F(Ab').sub.2, Fv, disulphide linked Fv, scFv, diabody) whether
derived from any species naturally producing an antibody, or
created by engineered DNA technology (for example fluorobodies,
green fluorescently labelled antibodies); whether isolated from
serum, B-cells, hybridomas, transfectomas, yeast or bacteria). It
also includes within its scope, non-protein binding agents which
comprise the binding specificity of an antibody molecule, or a
binding capacity in general for a determinant or sub-determinant of
a component of the protein structure/glycoprotein structure of a CD
molecule.
BRIEF DESCRIPTION OF THE FIGURES
[0081] FIG. 1. Disease Groups. Multiples of upper limit of normal
(ULN). All sCD's included
[0082] The limits indicated by each point are: [0083] No shading
.ltoreq.1.times.ULN [0084] Lightly shaded 1-2.times.ULN [0085]
Darkly shaded >2.times.ULN [0086] A white slash in the box
indicates no data available.
[0087] FIG. 2. All sCD's that appear not to discriminate from the
normals (sCD's 21; 102; 117, 126; 130; 26; 44v5; 44v6; 62P).
[0088] FIG. 3. Disease Groups. Mode of Response for Remaining 20
sCD's. To simplify the data further the modal response for each
disease group was plotted.
[0089] FIG. 4. Disease Groups. Mode of Response for remaining
sCD's. Data has been ranked in order of increased expression.
[0090] FIG. 5. Disease Groups. Mode of Response for remaining
sCD's.
[0091] FIG. 6. Disease Groups. Mode of Response for all sCD's. As
for FIG. 3 (except all sCD's are included). The limits indicated by
each point are: [0092] No shading .ltoreq.1.times.ULN [0093]
Lightly shaded 1-2.times.ULN [0094] Darkly shaded
>2.times.ULN
[0095] A white slash in the box indicates no data available.
[0096] FIG. 7. Disease Groups. Mode of Response for all sCD's.
[0097] FIG. 8. Disease Groups. Mode of Response for all sCD's.
[0098] FIG. 9. Shows the patterns of levels of sCDs during various
infectious disease states as compared with a group of `normal`
non-diseased individuals. [0099] Key: Lightly shaded box-sCD levels
unchanged Darkly shaded box-sCD levels increased Shaded box with
diagonal line-sCD levels decreased.
[0100] FIG. 10. Shows the patterns of levels of sCDs during various
inflammatory/autoimmune diseases as compared with a group of
`normal` non-diseased individuals. [0101] Key: Lightly shaded
box-sCD levels unchanged Darkly shaded box-sCD levels increased
Shaded box with diagonal line-sCD levels
[0102] FIG. 11. Shows the patterns of levels of sCDs during various
`other diseases` as compared with a group of `normal` non-diseased
individuals. [0103] Key: Lightly shaded box-sCD levels unchanged
Darkly shaded box-sCD levels increased Shaded box with diagonal
line-sCD levels decreased
[0104] FIG. 12. Shows the patterns of levels of sCDs during various
neoplastic disease states as compared with a group of `normal`
non-diseased individuals. [0105] Key: Lightly shaded box-sCD levels
unchanged Darkly shaded box-sCD levels increased Shaded box with
diagonal line-sCD levels decreased
[0106] FIG. 13. Shows the patterns of levels of sCDs during various
cardiovascular diseases as compared with a group of `normal`
non-diseased individuals. [0107] Key: Lightly shaded box-sCD levels
unchanged Darkly shaded box-sCD levels increased Shaded box with
diagonal line-sCD levels decreased
[0108] FIG. 14. Shows the patterns of levels of sCDs during various
metabolic and haematological diseases as compared with a group of
`normal` non-diseased individuals. [0109] Key: Lightly shaded
box-sCD levels unchanged Darkly shaded box-sCD levels increased
Shaded box with diagonal line-sCD levels decreased
[0110] FIG. 15. Shows the patterns of levels of sCDs during various
haematological malignancies as compared with a group of `normal`
non-diseased individuals. [0111] Key: Lightly shaded box-sCD levels
unchanged Darkly shaded box-sCD levels increased Shaded box with
diagonal line-sCD levels decreased
DETAILED DESCRIPTION OF THE INVENTION
General Techniques
[0112] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art (e.g., in cell culture, molecular
genetics, nucleic acid chemistry, hybridisation techniques and
biochemistry). Standard techniques are used for molecular, genetic
and biochemical methods (see generally, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,
Short Protocols in Molecular Biology (1999) 4.sup.th Ed, John Wiley
& Sons, Inc. which are incorporated herein by reference) and
chemical methods. In addition Harlow & Lane., A Laboratory
Manual Cold Spring Harbor, N.Y., is referred to for standard
Immunological Techniques.
sCD Molecules According To The Invention
[0113] In a first aspect, the present invention provides a shed CD
(sCD) fingerprint (sCD print) of one or more disease states.
[0114] In the context of the present invention, the term `CD`
refers to a different cell surface leukocyte molecule recognised by
a given monoclonal or group of monoclonal antibodies which
specifically `cluster` to the antigen/molecule in question. Many,
if not all of these molecules produce forms which are released from
the cell surface by alternative splicing, proteolytic cleavage,
dissociation or other mechanisms.
[0115] Thus in the context of the present invention, the term `shed
CD molecule (sCD)` refers to a released form of a cell surface
leukocyte molecule in which at least a portion of that molecule
recognised by a given monoclonal or group of monoclonal antibodies
as herein described.
Location of sCD Molecules.
[0116] Although first identified on leukocytes. CD antigens have
been located on other blood cells and non-blood cells. CD molecules
have been found on many different blood cell types including the
following blood cell types: erythroid, dendritic cells, B cells,
pre-B cells, T cells (cytotoxic, suppressor and helper subtypes),
monocytes, myeloid cells, endothelial cells, platelets, NK cells
(natural killer cells), red blood cells, thymocytes. Those skilled
in the art will appreciate that this list is not intended to be
exhaustive.
[0117] In addition, CD antigens have been found on the following
non-immune cells myocytes, peripheral nerve, liver, platelet
precursors, lung cells, cerebellum, cortex, glia, neuroepithelium,
placenta, prostate, spinal cord, brain, muscle, kidney, salivary
glands, muscle, melanoms, leukemias, lymphoma, hematopoietic cells,
lymphoid progenitor cells, breast, astrocytes, thyroid, lung,
pancreas, trachea, schwann cells, trophoblast, erthroblast,
microglia.
[0118] In the context of the present invention it is important to
note that the CD nomenclature is a simple method for representing a
whole range of molecules. For example: CD14 is the
lipopolysaccharide receptor, LP5-R; CD21 is the EBV receptor; CD 25
is the IL-2Ralpha receptor, CD 31 is PECAM-1; CD 44 is H-CAM; CD 50
is ICAM-3; CD 54 ICAM-1; CD 62E is LECAM-2; CD 62L is LECAM-1; CD
86 is B 70; CD 95 is FAS apoptosis antigen; CD 102 is ICAM-2; CD
106 is VCAM-1; CD 116 is GM-CSFR alpha; CD 117 is c-kit stem cell
factor receptor; CD 124 is IL-4R alpha; CD 126 is IL-6Ralpha; CD
130 is gp 130; CD 138 is syndican-1; CD 141 is thrombomodulin; CD8
is Lin 2; CD 27 is low affinity IgE-R; Cd 30 is Ki-1;
[0119] The present inventors realised that sCDs act as
representatives/ambassadors for the molecules from which they are
shed. Furthermore they realised that cell behaviour can be
interrogated on the basis of the patterns of sCD molecules shed by
cells.
[0120] Generation of a Fingerprint of One or More Disease
States
[0121] In a first aspect, the present invention provides a shed CD
(sCD) fingerprint of one or more disease states.
[0122] Clinical signs and symptoms and various biochemical
indicators of disease are used to identify individuals with one or
more defined disease states. sCD levels are then measured for a
number of sCDs present in one or more body fluid samples from each
individual, preferably in a number of individuals using methods
known to those skilled in the art and described herein, in order to
generate a reference disease state or reference composite disease
state fingerprint for those one or more given disease states.
(A) Diagnosis of Disease States
[0123] i. Diagnostic Indicators Used.
Appendicitis
[0124] Request for Amylase at admission A&E/MAU Subsequent
Histopathological Diagnosis
Bence Jones Proteinuria
[0124] [0125] Multiple myeloma in which the malignant plasma cells
excrete only light chains of one type (either 2 or 3); lytic bone
lesions occur in about 60% of the cases, and light chains (Bence
Jones protein) can be detected in the urine [0126] Positive
finding
Chronic Myeloid Leukaemia
[0126] [0127] Histopathological Diagnosis
Colorectal Carcinoma
[0127] [0128] Histopathological Diagnosis
Chronic Renal Failure
[0128] [0129] Prolonged elevation of serum creatinine.
Crohn's Disease
[0129] [0130] Histopathological diagnosis
Diabetic Nephropathy
[0130] [0131] Identified by abnormal urine Albumin/Creatinine ratio
from subjects attending diabetic clinic.
Cardiac Pathology
[0131] [0132] MI (as diagnosed by increased CK, symptoms and ECG
changes).
Infection
[0132] [0133] CRP (C reactive protein)>250 g/l (e.g.
Staphylococcus aureus infections).
Liver Damage
[0133] [0134] Clinical Details Alcoholic Liver Disease/Poisoning.
Abnormal liver function tests.
Lymphoma
[0134] [0135] Histopathological Diagnosis
Macrocytic Anaemia
[0135] [0136] Diagnosed by haematological parameters. Hb<10
g/dL; MCV>100 fL
Oligoclonal Banding
[0136] [0137] small discrete bands in the gamma globulin region of
the spinal fluid electrophoresis, indicating local central nervous
system production of IgG; bands are frequently seen in patients
with multiple sclerosis but can also be found in other diseases of
the central nervous system including syphilis, sarcoidosis, and
chronic infection or inflammation. VO (pulmonary angiogram)
Pulmonary Embolism/Deep Vein Thrombosis
[0137] [0138] ultrasound VQ or CT pulmonary angiogram scan
(ventilation perfusion mismatch)
Prostate Carcinoma
[0138] [0139] Histopathological diagnosis and elevated PSA.
[0140] In general, a combination of the patient's history, medical
examination, general health and indicators provided from
biochemical, histochemical, radiochemical and other types of tests
and disease and/or clinical signs of disease will be used in the
diagnosis of disease. For the avoidance of doubt, the term
`clinical signs and symptoms of disease` means the same as
`clinical details` of disease.
(B) Samples of Body Fluids from Disease States
[0141] For each sCD the following information is generally obtained
a) the dynamic range of the assay b) the range of concentrations
expected in health c) the range of concentrations expected in
disease. From this information an approximate dilution factor for
each assay may be obtained, allowing maximum use of subject
samples. One skilled will appreciate thought that in some
circumstances body fluid samples may not be diluted for
testing.
[0142] Suitable body fluids for measuring sCD levels as herein
defined include whole blood, serum, urine, tissue fluid,
cerebrospinal fluid, lymphatic fluid, synovial fluid, aspirate,
bone marrow aspirate, mucus or other tissue or body fluid. One
skilled in the art will appreciate that this list is not intended
to be exhaustive. Preferably sCD levels are measured in serum which
is prepared from whole blood using methods familiar to those
skilled in the art At least 1.5 ml of sample is required for
testing of all the sCDs. Advantageously, less than 1.5 mls of
sample is required for such testing. More advantageously, much
smaller volumes of sample will be needed for such testing. In
addition, the present inventors have shown that haemolysis and
lipaemia can interfere with some immunoassays used for detecting
sCDs and therefore samples are used which exhibit minimal
haemolysis and lipaemia.
[0143] Body fluid samples may be diluted in order to measure the
sCD levels and the dilution factor for each sCD should be the same
for the generation of the fingerprints for all disease states
tested. One skilled in the art will appreciate that the dilution
factor may be adjusted in order to focus on either high or low
concentrations of sCDs. Advantageously, the dilution factor will be
adjusted to focus on high concentrations of sCDs.
(C) Methods of Measuring sCD Levels.
[0144] Suitable methods for measuring levels of sCDs in body fluids
include flow cytometry, in particular multiplexed particle flow
cytometry, immunoassay and microarray technologies utilising
antibody or ligand interactions. Advantageously, sCDs levels are
measured using multiplexed particle flow cytometry and/or chip
based monoclonal antibody technology, engineered antibody molecules
or non-antibody or non-protein molecules that recognise sCD
antigens. These methods will be familiar to those skilled in the
art.
(i) Immunoassays
[0145] Immunoassays such as immunoblotting (detecting
membrane-bound and soluble proteins), and enzyme linked
immunoassays (ELISA) provide a sensitive and specific means of
detecting target substances.
[0146] Although the various types of immunoassays are performed
differently, they have one thing in common-they all involve
antibodies. Used in an appropriate immunoassay system, specificity
leads to sensitivity. As herein defined the term `antibodies`
includes antibody fragments, engineered immunoglobulin folds or
scaffolds which have a binding affinity for soluble CD
molecules.
[0147] One skilled in the art will appreciate that the `immunoassay
technique` may be adapted to use other molecules which selectively
bind sCDs. Those skilled in the art will be aware of such
molecules.
[0148] In a direct immunoassay, the antibody used as the primary
reagent is advantageously given a fluorescent, enzymatic, or
radio-active detection means. In indirect immunoassays, the
secondary antibody-usually polyclonal antisera produced by a goat
or a rabbit against human immunoglobulins-carries the detection
means. When a secondary antibody is used, the initial immune
reaction between the primary antibody and the target antigen is
amplified, producing a more readily detectable signal.
[0149] Western blots of electrophoretically separated proteins
(immunoblots), on the other hand, are generally probed with
antibodies labeled with an enzyme or a radioisotope such as 125I.
chromogenic or chemiluminescent substrates can also be used. For
example, enzymes such as HRP and AP catalyze chromagenic reactions,
in which a colourless substrate is converted into a coloured
compound, and also chemiluminescent reactions where light is
emitted.
[0150] Chromogenic substrate kits are commercially available and
include but are not limited to for example alkaline phosphatase,
horseradish peroxidase, and TMB peroxidase (TMB is
tetramethylbenzidine, the substrate in this case). Boehringer
Mannheim also has several enzyme substrates for immunoassays
available. They include but are not limited to for example ABTS
(2,2+-azino-di-3-ethylbenzthiazoline sulfonate) and TMB
(tetramethylbenzidine), which are used with HRP; and 4-nitrophenyl
phosphate and 5-bromo4-chloro-3-indolyl phosphate (BCIP) for
immunoassays in which an alkaline phosphatase-conjugated antibody
is used.
[0151] Chemiluminescent substrates are available from companies
such as Pierce, which for example, produces the SuperSignal CL-HRP
Substrate, an enhanced chemiluminescent substrate for horseradish
peroxidase. This system detects specific proteins on immunoblots
with a sensitivity that rivals radioactivity (reportedly to
picogram levels). When the chemiluminescent substrate is applied to
membrane-bound proteins on an immunoblot, an instantaneous but
long-lasting flash of light is produced.
[0152] Commercially available immunoassay kits for measuring sCD
levels include those from Diaclone 1, Bd A. Fleming BP 1985 F-25020
Besancon Cedex-France which provides kits for the measurement of a
number of CD molecules including CD 14, CD21, CD25, CD31, CD44,
CD50, CD54, CD62E, CD62L, CD86, CD95, CD102, CD106, CD116, CD117,
CD124, CD126, CD130, CD138, CD141, CD40L. Medsystems diagnostics
GmbH, Rennweg 95b, A-1030 Vienna Austria, also provides kits which
measure sCD levels.
(ii) Flow Cytometry
[0153] Techniques for carrying out flow cytometry are familiar to
those skilled in the art and are described in Flow Cytometry: A
Practical Approach. Edited by MG Ormerod. IRL
[0154] Press, Oxford. 1994. ISBN 0-19 963461-0. Practical Flow
Cytometry. 3rd Edition. Howard M Shapiro. Alan R Liss, Inc. ISBN
0471-30376-3. Flow Cytometry. First Principles. Alice Longobardi
Givan. Wiley-Liss, New York, 1992. ISBN 0471-56095-2. Handbook of
Flow Cytometry Methods. Edited by J Paul Robinson. Wiley-Liss, New
York, 1993. ISBN 0-471-59634-5.
(iii) Multiplexed Particle Flow Cytometry Assay
[0155] Methods for simultaneously assaying different proteins in
individual samples are commercially available. Some of those
commercially available are detailed below:
[0156] The versatile laboratory multianalyte profiling (LabMAP.TM.)
system developed by Luminex Corp. of Austin, Tex., can be used for
virtually any bioassay that is based on the specific binding of one
molecule to another, for example a monoclonal antibody raised
against a sCD and a CD molecule.
[0157] LabMAP assays for a sCD molecule can be based on the
immunological detection, and/or may follow the gain or loss of
fluorescence (e.g., when a mAb raised against a sCD binds to a sCD
target). LabMAP assays employ three different fluorochromes: two to
create color-coded microspheres, and the third for quantifying the
reaction. Polystyrene microspheres are internally dyed with precise
ratios of two spectrally distinct fluorochromes. This ratio confers
a unique identifying "signature" or "spectral address" to each
microsphere set.
[0158] Bioassays are conducted on the surfaces of the microspheres.
Each capture probe (e.g., sCD-specific antibody or other
affirmative detection reagent) is immobilized onto a color-coded
set of microspheres using any of a variety of different surface
chemistries. Luminex offers microspheres bearing Lumavidin.TM. (an
avidin derivative), for immobilizing biotinylated molecules, or
carboxyl groups, for covalently coupling protein. The binding of
analyte to an immobilized probe is detected via a detection reagent
labeled with the third fluorochrome. Luminex currently offers 100
different microsphere sets, each of which can be used for the
simultaneous measurement of a different analyte. Thus, in theory,
up to 100 different species can be simultaneously measured in a
single tube or microplate well.
[0159] The Luminex microsphere product line is designed
specifically to work with the instruments available from Luminex or
their partners. The Luminex 100.TM. instrument uses microfluidics
to align the microspheres in single file and employs two lasers,
one for the detection of the fluorescent microsphere itself, and
the other for the reporter reagent. The colour signals are captured
by an optics system and translated into binding data via digital
signal processing.
[0160] Instrumentation, reagents, and custom services for LabMAP
technology users are also available from companies other than
Luminex. For example, Bio-Rad Laboratories of Hercules, Calif.,
introduced the Bio-Plex.TM. Protein Array System. This system
combines a fluorescent reader with the software, protocols, and
supplies needed for performing LabMAP-based assays in a 96-well
microplate format. The primary benefits of the Bio-Plex Protein
Array System, other than up to a 100-fold increase in data, include
significantly reduced sample requirements.
[0161] The Cytometric Bead Array (CBA) system from BD Biosciences
of San Diego is flexible in that it accommodates multiple sizes and
fluorescent intensities of particles. The system includes
everything the researcher needs to implement this technology,
including a cytometer setup kit with the requisite software,
reagents and standards. The company's CBA assay kits employ their
proprietary bead sets, which are internally dyed with varying
intensities of a proprietary fluorophore. These sets are
distinguished via one fluorescence parameter and two size
discriminators. However, the system is also capable of handling
assays based on the use of other types of spectrally distinct
microsphere sets. The CBA analysis software is an "add-in" for
Microsoft Excel.RTM., and is compatible with contemporary data
acquisition software such as CellQuest.TM.. Researchers can employ
a variety of preset configurations for generating standard dilution
series and calibration curves, and data reports can be generated at
each step in the process.
(iv) "Antibody Chip" Array Technology.
[0162] The array format has revolutionised biomedical
experimentation and diagnostics, enabling ordered high-throughput
analysis. During the past decade, classic solid phase substrates,
such as microtitre plates, membrane filters and microscopic slides,
have been turned into high-density, chip-like structures.
[0163] Protein array technology allows high throughput screening
for gene expression and molecular interactions. Protein arrays
appear as new and versatile tools in functional genomics, enabling
the translation of gene expression patterns of normal and diseased
tissues into protein product. Protein function, such as enzyme
activity, antibody specificity, and other ligand-receptor
interactions and binding of nucleic acids or small molecules can be
analysed on a whole genome level.
[0164] As the array technology develops, an ever increasing variety
of formats become available (eg nanoplates, patterned arrays,
three-dimensional pads, flat-surface spot arrays, microfluidic
chips), and proteins can be arrayed onto different surfaces (e.g,
membrane filters, polystyrene film, glass, silane, gold). Various
techniques are being developed for producing arrays. The emerging
future array systems will be used for high-throughput functional
annotation of gene products.
[0165] Protein microarrays are particularly useful in molecular
diagnostics The concept of the array library was central to this
development which now extends from DNA to protein. Similar to the
gene chip arrays measuring mRNA levels on a genome wide scale, the
protein product of expressed cells can be used for the simultaneous
assessment of protein levels on a proteome wide scale.
Additionally, protein specific antibodies can be arrayed to produce
"antibody chip arrays" (Cahill D., 2001, J. Imm. Meth. 250, 81-91).
The availability of such antibody chip arrays can be used to
simultaneously analyse numerous interactions within a single
sample. The "antibody chip" can be used to demonstrate
antibody-protein interactions by incubating the chip with target
proteins which have been labelled with a traceable marker
(ProteinChip, Ciphergen Biosystems, Fremont, Calif., USA; BIAcore
chips, Biacore, Upsala, Sweden) or by incubating the chip with
protein molecules or fragments thereof and detecting association
between antibody and protein molecule or fragment thereof using
ELISA type assays (Caliper Technologies, Mountain View, Calif.,
USA; Orchid Biocomputer Inc., Princeton, N.J., USA). It should be
noted that sCDs according to the invention may be found complexed
with a ligand and thus chip based technology described herein may
be used to measure/demonstrate interactions of ligand bound sCD
with other molecules.
[0166] Techniques for preparing antibody arrays are described
below:
[0167] The antibodies may be covalently linked to a suitable
membrane such as an Immobilon P membrane (PVDF; Millipore
Corporation) Subsequent blocking with an excess of a protein
solution such as a skim milk preparation is preferred. A blocking
agent is designed to eliminate non-specific binding on the binding
surface Other suitable blocking agents are Irish moss extract or
other source of carrageenan or gelatin. The antibodies are also
adsorbed to a nitrocellulose film on a glass microscope slide
(Schleicher and Schuell, N.H., USA) and the unbound nitrocellulose
is then blocked with skim milk. Antibodies are also adsorbed to
Nylon membranes. To increase the accessibility of bound anti-CD
antibodies to antigens on cells, the solid support used for the
array is initially coated with a recombinant, truncated form of
Protein G from Streptococcus which retains its affinity for the Fc
portion of IgG lacks albumin and Fab binding sites, and
membrane-binding regions (Goward et al., 1990). Antibodies are
applied to this coat of Protein G and bind via their domains
leaving the Fab domains free to interact with cells. The Fab
domains are also further from the solid support providing greater
accessibility of CD antigens on cell membranes to antibodies.
[0168] The array of antibodies is also constructed on a membrane or
a coverslip. In this case, the antibodies are covalently linked to
the membrane as duplicate spots in a two dimensional matrix. The
spots are arranged in a matrix such as but not limited to a
15.times.15 matrix.
[0169] The antibodies are advantageously monoclonal and are
specific for the cluster of differentiation (cluster designation)
antigens (CD antigens). Details of CD antigens are available at
http://www.ncbi.nlm.nihgov/prow/cd/index molecule.htm. The spots
are of microscopic size and are produced by the application of a
drop (-10 nanolitres) of antibody solution (e.g. 10/.tg protein/ml)
on designated portions of a membrane or glass surface such as a
coverslip, first washed with a non-specific protein absorbent such
as 30% w/v skim milk (Dutch Jug, Bonlac Foods Ltd, Melbourne,
Australia) and then rinsed. Other protein solutions and other
brands of skim milk may also be employed. The antibodies may be
covalently coupled to the solid support such as through amino
groups of lysine residues, the carboxylate groups of aspartate or
glutamic acid residues or the sulthydryl groups of cysteine
residues. The array of antibodies selectively binds cells from body
fluids which express the respective antigens or may bind free
antigens. A positive and/or negative control is included such as an
antibody for surface molecules or soluble molecules known to be
present in the sample. An example of one form of the assay device
is shown in FIG. 3. The solid support is conveniently of similar
size and shape to a microscope slide and may be constructed of
glass or other polymeric material.
[0170] A wall around the microscope slide may be separately added
or moulded with the slide and this facilitates retention of fluid
material. The present invention extends to any other device capable
of fulfilling the method of the present invention.
[0171] In the case of nitrocellulose based antibody arrays are
preferably constructed using a Biodot Aspirate and Dispense System
(Cartesian Technologies) where 5 nL dots are applied to a
nitrocellulose film. on glass microscope slides (Schleicher and
Schuell, Cat. No. 10484182). Purified monoclonal antibodies
(Beckman Coulter, Becton Dickinson or Biosource International) are
used at concentrations recommended for flow cytometric analysis and
are applied in the same buffers as supplied by the manufacturers.
The nitrocellulose is then blocked by incubation with 5% w/v skim
milk (Dutch Jug) for 1.5 h at 37.degree. C. These blocking
conditions are chosen to minimize background binding.
[0172] The stability of the arrays is further enhanced by adding
protein stabilizing agents to the antibodies (e.g. polyethylene
glycol or stabilizer products commercially available from
Surmodics, Minn., USA).
[0173] The following description provides a preferred method for
preparing the antibody arrays: The panel of antibodies is generally
used to construct antibody arrays with the Cartesian Technologies
PixSys.TM. 3200 Aspirate and Dispense System. The antibodies are
chosen for use in a particular diagnosis or detection protocol.
Each antibody is generally applied in the volume of from about 1 to
about 10 nanoliters in a dot format at approximately from 0.5 to
1.5 min intervals to create an appropriate array on a
nitrocellulose film generally laid on a solid support such as but
not limited to a microscope slide, plastic or gold support. After
dotting, the supports are assessed on a light box and the corners
of the arrays marked gently using, for example, a lead pencil. The
antibody arrays are then immersed in a blocking agent such as but
not limited to skim milk, Irish moss extract or other source of
carrageenan or gelatin. From about 2% to about 15% w/v skim milk
powder in PBS at 4.degree. C. overnight or at 37.degree. C. for
from about 60-120 minutes is particularly useful. After application
of the blocking agent, the solid supports are washed gently with
purified water and allowed to dry at room temperature for a period
of time from about 60-120 minutes.
[0174] The solid supports are then stored in an airtight bag at
4.degree. C. in the dark
[0175] An alternative method of detection is the use of acoustic
detection based methods, such as those described by Akubio
Technology. (DDT vol 7, No 5 (Suppl.) 2002). This form of detection
technology is based on the sound made as molecular interactions are
disrupted. It does not use any form of electromagnetic radiation.
Very high accelerations, millions of times the force of gravity are
used to disrupt such interactions. Such forces are generated by an
acoustic wave device such as a quartz crystal resonator. By
monitoring the change in resonant frequency of the crystal, which
occurs on adsorption of mass to the surface, quartz crystal
resonators can be used together with appropriate surface chemistry
and fluidics to detect the adsorption of proteins,
oligonucleotides, cells and other particles to surface-bound
receptors. This allows the label-free determination of interaction
affinities and kinetics in real time. (Janshoff A et al (2000)
Piezoelectric mass-sensing devices as biosensors. An alternative to
optical biosensors. Angew. Chem., 39, 4004-4032); Ward et al,
(1990) In situ interfacial mass detection with piezoelectric
transducers, Science 249, 1000-1007).
(D) A sCD Fingerprint for One or More Disease States According to
the Present Invention
[0176] Advantageously, levels of sCDs are measured in diseased
individuals and absolute values may be divided by the upper limit
of normal (ULN) obtained from healthy individuals. The data is
collated and the resultant pattern of values obtained for each sCD
for one or more given disease states, from one or more individuals
forms the basis of a sCD fingerprint of one or more given disease
states.
[0177] The statistical significance of the increases or decreases
in sCD levels found in various disease states can be assessed using
a number of methods.
[0178] The sCD fingerprint can advantageously be simplified by
removing those sCDs whose levels do not generally change
significantly during one or more given disease states. Examples of
such sCDs include but are not limited to CD21, CD102, CD117, CD
126, CD130, CD 26, CD44v5, CDv6, CD62P. For example see FIG. 2.
[0179] To simplify the fingerprint further, instead of showing the
data from each individual for any given sCD during a disease, a
modal value for each sCD calculated from the group of individuals
may be plotted. The rational of this is demonstrated in FIGS. 3, 5,
6, 7, 8. This provides an easily readable and simple `fingerprint`
of a disease.
[0180] One skilled in the art will appreciate that there are many
methods suitable for the statistical analysis of the sCD level data
measured as herein described. These include but are not limited to
cluster analysis and other statistical methods for the detection of
patterns.
Statistical Analysis
[0181] Methods for the statistical analysis of data are known to
those skilled in the art. An example of a suitable protocol for the
analysis of data is outlined below:
(i) The statistical analysis procedures described here relate to
immunoprecipitation assays of soluble CD molecules in samples
arrayed in 96-well microtitre trays.
[0182] Statistical analysis of the proposed antibody-chip arrays
could essentially follow the same procedures, although
modifications would inevitably need to be made to account for
experimental design and data quality issues specific to that
technology.
[0183] In the present analysis, each tray was designed to measure
the concentration of a specific soluble CD molecule in a number of
samples. Trays were prepared for each of 29 soluble CD molecules,
for each of three sets of 37 patients. In all, the three sets of
patients represented 17 disparate disease groups, including one
group of healthy individuals. The 96 wells on each tray contained:
sera from each of the 37 patients of one set (duplicated); a
standard control preparation containing the target antigen at each
of 7 different concentrations (duplicated); pooled sera from
`normal` control patients (replicated 3 times); and the same pooled
sera spiked with the target antigen (replicated 3 times).
[0184] Each tray was washed with a solution containing
fluorescently labelled monoclonal antibody specific to its
designated target antigen, and exposed to light. For each well, a
measurement of light absorption was recorded.
(ii) Statistical Analysis Using Artificial Intelligence Type Neural
Networking
[0185] This method describes the ability of the computer system
which analyses the data to learn to recognise patterns in data.
Thus the more the system is used, the better able the system is to
recognise patterns. Such a system is described in amongst other
documents Sven Olsson et al (2002), Clin Physiol & Func In
(2002) 22, pp 295-299; Sijo Perekattil et al (March 2003), Journal
of Virology, vol 169, pg 917-920. This approach is reviewed in PJG
Lisboa, (2002), Neural Networks 15, 11-39. All of these documents
are herein incorporated by reference.
Data Preparation
[0186] The recorded light absorptions were mathematically
transformed, as follows. [0187] Absorptions were converted to
antigen concentrations, via a calibration curve based on the
absorptions recorded for the antigen standards in the same tray.
Absorptions of dubious quality were flagged missing, and omitted
from subsequent analysis. [0188] Concentrations exceeding that of
the most concentrated antigen standard of the tray were flagged as
high. Concentrations corresponding to absorptions at the limit of
instrumentation were similarly flagged. [0189] Each flagged
concentration was replaced by the concentration of its duplicate in
the tray, provided the duplicate concentration was not also
flagged. [0190] The average of each concentration with its
duplicate was computed. We refer to these as mean concentrations.
[0191] For each CD, the 10th and 90th centiles of the distribution
of mean concentrations in normal individuals was computed. We refer
to these as CD.sub.10 and CD.sub.90. Subtracting CD.sub.10 from
CD.sub.90 gives a range, which we denote CD.sub.range.
Concentration categories were defined for each CD as follows:
TABLE-US-00001 [0191] Category from to -1 0 CD.sub.10 0 CD.sub.10
CD.sub.90 1 CD.sub.90 CD.sub.90 + CD.sub.range 2 CD.sub.90 +
CD.sub.range CD.sub.90 + 2 .times. CD.sub.range 3 CD.sub.90 + 2
.times. CD.sub.range CD.sub.90 + 3 .times. CD.sub.range 4 CD.sub.90
+ 3 .times. CD.sub.range --
[0192] To control for differences in the scale of reactivity of
different CDs, to stabilise variability between duplicates across
the range of concentrations, and to relate concentrations to values
obtained for normal individuals, each mean concentration was put
into the appropriate concentration category of its CD. All
concentrations flagged high were put into the highest category. We
refer to these as categorised concentrations.
Data Analyses
Cluster Analysis
[0193] A cluster analysis was performed to identify clusters of CDs
having similar patterns of reactivity across the patients,
standards and controls. Likewise, a second cluster analysis was
performed to identify clusters of patients with similar profiles of
reactivity across the CDs. Cluster analyses were hierarchical and
based on the categorised concentrations, described above, using a
Euclidean distance metric and the average linkage criterion for
cluster merging.
[0194] The results from the cluster analyses were displayed in the
form of a level plot, a part of which is given in FIG. 16. In this
level plot, the rows correspond to CDs and the columns correspond
to patients, standards and controls. Rows and columns are ordered
so that those in the same cluster are adjacent. Also, dendrograms
were produced showing the hierarchy of clusters, separately for the
clustering of CDs and for the clustering of patients, standards and
controls. Each cell in the level plot is toned to indicate its
categorised concentration, deep blue corresponding to Category -1
(below normal), and deep red corresponding to Category 4 (high).
Missing concentrations are denoted by an `X`.
A sCD Fingerprint Database According to the Invention
[0195] In a further aspect still, the present invention provides a
sCD reference database comprising pathological and/or normal sCD
fingerprint patterns.
[0196] As herein described the term `a database` refers to a
collection of sCD fingerprints from normal `non-diseased` and/or
diseased individuals. Advantageously, the database is computer
generated and/or stored. Advantageously the data from more than 5
individuals is present in the database. More advantageously the
data from more than 10, 100, or 1000 individuals comprises the
database. More advantageously still the data from more than 10,000
or more than 50,000 individuals comprises the database. Most
advantageously the data from more than 100,000 individuals
comprises the database.
[0197] Advantageously the database, in addition to sCD data also
comprises clinical information relating to various patients and/or
disease conditions. Alternatively or in addition, a database
according to the present invention comprises genomic information
and/or sCD profiles relating to specific disease states and other
pathological states and clinical data. Thus the inventors
contemplate the use of a database in which sCD data and other data
may be integrated and used to obtain a more complete analysis of
one or more disease states.
Uses of One or More `Fingerprints of Disease` According to the
Present Invention
[0198] In a further aspect, the present invention provides a method
for predicting the presence of one or more disease states in an
individual comprising the step of analysing the sCD fingerprint in
that individual.
[0199] In a further aspect still, the present invention provides a
method for detecting the presence of one or more disease states in
an individual comprising the step of analysing the pattern/s of
shed CD levels of more than one shed CD which is present in that
individual.
[0200] In a further aspect still, the present invention provides a
method for detecting the extent of one or more disease states in an
individual comprising the step of comparing one or more sCD
fingerprint/s generated from that individual with one or more
reference sCD fingerprint/s.
[0201] In yet a further aspect, the present invention provides a
method for assessing the progression of a disease state in an
individual comprising the step of comparing the sCD fingerprint of
an individual at two or more periods during the occurrence of the
disease.
[0202] In a further aspect still, the present invention provides a
method for assessing the effect of one or more agents on one or
more disease states in an individual comprising the step of
comparing a sCD fingerprint of an individual at two or more time
periods.
[0203] In a further aspect, the data generated by the present
invention is used to compile a reference database, comprising
pathological and/or normal sCD fingerprints, against which the
expression sCD pattern of any individual will be compared.
[0204] In a further aspect still, the present invention provides
the use of a sCD fingerprint to assess the effect of one or more
agents in an individual.
[0205] An embodiment of the present invention is its use as a tool
for assessing the affect different diet and exercise regimes may
have on human or other mammals.
[0206] Additionally, the present invention may be used to construct
sub-categories of sCD fingerprint profiles, suitable for common
therapeutic treatment.
The Therapeutic Inhibition of the Production of One or More sCDs
According to the Invention
[0207] Recent studies which have investigated the impaired
expression of NKG2D and T-cell activation by tumour-derived soluble
MHC ligands (Nature, vol 419, 17 Oct. 2002). Studies have shown
that tumours release large amounts of the MHC class I homologue MHC
into the serum. Activation of the NKG2D receptor on natural T cells
is known to stimulate their ability to destroy tumours, but the
high levels of tumour derived MIC seem to downregulate the NKG2D
receptor and block the antitumour effect. (Nature, vol 419, 17 Oct.
2002, p 679, pg 734). These soluble forms of MHC are produced
either enzymatic cleavage or by alternative splicing (Nature, vol
419, 17 Oct. 2002, p 679, pg 734).
[0208] It is apparent from the invention described herein that sCDs
may be produced by alternative splicing (Woolfson and Milstein,
PNAS Vol 91, pp 6683-6687), enzymatic cleavage or other mechanisms.
The present inventors consider that sCD molecules may also bind to
a ligand/receptor and thereby block down stream effects. Thus, the
present inventors have realised that the blockage of the production
of sCD molecules via the inhibition of any of the methods of sCD
generation described herein, may be a therapeutically useful method
for the prophylaxis or treatment of one or more diseases in
particular tumourigenesis.
[0209] Thus in a further aspect still, the present invention
provides a method for treating one or more diseases comprising the
step of inhibiting the production of one or more sCDs within an
individual.
[0210] In a further aspect still, the present invention provides
the use of an inhibitor of the production of one or more sCDs in
the preparation of a medicament for the treatment of disease.
[0211] According to the above aspect of the invention, the term `an
inhibitor of the production of one or more sCDs` refers to one or
more agents which inhibit the production of a shed form of sCD as
herein defined. In reducing the amount of shed molecule produced,
the level of cell surface molecule should increase correspondingly.
This should be advantageous to the cell. Advantageously, the
inhibitor is a specific inhibitor of those one or more sCDs.
Suitable inhibitors include alternative splicing inhibitors and/or
enzymatic cleavage inhibitors. Advantageously, the inhibitor is an
alternative splicing inhibitor. Such alternative splicing
inhibitors are include for example inhibitors of exonic splicing
enhancers (Fairbrother et al, Science, vol 297, 9Aug. 2002).
[0212] According to the above aspects of the invention, the
production of any one or more sCDs present in the body fluid of an
individual may be inhibited. Advantageously, the one or more sCDs
are any one of those selected from the group consisting of the
following: CD14, CD25, CD31, CD44, CD50, CD54, CD62E, CD62L, CD86,
CD95, CD106, CD116, CD124, CD138, CD141, CD40L, CDS, CD23, CD30,
CD40. More advantageously the sCD is CD1. Advantageously, the sCD
is CD1 and the inhibitory agent is an alternative splicing
inhibitor.
[0213] According to the above aspects of the invention, one or more
inhibitory agents may be used for the prophylaxis or treatment of
any one or more disease states selected from the group consisting
of the following: infections, autoimmune disease, neoplastic,
vascular endocrinological, metabolic, inflammatory degenerative,
psychiatric psychological, traumatic, drug/toxin-related,
bacterial, fungal, protozoan and viral infections, non-neoplastic
disorders; pain; diabetes, obesity; anorexia; bulimia; asthma;
pregnancy; endocrine; vascular; metabolic; gastrointestinal;
iatrogenic; psychiatric; psychoclogical; exercise-induced;
diet-related; ME; degenerative; Parkinson's disease; thrombosis;
atherosclerosis; acute heart failure; hypotension; hypertension;
erectile dysfunction; urinary retention; metabolic bone diseases
such as osteoporosis; angina pectoris; hepatitis; myocardial
infarction; ulcers; allergies; rheumatoid arthritis; inflammatory
bowel disease; irritable bowel syndrome benign prostatic
hypertrophy; psychosis; psychiatric disorders; including anxiety;
schizophrenia; manic depression; delirium; dementia; severe mental
retardation and dyskinesias, such as Huntington's disease or Gilles
de la Tourett's syndrome; and preferably tumours which can be
benign or malignant cancers; breast cancer; myeloma; melanoma;
bladder cancer; leukaemia; plasmocytoma and others, but most
preferably appendicitis; Bence Jones Proteinuria; Chronic Myoeloid
Leukaemia; Colorectal cancer; chronic renal failure; Crohn's
Disease; Diabetic Nephropathy; Cardiac pathology; Infection; Liver
damage; Lymphoma; macrocytic anaemia; Prostate Cancer; Oligoclonal
Banding and PE/DVT. Advantageously, the disease is
tumourigenesis.
[0214] The invention will now be described by the following
examples which are in no way limiting of the invention.
EXAMPLE 1
FIG. 1. Disease Groups. Multiples of ULN All sCD's included
[0215] Two values obtained (CD40L and CD30) for the individual,
classified as normal, with a suspected drug overdose were omitted
from the calculation of the upper limit of normal. The dilution
factor for each sCD was fixed throughout the study. The results
obtained are those of the diluted sample and have not been
multiplied by the dilution factor. The absolute value of each data
point was divided by the upper limit of normal (ULN) as defined
above. Where the absolute value was greater than the dynamic range
of the assay the result [9999] was recorded.
[0216] The limits indicated by each point are: [0217] Green
.ltoreq.1.times.ULN [0218] Blue 1-2.times.ULN [0219] Red
>2.times.ULN [0220] A white block indicates no data
available.
EXAMPLE 2
FIG. 2 Remove all sCD's that Appear not to Discriminate from the
Normals (sCD's 21; 102; 117; 126; 130; 26; 44v5; 44v6; 62P)
[0221] To simplify the diagram the above sCD plots were
removed.
[0222] The data suggests (FIG. 1.) that the concentration of some
of these sCD may actually be lowered in disease. As we initially
worked on the premise that there would be over-expression of these
molecules in disease, samples have been diluted optimally to focus
on high, rather than low concentrations.
EXAMPLE 3
FIG. 3. Disease Groups. Mode of Response for Remaining 20 sCD's
[0223] To simplify the data further the modal response for each
disease group has been plotted. As the lymphoma and
"oligoclonal-banding positive" group contain only a single subject,
they have been omitted. Where there is no clear mode, both
responses have been shown.
EXAMPLE 4
FIG. 4. Disease Groups. Mode of Response for Remaining sCD's
[0224] Data has been ranked in order of increased expression.
EXAMPLE 5
FIG. 5. Disease Groups. Mode of Response for Remaining sCD's
[0225] As for FIG. 4. except responses have been classified as
"normal" and "abnormal". Values >1 ULN have been classified as
abnormal.
[0226] Both FIGS. 4 and 5 suggest that each disease state exhibit a
unique pattern of elevated sCD expression.
EXAMPLE 6
FIG. 6. Disease Groups. Mode of Response for all sCD's
[0227] As for FIG. 3 (except all sCD's are included).
[0228] The limits indicated by each point are: [0229] Green
.ltoreq.1.times.MoM [0230] Blue 1-2.times.MoM [0231] Red
>2.times.MoM [0232] A white block indicates no data
available.
EXAMPLE 7
FIG. 7. Disease Groups. Mode of Response for all sCD's
[0233] As for FIG. 4.
EXAMPLE 8
FIG. 8. Disease Groups. Mode of Response for all sCD's
[0234] As for FIG. 5. (except that values >2 MoM have been
classified as abnormal).
[0235] Comparison of FIGS. 5 and 8 illustrate the importance of
determining the cut-off threshold values in order to obtain a
defined pattern.
[0236] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the present invention.
Although the present invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in biochemistry, molecular biology and biotechnology
or related fields are intended to be within the scope of the
following claims.
* * * * *
References