U.S. patent application number 14/083874 was filed with the patent office on 2014-03-20 for tumour marker proteins and uses thereof.
This patent application is currently assigned to Onclmmune Limited. The applicant listed for this patent is Onclmmune Limited. Invention is credited to Catherine Rosamund Louise Graves, John Forsyth Russell Robertson.
Application Number | 20140080736 14/083874 |
Document ID | / |
Family ID | 9947849 |
Filed Date | 2014-03-20 |
United States Patent
Application |
20140080736 |
Kind Code |
A1 |
Robertson; John Forsyth Russell ;
et al. |
March 20, 2014 |
TUMOUR MARKER PROTEINS AND USES THEREOF
Abstract
Tumour marker proteins and their preparation from fluids from
one or more cancer patients, wherein said fluids are those which
collect in a body cavity or space which is naturally occurring or
which is the result of cancer or medical intervention for cancer.
The present application also relates to preparation of tumour
marker proteins from excretions taken from patients with cancer.
The tumour marker proteins are useful as immunoassay reagents in
the detection of cancer-associated anti-tumour marker
autoantibodies.
Inventors: |
Robertson; John Forsyth
Russell; (Nottingham, GB) ; Graves; Catherine
Rosamund Louise; (Nottingham, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Onclmmune Limited |
Derby |
|
GB |
|
|
Assignee: |
Onclmmune Limited
Derby
GB
|
Family ID: |
9947849 |
Appl. No.: |
14/083874 |
Filed: |
November 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10534773 |
May 13, 2005 |
8592169 |
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PCT/GB2003/004950 |
Nov 13, 2003 |
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14083874 |
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Current U.S.
Class: |
506/9 ; 435/7.92;
530/350; 530/352; 530/395; 530/412 |
Current CPC
Class: |
G01N 33/57488 20130101;
C07K 1/36 20130101; G01N 2800/24 20130101; G01N 33/6854 20130101;
G01N 33/564 20130101; A61P 35/00 20180101 |
Class at
Publication: |
506/9 ; 530/352;
530/412; 530/350; 530/395; 435/7.92 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 1/36 20060101 C07K001/36 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 14, 2002 |
GB |
0226622.9 |
Claims
1. A method of detecting cancer-associated anti-tumor
autoantibodies in a sample from an individual, comprising:
contacting the sample with an immunoassay reagent; and detecting a
presence of complexes formed by specific binding of the immunoassay
reagent to any cancer-associated anti-tumor autoantibodies present
in the sample, wherein the immunoassay reagent comprises two or
more tumor marker proteins, one or more of which having been
prepared from a tumor-induced bodily fluid produced in a body
cavity or space in the presence of the tumor of one or more cancer
patients, wherein the bodily fluid contains more cancer-associated
forms of the tumor marker protein than a non-tumor-induced bodily
fluid in the same patient, and the bodily fluid is not a fluid from
the systemic circulation, wherein the one or more tumor marker
proteins prepared from a bodily fluid exhibit selective reactivity
with cancer-associated anti-tumor autoantibodies, wherein the tumor
marker proteins are over-expressed or altered forms of wild-type
proteins, and wherein detection of complexes indicates the presence
of cancer-associated anti-tumor autoantibodies in the
individual.
2. The method of claim 1, further comprising detecting and/or
quantitatively measuring the presence of two or more types of
autoantibodies, wherein each one of the two or more types of the
autoantibodies is immunologically specific to a different tumor
marker protein or to different epitopes of the same tumor marker
protein, wherein the immunoassay is carried out using a panel of
the two or more tumor marker proteins.
3. The method of claim 2, wherein the immunoassay is carried out
using a panel of two or more immunoassay reagents, wherein each one
of the two or more immunoassay reagents is for detection of a
different tumor marker protein, wherein the sample is obtained from
a patient, the method further comprising: determining relative
strength of immune response of the patient to each one of the two
or more different tumor marker proteins; and selecting one or more
tumor marker proteins to form a basis of an anti-cancer vaccine for
use in the patient, wherein the one or more tumor marker proteins
to which the patient has strongest immune response is selected out
of the two or more different tumor marker proteins.
4. The method of claim 1, wherein the bodily fluid is ascites
fluid, pleural effusion, seroma, hydrocoele or wound drainage
fluid.
5. The method of claim 1, wherein the excretion is urine, faeces or
seminal fluid.
6. The method of claim 1, wherein the one or more tumor marker
proteins prepared from the bodily fluid is MUC1, MUC16, c-myc,
c-erbB2, p53, ras, BRCA1, BRCA2, APC, PSA, CEA, or CA19.9.
7. A method of determining whether a vaccination procedure,
comprising challenging a patient with an immunogenic preparation
comprising a tumor marker protein or an antigenic fragment thereof
or with a nucleic acid sequence expressing the tumor marker
protein, has been successful in eliciting cancer-associated
antibodies to the tumor marker protein in the patient, wherein the
method is an immunoassay, comprising: contacting a sample of bodily
fluid from the patient with an immunoassay reagent; and detecting
presence of complexes formed by specific binding of the immunoassay
reagent to any cancer-associated antibodies present in the sample,
wherein the immunoassay reagent comprises a sample of the tumor
marker protein, wherein the tumor marker protein is prepared from a
bodily fluid derived from one or more of a body cavity or in which
a tumor is or was present or with which a tumour is or was
associated, of one or more cancer patients, or from an excretion
from one or more cancer patients, wherein the tumor marker protein
exhibits selective reactivity with cancer-associated anti-tumor
antibodies.
8. A method of preparing a tumour marker protein wherein the method
comprises isolating the tumour marker protein from bodily fluid
wherein the fluid is: (i) collected from a body cavity or space in
which a tumour is or was present or with which a tumour is or was
associated, and (ii) the fluid represents the pooled fluid samples
from two or more cancer patients.
9. The method of claim 8 wherein the fluid is ascites, pleural
effusion, seroma, hydrocoele or wound drainage fluid or a mixture
thereof.
10. The method of claim 8 wherein the tumour marker protein is
MUC1, c-erbB2, p53, ras, BRCA1, BRCA2, APC, PSA, CEA, CA19.9, MUC16
or c-myc.
11. A method of preparing a tumour marker protein wherein the
method comprises isolating the tumour marker protein from a bodily
fluid collected from a body cavity or space in which a tumour is or
was present or with which a tumour is or was associated, and
wherein the bodily fluid is wound drainage fluid, seroma,
hydrocoele or a mixture thereof.
12. A method of preparing a tumour marker protein wherein the
method comprises isolating the tumour marker protein from an
excretion wherein: (i) the excretion or any component thereof has
been in contact with a tumour or tumour cells, and (ii) the
excretion represents pooled excretion samples from two or more
cancer patients.
13. The method of claim 12 wherein the excretion is urine, faeces
or seminal fluid.
14. The method of claim 12 wherein the relevant component of the
excretion is bile.
15. The method of claim 12, wherein the tumour marker protein is
MUC1, c-erbB2, p53, ras, BRCA1, BRCA2, APC, PSA, CEA, CA19.9, MUC16
or c-myc.
16. The method of claim 12 wherein the tumour marker is purified
from the fluid or excretion by affinity chromatography.
17. The method of claim 12, further comprising removing
contaminating immunoglobulin from the tumour marker protein.
18. The method of claim 12, further comprising immobilizing the
isolated tumour marker protein on a solid support.
19. A tumour marker protein prepared by the method of claim 12,
wherein the tumour marker protein is substantially immunoglobulin
free.
20. A kit or reagent suitable for carrying out an immunoassay,
wherein the kit or reagent comprises the tumour marker protein of
claim 19 immobilized on a solid support.
21. The kit or reagent of claim 20, wherein the solid support is
the surface of a well of a multiwell plate or a bead.
22. The kit or reagent of claim 20, wherein the immobilized tumour
marker protein is absorbed, adsorbed or covalently attached to the
solid support.
23. A method of calibrating an assay for measurement or detection
of a tumour marker protein in a clinical sample, wherein the method
comprises the steps of: a) preparing at least two samples of the
tumour marker protein of claim 19, each of which comprises the
given tumour marker protein and each of which has a different
tumour marker protein concentration to each of the other the
samples: b) carrying out a quantitative measurement of the
concentration of the tumour marker protein in each of the samples
using (i) a spectrophotometric method and/or, (ii) an antibody
reagent to the tumour marker protein, and c) constructing a
standard curve for a tumour marker protein concentration based on
the measurements obtained in step (b).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 10/534,773, filed May 13, 2005, which is the
U.S. national phase of International Application No.
PCT/GB2003/004950 filed on Nov. 13, 2003 and published in English
on May 27, 2004 as International Publication No. WO 2004/044950,
which application claims priority to GB patent Application No.
0226622.9 filed on Nov. 14, 2002, the contents of which are
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to tumour marker proteins and their
preparation from fluids from one or more cancer patients, wherein
said fluids are those which collect in a body cavity or space which
is naturally occurring or which is the result of cancer or medical
intervention for cancer. Exemplary fluids are ascites, pleural
effusion, seroma, hydrocoele and wound drainage fluid. The
invention also relates to preparation of tumour marker proteins
from excretions taken from patients with cancer.
[0003] The said tumour marker proteins are useful in cancer
detection methods which involve detecting or quantitatively
measuring autoantibodies to circulating tumour markers or markers
expressed on or in tumour cells and in various research
applications. The invention is also directed to such uses.
BACKGROUND TO THE INVENTION
[0004] The development and progression of cancer in a patient is
generally found to be associated with the presence of markers in
the bodily fluid of the patient, these "tumour markers" reflecting
different aspects of the biology of the cancer (see Fateh-Maghadam,
A. & Steilber, P. (1993) Sensible use of tumour markers.
Published by Verlag GMBH, ISBN 3-926725-07-9). Tumour markers are
often found to be altered forms of wild-type proteins expressed by
"normal" cells, in which case the alteration may be a change in
primary amino acid sequence, a change in secondary, tertiary or
quaternary structure or a change in post-translational
modification, for example, abnormal glycosylation. In addition,
wild-type proteins which are up-regulated or over-expressed in
tumour cells, possibly as a result of gene amplification or
abnormal transcriptional regulation, may also be tumour
markers.
[0005] Established assays for tumour markers present in bodily
fluids tend to focus on the detection of tumour markers which
reflect tumour bulk and as such are of value late in the disease
process, for example in the diagnosis of metastatic disease. The
most widely used of these markers include carcinoembryonic antigen
(CEA) and the glycoprotein termed CA 15.3, both of which have been
useful mainly as indicators of systemic disease burden and of
relapse following therapy (Molina, R., Zanon, G., Filella, X. et
al. Use of serial carcinoembryonic antigen and CA 15.3 assays in
detecting relapses in breast cancer patients. (1995) Breast Cancer
Res Treat 36: 41-48). These markers are of limited use earlier in
the course of the disease, for example in early detection or in the
screening of asymptomatic patients. Thus, in the search for tumour
markers present in bodily fluid that are of use in assisting
diagnosis earlier in the disease process the present inventors have
sought to identify markers which do not depend on tumour bulk per
se.
[0006] Differences between a wild type protein expressed by
"normal" cells and a corresponding tumour marker protein may, in
some instances, lead to the tumour marker protein being recognized
by an individual's immune system as "non-self" and thus eliciting
an immune response in that individual. This may be a humoral (i.e B
cell-mediated) immune response leading to the production of
autoantibodies immunologically specific to the tumour marker
protein. Autoantibodies are naturally occurring antibodies directed
to an antigen which an individual's immune system recognizes as
foreign even though that antigen actually originated in the
individual. They may be present in the circulation as circulating
free autoantibodies or in the form of circulating immune complexes
consisting of autoantibodies bound to their target tumour marker
protein.
[0007] As an alternative to the direct measurement or detection of
tumour marker protein in bodily fluids, assays may be developed to
measure the immune response of the individual to the presence of
tumour marker protein in terms of autoantibody production. Such
assays essentially constitute indirect detection of the presence of
tumour marker protein. Because of the nature of the immune
response, it is likely that autoantibodies can be elicited by a
very small amount of circulating tumour marker protein and indirect
methods which rely on detecting the immune response to tumour
markers will consequently be more sensitive than methods for the
direct measurement of tumour markers in bodily fluids. Assay
methods based on the detection of autoantibodies may therefore be
of particular value early in the disease process and possibly also
in relation to screening of asymptomatic patients, for example in
screening to identify individuals "at risk" of developing disease
amongst a population of asymptomatic individuals. Furthermore, they
may be useful for earlier detection of recurrent disease.
[0008] Tumour marker proteins observed to elicit serum
autoantibodies include a particular class of mutant p53 protein,
described in U.S. Pat. No. 5,652,115, which can be defined by its
ability to bind to the 70 kd heat shock protein (hsp70). p53
autoantibodies can be detected in patients with a number of
different benign and malignant conditions (described in U.S. Pat.
No. 5,652,115) but are in each case present in only a subset of
patients. For example, one study utilizing an ELISA assay for
detection of autoantibodies directed against the p53 protein in the
serum of breast cancer patients reported that p53 autoantibodies
were produced by 26% of patients and 1.3% of control subjects
(Mudenda, B., Green, J. A., Green, B. et al. The relationship
between serum p53 autoantibodies and characteristics of human
breast cancer, (1994) Br J Cancer 69: 4445-4449). A second tumour
marker protein known to elicit serum autoantibodies is the
epithelial mucin MUC1 (Hinoda, Y. et al. (1993) Immunol Lett. 35:
163-168; Kotera, Y. et al. (1994) Cancer Res. 54: 2856-2860).
[0009] WO 99/58978 describes methods for use in the
detection/diagnosis of cancer which are based on evaluating the
immune response of an individual to two or more distinct tumour
markers. These methods generally involve contacting a sample of
bodily fluid taken from the individual with a panel of two or more
distinct tumour marker antigens, each derived from a separate
tumour marker protein, and detecting the formation of complexes of
the tumour marker antigens bound to circulating autoantibodies
immunologically specific for the tumour marker proteins. The
presence of such circulating autoantibodies is taken as an
indication of the presence of cancer.
[0010] Cancer detection methods based on detection of circulating
autoantibodies are frequently immunoassays utilizing an
"immunoassay reagent" reactive with the circulating autoantibodies.
Typically, the "reagents" used in such assays comprise recombinant
tumour marker proteins (expressed in bacterial, insect, yeast or
mammalian cells) or chemically synthesized tumour marker antigens,
which may comprise substantially whole tumour marker proteins, or
fragments thereof, such as short peptide antigens. Other potential
sources of tumour-associated proteins for use as the basis of
immunoassay reagents for the detection of anti-tumour
auto-antibodies include cultured tumour cells (and the spent media
used for their growth), tumour tissue, and serum from individuals
with neoplasia. The majority of these sources have significant
drawbacks, as discussed below.
[0011] With cultured tumour cells (and their spent media) the
amount of expressed protein can vary depending on growth phase at
the time of harvest, leading to variations in quality and quantity.
In addition, the desired protein is generally present at low
concentration; therefore it is time-consuming to purify sufficient
quantities of protein. Furthermore, the cell stock will be clonal,
unlike cell stock in a tumour which is likely to have become
heterogeneous in nature during the growth of the neoplasm,
therefore producing variations in protein (especially in the degree
of glycosylation).
[0012] Recombinant proteins expressed in bacterial cells are not
glycosylated, and thus significantly different from naturally
glycosylated proteins. In addition, refolding of recombinantly
expressed proteins may not be appropriate, thus giving an incorrect
conformation for auto-antibody recognition.
[0013] Tumour tissue is usually only available in small quantities
and the purification of proteins therefrom is laborious and time
consuming.
[0014] Serum samples are usually available only in small
quantities; therefore it is difficult to purify sufficient
quantities of protein.
[0015] The present inventors have now determined that significant
advantages can be gained by the use of tumour marker antigens
purified from bodily fluids derived from a body cavity or space in
which a tumour is present or with which it is or was associated,
such as ascites fluid, pleural effusion, seroma, hydrocoele or
wound drainage fluid, or from excretions, as the "reagent" in
auto-antibody immunoassays. In particular, the inventors have
observed that use of reagents comprising tumour marker antigens
purified from bodily fluids derived from the above defined body
cavities or spaces results in increased sensitivity (as compared to
the use of reagents derived from a "normal" body fluid) and
produces a more "clinically relevant" result. There are also
significant practical advantages to be gained from the use of such
fluids as a source of assay reagent.
SUMMARY OF THE INVENTION
[0016] In a first aspect the invention relates to a method of
detecting cancer-associated anti-tumour autoantibodies, which
method is an immunoassay comprising contacting a sample to be
tested for the presence of such autoantibodies with an immunoassay
reagent and detecting the presence of complexes formed by specific
binding of the immunoassay reagent to any cancer-associated
anti-tumour autoantibodies present in the sample, wherein the
immunoassay reagent comprises tumour marker protein prepared from
bodily fluid derived from a body cavity or space within which a
tumour is or was present or with which a tumour is or was
associated, from one or more cancer patients and/or tumour marker
protein prepared from an excretion from one or more cancer
patients, wherein said tumour marker protein exhibits selective
reactivity with cancer-associated anti-tumour autoantibodies.
[0017] In a second aspect the invention relates to use of tumour
marker protein prepared from bodily fluid derived from a body
cavity or space within which a tumour is or was present or with
which a tumour is or was associated, of one or more cancer patients
and/or tumour marker protein derived from an excretion of one or
more cancer patients in the manufacture of an immunoassay reagent
exhibiting selective reactivity with cancer-associated anti-tumour
autoantibodies.
[0018] In a third aspect, the invention relates to a method of
preparing a tumour marker protein which method comprises isolating
said tumour marker protein from bodily fluid wherein said fluid is:
[0019] (i) collected from a body cavity or space in which a tumour
is or was present or with which a tumour is or was associated, and
[0020] (ii) said fluid represents the pooled fluid samples from two
or more cancer patients.
[0021] In a fourth aspect, the invention relates to a method of
preparing a tumour marker protein which method comprises isolating
said tumour marker protein from an excretion wherein: [0022] (i)
said excretion or any component thereof has been in contact with a
tumour or tumour cells, and [0023] (ii) said excretion represents
pooled excretion samples from two or more cancer patients.
[0024] In a fifth aspect the invention relates to tumour marker
protein preparations prepared using the methods described above
which are substantially immunoglobulin free and to kits and
reagents comprising said preparations.
DETAILED DESCRIPTION OF THE INVENTION
[0025] In the first aspect, the invention relates to a method of
detecting "cancer-associated" anti-tumour autoantibodies.
[0026] The term "cancer-associated" anti-tumour autoantibodies
refers to autoantibodies which are characteristic of the cancer
disease state, and which are directed against epitopes present on
forms of tumour marker proteins which are preferentially expressed
in the cancer disease state.
[0027] The method of the invention comprises an immunoassay to
detect and/or quantitatively measure autoantibodies immunologically
specific for one or more tumour marker proteins, and is
characterized in that the "immunoassay reagent" used in the
immunoassay comprises tumour marker protein prepared from bodily
fluid derived from a body cavity or space in which a tumour is or
was present or with which a tumour is or was associated, from one
or more cancer patients and/or tumour marker protein prepared from
an excretion of one or more cancer patients. Generally, the
excretion will have passed through an organ in which cancer is
present wherein the excretion is in contact with said cancer, or
the excretion will include one or more components which have been
in contact with cancer elsewhere in the body. A particular example
is bile which may be in contact with cancer in the gall bladder but
will appear in the faeces.
[0028] The immunoassay reagent exhibits "selective reactivity" with
cancer-associated anti-tumour autoantibodies. As used herein
"selective reactivity" means a tumour marker protein has a greater
affinity for autoantibodies to the tumour-associated antigen than
it does for any antibody or autoantibody made to the same antigen
which exists in the normal i.e. non-tumour possessing state.
[0029] The term "body cavity or space" includes any body cavity or
space, whether it be a natural cavity or a space or cavity arising
as a result of diseases or medical intervention including collapsed
or former cavities. The fluid is derived from such a cavity or
space in which a tumour is or was present or with which a tumour is
or was associated. Preferably the "bodily fluid derived from a body
cavity" will be a tumour-induced body fluid, meaning a body fluid
which is produced during the disease process, for example in
response to or as a consequence of the presence of tumour cells.
Exemplary body fluids are ascites, pleural effusion, seroma,
hydrocoele and wound drainage fluid.
[0030] For the avoidance of doubt "bodily fluids derived from a
body cavity or space" do not include fluids derived from the
systemic circulation, such as whole blood or serum.
[0031] The term "excretion" includes, inter alia, urine, faeces,
and seminal fluid.
[0032] The general features of immunoassays, for example ELISA,
radioimmunoassays and the like, are well known to those skilled in
the art (see Immunoassay, E. Diamandis and T. Christopoulus,
Academic Press, Inc., San Diego, Calif., 1996). Immunoassays for
the detection of antibodies having a particular immunological
specificity (e.g. autoantibodies having immunological reactivity
with a given tumour marker protein) generally require the use of a
reagent that exhibits specific immunological reactivity with the
antibody under test. Depending on the format of the assay this
reagent may be immobilized on a solid support. A sample to be
tested for the presence of the antibody is brought into contact
with the reagent and if antibodies of the required immunological
reactivity are present in the sample they will immunologically
react with the reagent to form autoantibody-reagent complexes which
may then be detected or quantitatively measured.
[0033] Suitable samples of tumour marker protein for use as the
basis of the "immunoassay reagent" may be isolated from bodily
fluids derived from a body cavity or space from one or more cancer
patients and/or from excretions from one or more cancer patients
using standard protein purification techniques, such as are
generally known in the art. For example, tumour marker proteins may
be isolated by affinity chromatography using a suitable antibody
(or antibody fragment) immunologically specific for the tumour
marker protein. The inventors have shown in the accompanying
examples that several different tumour marker proteins may be
purified using purification methods based on affinity
chromatography. It would be apparent to the skilled reader that
analogous purification methods used for any other tumour marker
proteins, with the use of a suitable antibody or antibody
fragment.
[0034] The starting material of bodily fluids derived from a body
cavity and/or excretions is/are taken from one or more cancer
patients. In this context the term "cancer patient" includes an
individual previously diagnosed as having cancer. The
fluid/excretion may be taken from a single patient or samples from
two or more patients may be pooled together. Samples may be pooled
from two or more patients having the same or different stages of
the same or different types of cancers. Samples may also be pooled
from different types of bodily fluids or excretions from a single
or multiple patients. Advantageously, an immunoassay reagent
prepared from fluid and/or excretion taken from cancer patient(s)
with a particular type of cancer may be used to assist in the
diagnosis of the same types of cancers in the other
individuals.
[0035] In one embodiment the "cancer patient" from which the
fluid/excretion is taken may be the same patient which it is later
intended to test using the assay reagent. For example, a stock of
reagent prepared from a patient diagnosed with cancer may be used
at a later date to assess the immune status of the same patient,
for example to monitor disease progression and/or to assess the
effectiveness of a course of anti-cancer treatment in that
patient.
[0036] The "immunoassay reagent" or "tumour marker preparation" may
comprise substantially whole tumour marker protein, for example
tumour marker protein substantially in the form in which it is
isolated from the fluid/excretion, or it may comprise a fragment of
the tumour marker protein. To be effective as an immunoassay
reagent any such "fragment" must retain immunological reactivity
with the (auto)antibodies for which it is desired to test using the
reagent. Suitable fragments might, for example, be prepared by
chemical or enzymatic cleavage of the isolated tumour marker
protein.
[0037] Depending on the precise nature of the immunoassay in which
it will be used, the "reagent" or "tumour marker protein
preparation" may comprise a tumour marker protein, or fragment
thereof, linked to one or more further molecules which impart some
desirable characteristic not naturally present in the tumour marker
protein. For example, the tumour marker protein may be conjugated
to a revealing label,--such as a fluorescent label, coloured label,
luminescent label, radiolabel or heavy metal such as colloidal
gold.
[0038] The tumour marker protein as prepared by the method
described herein can also be immobilized for use on a solid support
such as a bead or surface of a well of a multiwell plate. The
immobilization may be by absorption or by covalent attachment.
[0039] The tumour marker protein (or assay reagent comprising such
protein) is preferably substantially immunoglobulin free by virtue
of the fact that following isolation, for example, by affinity
chromatography, the protein preparation is treated to specifically
remove contaminating immunoglobulins.
[0040] The use of an immunoassay reagent comprising a tumour marker
protein (or fragment thereof) isolated from body cavity fluids
and/or excretions taken from one or more cancer patients provides
significant advantages over the use of other reagents, such as
recombinantly expressed or chemically synthesized polypeptides, in
the clinical detection of cancer (including diagnosis, monitoring
of disease recurrence or disease progression, etc.).
[0041] It might be expected that the precise characteristics of
tumour marker proteins isolated from cancer patients could vary
depending upon the source material (e.g. tissue or fluid) from
which the tumour marker protein is isolated. For example, the
characteristics of proteins isolated from urine may be different to
those isolated from whole blood or serum, which may be different
again to those isolated from ascites or pleural effusion. This may
in turn affect the utility of the tumour marker protein as an assay
reagent.
[0042] In fact, the inventors have surprisingly observed that
reagents prepared from tumour marker proteins isolated from body
cavity-derived fluids or excretions from cancer patients,
particularly ascites fluid, pleural effusion, seroma or wound
drainage fluid are generally more specific for cancer-associated
autoantibodies than reagents based on the equivalent proteins
isolated from "normal" individuals. This increased specificity for
cancer-associated autoantibodies means that immunoassays based on
the use of reagents prepared from body cavity-derived fluids or
excretions from cancer patients produce results that are more
"clinically relevant" in the detection of an immune response to
cancer.
[0043] Prior to the present invention, it was not clear how
reagents comprising antigens prepared from body cavity-derived
fluids or excretions from cancer patients would perform as reagents
for immunological detection of autoantibodies. In particular, it
was not known whether such antigens would exhibit higher
specificity for cancer-associated autoantibodies. It could not be
predicted whether antigens from such sources would perform
similarly to or better than antigen prepared from blood or serum,
in terms of their ability to detect cancer-associated
autoantibodies. Whilst it was known that tumour marker proteins may
be present in fluids derived from body cavities and spaces, there
is generally more potential for the antigens in these body cavities
and spaces to be broken down. This in turn would mean that they
might not detect autoantibodies as well as serum-derived antigens.
Furthermore, it could not be concluded with certainty that antigens
derived from cavity-derived fluids and excretions are
immunologically similar to antigens derived from serum.
Accordingly, it was surprising to observe that antigens prepared
from cavity-derived fluids and excretions of cancer patients
perform well as immunoassay reagents.
[0044] The inventors postulate that the improved specificity
observed with the use of reagents prepared from fluids derived from
body cavities of cancer patients, such as ascites, pleural
effusion, seroma or wound drainage fluid, is due to the origin of
such fluids within the body cavities or spaces of cancer patients.
It is postulated that fluids originating in body cavities or spaces
due to the presence of a tumour in contact with the major organs
may pick up more "cancer-associated" forms of the tumour marker
protein, which are actually relevant to the cancer disease state,
and contain less of the corresponding "normal" proteins. Since it
is generally differences between "tumour" marker proteins and their
"normal" counterparts which trigger the development of an immune
response (i.e. autoantibody production), the inventors hypothesize
that reagents based on the use of tumour markers isolated from
cancer patients will be more specific for cancer autoantibodies
than the equivalent "normal" proteins. This is indeed the case with
tumour marker antigens isolated from ascites, pleural effusion or
seroma, as shown in the accompanying Examples.
[0045] There are further practical advantages associated with the
use of ascites fluid, pleural effusion, seroma, hydrocoele or wound
drainage fluid, as a source of tumour marker proteins. These fluids
may be readily removed from patients in relatively large volumes as
part of the therapeutic strategy. This material, which would
otherwise be discarded, is a valuable source of useful assay
reagent.
[0046] Given that fluids such as ascites fluid, pleural effusion,
seroma, hydrocoele or wound drainage fluid are produced in large
volumes, there was doubt as to whether the concentration of tumour
marker proteins in such fluids would be high enough to enable such
fluids to be used as a practical source of antigens. One might
reasonably expect the concentration of tumour marker proteins to be
more dilute in such fluids as compared to blood or serum.
Surprisingly, the inventors observed that the concentrations of
tumour marker proteins in such fluids are in fact significantly
higher than in serum. Accordingly, there are substantial benefits
to be gained in terms of yield in recovering tumour marker proteins
from such fluids.
[0047] Furthermore, it has also been observed by the inventors that
additional significant advantages can be secured by pooling body
cavity fluid samples or excretions from two or more patients. Apart
from increasing protein yield, the product secures at least as good
a detection rate as marker protein from an individual sample while,
at the same time, being more consistent in its characteristics from
batch to batch. Thus, adequate affinity of the antigen can be
relied upon every time.
[0048] In particular embodiments the methods of the invention may
comprise immunoassays to (simultaneously) detect two or more types
of autoantibodies, each having specificity for different tumour
marker proteins or for different epitopes on the same tumour marker
proteins. These methods will typically involve use of a panel of
two or more assay reagents, each reagent comprising a different
tumour marker protein. These methods, which may be hereinafter
referred to as "panel assays", utilize a panel of two or more
reagents to monitor the overall immune response of an individual to
a tumour or other carcinogenic/neoplastic change. These methods
thus detect a "profile" of the immune response in a given
individual, indicating which tumour markers elicit an immune
response resulting in autoantibody production. The use of a panel
of two or more reagents to monitor production of autoantibodies
against two or more different tumour markers is generally more
sensitive than the detection of autoantibodies to single markers
and gives a much lower frequency of false negative results.
[0049] The methods of the invention are preferred for the detection
of circulating free autoantibodies, but may be adapted for
detection of autoantibodies present in immune complexes, as would
be appreciated by the skilled reader, for example by the
competitive use of labelled tumour marker.
[0050] In preferred applications the method of the invention will
be used to detect the presence of cancer-associated anti-tumour
autoantibodies in human subjects or patients, and will most
preferably take the form of an in vitro immunoassay, performed on
samples of bodily fluid taken from the subject/patient. Such in
vitro immunoassays are non-invasive and can be repeated as often as
is thought necessary to build up a profile of autoantibody
production in a patient, either prior to the onset of disease, as
in the screening of "at risk" individuals, or throughout the course
of disease (further discussed below in relation to preferred
applications of the method). As used herein the term "bodily
fluid", when referring to the material to be tested for the
presence of autoantibodies by immunoassay, includes inter alia
plasma, serum, whole blood, urine, sweat, lymph, faeces,
cerebrospinal fluid, ascites, pleural effusion, seminal fluid,
sputum or nipple aspirate. The type of bodily fluid used may vary
depending upon the type of cancer involved and the clinical
situation in which the assay is used. In general, it is preferred
to perform the assays on samples of serum or plasma.
[0051] As aforesaid, the "immunoassay" used to detect/quantitate
cancer-associated autoantibodies may be carried out according to
standard techniques known in the art. In a most preferred
embodiment the immunoassay may be an ELISA. ELISAs are generally
well known in the art. In a typical "sandwich" ELISA a reagent
having specificity for the autoantibodies under test is immobilized
on a solid surface (e.g. the wells of a standard microtiter assay
plate, or the surface of a microbead) and a sample of body fluid to
be tested for the presence of autoantibodies is brought into
contact with the immobilized reagent. Any autoantibodies of the
desired specificity present in the sample will bind to the
immobilized reagent. The bound autoantibody/reagent complexes may
then be detected using any suitable method. In one preferred
embodiment a labelled secondary anti-human immunoglobulin antibody,
which specifically recognizes an epitope common to one or more
classes of human immunoglobulins, is used to detect the
autoantibody/reagent complexes. Typically the secondary antibody
will be anti-IgG or anti-IgM. The secondary antibody is usually
labelled with a detectable marker, typically an enzyme marker such
as, for example, peroxidase or alkaline phosphatase, allowing
quantitative detection by the addition of a substrate for the
enzyme which generates a detectable product, for example a
coloured, chemiluminescent or fluorescent product. Other types of
detectable labels known in the art may be used with equivalent
effect.
[0052] ELISAs may be performed in a qualitative format, in which
the objective is merely to determine the presence or absence of
autoantibodies in the sample, or in a quantitative format, which
provides a measurement of the quantity of autoantibodies present in
the sample. For quantitative assays, a standard curve may be
generated by measuring the signal obtained (using the same
detection reaction as will be used for the assay) from a series of
standard samples containing known concentrations of antibodies
having similar specificity as the autoantibodies under test. The
quantity of autoantibodies present in the sample under test may
then be interpolated from the standard curve.
[0053] Panel assays may be performed in a multi-well format in
which each one of the two or more assay reagents is placed in a
separate well of a multi-well assay plate or, alternatively, in a
single-pot format in which the two or more assay reagents are
placed in a single container.
[0054] The method of the invention may be adapted for use in the
detection of autoantibodies to essentially any tumour marker
protein for which a suitable "assay reagent" may be prepared from
bodily fluid derived from a body cavity and/or from an excretion
from a cancer patient. In particular, the method may be adapted to
detect/measure autoantibodies to the epidermal growth factor
receptor-related protein c-erbB2 (Dsouza, B. et al. (1993)
Oncogene. 8: 1797-1806), the glycoprotein MUC1 (Batra, S. K. et al.
(1992) Int. J. Pancreatology. 12: 271-283) and the signal
transduction/cell cycle regulatory proteins Myc (Blackwood, E. M.
et al. (1994) Molecular Biology of the Cell 5: 597-609), p53
(Matlashewski, G. et al. (1984) EMBO J. 3: 3257-3262; Wolf, D. et
al. (1985) Mol. Cell. Biol. 5: 1887-1893) and ras (or Ras)
(Capella, G. et al. (1991) Environ Health Perspectives. 93:
125-131), and also BRCA1 (Scully, R. et al. (1997) PNAS 94:
5605-10), BRCA2 (Sharan, S. K. et al. (1997) Nature. 386: 804-810),
APC (Su, L. K. et al. (1993) Cancer Res. 53: 2728-2731; Munemitsu,
S. et al. (1995) PNAS 92: 3046-50), CA125 (Nouwen, E. J. et al.
(1990) Differentiation. 45: 192-8), PSA (Rosenberg, R. S. et al.
(1998) Biochem Biophys Res Commun. 248: 935-939), carcinoembryonic
antigen CEA (Duffy, M. J. (2001) Clin Chem, April 47(4):624-30),
and CA19.9 (Haga, Y. et al (1989) Clin Biochem (1989) October
22(5): 363-8). However, the invention is not intended to be limited
to the detection of autoantibodies to these particular tumour
markers.
[0055] The assay method of the invention may be employed in a
variety of different clinical situations. In particular, the method
may be used in the detection or diagnosis of cancer, in monitoring
the progress of cancer or other neoplastic disease in a patient, in
detecting early neoplastic or early carcinogenic change in an
asymptomatic human subject, in screening a population of
asymptomatic human subjects in order to identify those subjects who
are at increased risk of developing cancer, in monitoring the
response of a cancer patient to anti-cancer treatment, in the
detection of recurrent disease in a patient previously diagnosed as
having cancer who has undergone anti-cancer treatment to reduce the
amount of cancer present, or in the selection of an anti-cancer
vaccine for use in a particular patient.
[0056] The inventors have generally observed that levels of
cancer-associated autoantibodies show a positive correlation with
disease state (see also WO 99/58979, the contents of which are
incorporated herein by reference). Hence, when the method of the
invention is used in clinical applications increased levels of
anti-tumour marker autoantibodies, as compared to suitable
controls, are generally taken as an indication of the cancer
disease state.
[0057] For example, when the immunoassays are used in the diagnosis
of cancer, the presence of an elevated level of autoantibodies, as
compared to "normal" control individuals, is taken as an indication
that the individual has cancer. The "normal" control individuals
will preferably be age-matched controls not having any diagnosis of
cancer based on clinical, imaging and/or biochemical criteria.
[0058] When the immunoassays are used in monitoring the progress of
cancer or other neoplastic disease in a patient, the presence of an
elevated level of autoantibodies, as compared to a "normal
control", is taken as an indication of the presence of cancer in
the patient. The "normal control" may be levels of autoantibodies
present in control individuals, preferably age-matched, not having
any diagnosis of cancer based on clinical, imaging and/or
biochemical criteria. Alternatively, the "normal control" may be a
"base-line" level established for the particular patient under
test. The "base-line" level may be, for example, the level of
autoantibodies present when either a first diagnosis of cancer or a
diagnosis of recurrent cancer was made. Any increase above the
base-line level would be taken as an indication that the amount of
cancer present in the patient has increased, whereas any decrease
below the base-line would be taken as an indication that the amount
of cancer present in the patient has decreased. The "base-line"
value may also be, for example, the level before a new treatment is
commenced. A change in the level of autoantibodies would be taken
as an indication of the effectiveness of the therapy. The direction
of the "change" (i.e. increase verses decrease) indicating a
positive response to treatment will be dependent upon the precise
nature of the treatment. For any given treatment the direction of
the "change" in autoantibody levels indicating a positive result
may be readily determined, for example by monitoring autoantibody
levels in comparison to other clinical or biochemical indicators of
response to the treatment.
[0059] When the immunoassays are used in screening a population of
asymptomatic human subjects to identify those subjects who are at
increased risk of developing cancer, individuals having an elevated
level of autoantibodies, as compared to "normal" control
individuals, are identified as being "at risk" of developing
cancer. The "normal" control individuals will preferably be
age-matched controls not identified as having any predisposition to
developing cancer or any significant elevated risk of developing
cancer. An exception to this may be where age itself is a major
risk factor.
[0060] When the immunoassays are used in monitoring the response of
a cancer patient to anti-cancer treatment, the presence of a
decreased level of autoantibodies after treatment is taken as an
indication that the patient has responded positively to the
treatment. A base-line level of autoantibodies taken before
treatment is commenced may be used for comparison purposes in order
to determine whether treatment results in a "decrease" in
autoantibody levels.
[0061] When the immunoassays are used in detection of recurrent
disease, the presence of an increased level of autoantibodies in
the patient, as compared to a "normal control", is taken as an
indication that disease has recurred. The "normal control" may be
levels of autoantibodies present in control individuals, preferably
age-matched not having any diagnosis of cancer based on clinical,
imaging and/or biochemical criteria. Alternatively, the "normal
control" may be a "base-line" level established for the particular
patient under test. The "base-line" level may be, for example, the
level of autoantibodies present during a period of remission from
disease based on clinical, imaging and/or biochemical criteria.
[0062] The assay method of the invention may be applied in the
detection of many different types of cancer, of which examples are
breast, bladder, colorectal, prostate and ovarian cancers. The
assays may complement existing methods of screening and
surveillance. For example, in the case of primary breast cancer
immunoassays for autoantibodies could be used to alert clinicians
to biopsy small lesions on mammograms which radiographically do not
appear suspicious or to carry out breast imaging or to repeat
imaging earlier than planned. In the clinic, the assay methods of
the invention are expected to be more objective and reproducible
compared to current imaging techniques (i.e. mammography and
ultrasound), the success of which can be operator-dependent.
[0063] "Panel assays" may be tailored having regard to the
particular clinical application. A panel of reagents for detection
of autoantibodies to at least p53 and c-erbB2 is particularly
useful for many types of cancer and can optionally be supplemented
with other markers having a known association with the particular
cancer, or a stage of the particular cancer, to be detected. For
example for breast cancer the panel might include MUC1 and/or c-myc
and/or BRCA1 and/or BRCA2 and/or PSA whereas bladder cancer the
panel might optionally include MUC1 and/or c-myc, for colorectal
cancer ras and/or APC, for prostate cancer PSA and/or BRCA1 and/or
BRCA2 or for ovarian cancer BRCA1 and/or BRCA2 and/or CA125. There
are other preferred embodiments in which p53 or c-erbB2 are not
necessarily essential. For example, in the case of breast cancer
suitable panels could be selected from the following:
p53 and MUC1 with optional c-erbB2 and/or c-myc, and/or BRCA1
and/or BRCA2 and/or PSA; p53 and c-myc with optional c-erbB2 and/or
MUC1 and/or BRCA1 and/or BRCA2 and/or PSA; p53 and BRCA1 with
optional c-erbB2 and/or MUC1 and/or c-myc and/or BRCA2 and/or PSA;
p53 and BRCA2 with optional c-erbB2 and/or MUC1 and/or c-myc and/or
BRCA1 and/or PSA; c-erbB2 and MUC1 with optional p53 and/or c-myc,
and/or BRCA1 and/or BRCA2 and/or PSA; c-erbB2 and c-myc with
optional p53 and/or MUC1 and/or BRCA1 and/or BRCA2 and/or PSA;
c-erbB2 and BRCA1 with optional p53 and/or MUC1 and/or c-myc and/or
BRCA2 and/or PSA; c-erbB2 and BRCA2 with optional p53 and/or MUC1
and/or c-myc and/or BRCA1 and/or PSA.
[0064] In the case of colorectal cancer suitable panels could be
selected for example from the following:
p53 and ras with optional c-erbB2 and/or APC; p53 and APC with
optional c-erbB2 and/or Ras; Ras and APC with optional p53 and/or
c-erbB2 Such panels might also include CEA or CA19-9.
[0065] In the case of prostate cancer suitable panels could be
selected for example from the following:
p53 and PSA with optional BRCA1 and/or BRCA2 and/or c-erbB2;
c-erbB2 and PSA with optional p53 and/or BRCA1 and/or BRCA2.
[0066] In the case of ovarian cancer suitable panels could be
selected for example from the following:
p53 and CA125 with optional c-erbB2 and/or BRCA1 and/or BRCA2;
c-erbB2 and CA125 with optional p53 and/or BRCA1 and/or BRCA2.
[0067] In a further embodiment, the immunoassay method of the
invention may be used in the selection of an anti-cancer vaccine
for use in a particular patient. In this embodiment a sample of
bodily fluid taken from the patient is tested using a panel of two
or more immunoassay reagents, each corresponding to a different
tumour marker protein, in order to determine the relative strength
of the patient's immune response to each of the different tumour
marker proteins. The "strength of immune response" to a given
tumour marker protein or proteins is indicated by the presence
and/or the amount of cancer-associated autoantibodies specific to
that tumour marker protein detected using the immunoassay; where
autoantibodies are quantified, the greater the level of
cancer-associated auto-antibodies, the stronger the immune
response. The tumour marker protein or proteins identified as
eliciting the strongest immune response or responses in the patient
(i.e. the highest level of autoantibodies) is or are then selected
to form the basis of an anti-cancer vaccine for use in the
patient.
[0068] In a further embodiment, the invention provides a method of
monitoring whether vaccination of a subject with an anti-cancer
vaccine based on a particular tumour marker protein has been
successful in eliciting a humoral immune response (i.e. antibodies
against the said tumour marker protein). This method is based on
the same immunoassay methodology used to measure cancer-associated
anti-tumour autoantibodies (i.e. use of an immunoassay reagent
based on tumour marker protein purified from a body cavity fluid or
an excretion taken from a cancer patient), the only difference
being what is measured in the assay is an antibody response rather
than an autoantibody response.
[0069] In this embodiment a sample of bodily fluid taken from a
patient previously treated with the anti-cancer vaccine (e.g. an
immunogenic preparation comprising the relevant tumour marker
protein, or an antigenic fragment thereof or a vaccine comprising a
nucleic acid encoding said relevant tumour marker protein) is
contacted with an immunoassay reagent and complexes formed by
specific binding of the immunoassay reagent to cancer-associated
antibodies present in the sample are detected. The immunoassay
reagent again comprises a sample of the said tumour marker protein
prepared from bodily fluid derived from a body cavity or space as
defined herein from one or more cancer patients and/or tumour
marker protein prepared from an excretion from one or more cancer
patients.
[0070] In addition to clinical applications in the detection of
cancer, etc., the method of the invention may be used in any
application where it is desired to test for the presence of
cancer-associated anti-tumour autoantibodies. For example, the
method of the invention may have applications in the laboratory as
a research tool.
[0071] The tumour marker protein preparations provided by the
invention are advantageously used as (components of) immunoassay
reagents for use in the assay methods of the invention. However,
the utility of the tumour marker protein preparations is not
limited to such use. For example, they too may have applications in
the laboratory as research tools. Moreover, it is possible for
tumour marker proteins to have utility as therapeutic agents. The
availability of large quantities of protein as provided by the
bodily fluids defined herein allows pre-clinical and clinical
testing, either in vitro or in vivo in humans or non-human animals,
to determine efficacy of particular tumour marker proteins as
therapeutic agents. Such testing methods would be applicable to
each or all of the various tumour marker proteins described
herein.
[0072] Another utility for tumour marker preparations of the
invention is as a calibration material to be used in conjunction
with the development of diagnostic tests for the presence of cancer
or risk of cancer, which tests are based upon determination of the
presence and/or level of any particular tumour marker protein in a
clinical sample from a patient. The tumour marker protein
preparations of the invention can be used to construct calibration
curves for such tests. In particular this aspect of the invention
includes:
[0073] A method of calibrating an assay for measurement or
detection of a given tumour marker protein in a clinical sample
which method comprises the steps of:
[0074] a) preparing at least two samples of a tumour marker protein
prepared according to the method of the invention, each of which
comprises said given tumour marker protein and each of which has a
different tumour marker protein concentration to each of the other
said samples:
[0075] b) carrying out a quantitative measurement of the
concentration of said tumour marker protein in each of said samples
using: [0076] i) a spectrometric or spectrophotometric method
and/or, [0077] ii) an antibody reagent to said tumour marker
protein, and
[0078] c) constructing a standard curve for tumour marker protein
concentration based on the measurements obtained in step (b).
[0079] Such standard curves may be constructed for any or all of
the specific tumour marker proteins described herein.
[0080] The invention will be further understood with reference to
the following experimental Examples, together with the accompanying
Figures in which
[0081] FIG. 1 shows a post.about.Ig disruption gel filtration
chromatogram of a preparation of MUC16 (CA125) from ascites;
[0082] FIG. 2 shows a silver stained gel of c-myc purification from
ascitic fluid, post immunoaffinity chromatography;
[0083] FIG. 3 shows an immunoprobed blot, c-myc purification from
ascitic fluid, post immunoaffinity chromatography;
[0084] FIG. 4 shows a comparison of patient serum (patients with no
evidence of breast cancer themselves but with a family history of
breast cancer and those with primary breast cancer) auto-antibody
reactivity against MUC1 isolated from various body fluids: urine
(from "normal" individuals), pleural effusion from a cancer patient
and serum from advanced breast cancer patients (ABC serum);
[0085] FIG. 5 shows autoantibody reactivity in serum from normal
individuals against MUC1 from various body fluids: urinary MUC1
(normal), pleural effusion from a cancer patient and from advanced
breast cancer patients (ABC serum);
[0086] FIG. 6 shows the autoantibody reactivity in serum samples
from pre-operative patients with ovarian masses against normal
MUC16 (CA125) and against tumour-associated MUC16 from ascites;
[0087] FIG. 7 shows the cancer-associated MUC1 concentration in
sera, pleural effusion and ascitic fluid;
[0088] FIG. 8 shows the cancer-associated MUC1 concentration in
serum, wound drainage fluid and in seroma;
[0089] FIG. 9 shows the reactivity of purified autoantibodies from
seroma of patient M with cancer against purified urinary MUC1 from
patient M taken two years prior to cancer diagnosis, MUC1 derived
from the seroma of patient M, after diagnosis with cancer and
bovine serum albumen conjugated to MUC1 protein core peptide;
[0090] FIG. 10 shows serum autoantibody reactivity against MUC1
purified from pooled ascites fluid and against MUC1 purified from
individual ascites samples from cancer patients;
[0091] FIG. 11 shows serum autoantibody reactivity against MUC1
purified from pooled pleural effusions and against MUC1 purified
from individuals pleural effusion samples from cancer patients;
and
[0092] FIG. 12 shows a calibration curve prepared from MUC1 from a
pleural effusion.
EXAMPLE 1
General Protocol for Purification of MUC1 Antigen
[0093] Monoclonal anti-MUC1 antibody B55 (also known as NCRC 11,
Xoma Corporation) is conjugated to CNBr-sepharose beads. Other
anti-MUC1 monoclonal antibodies may be substituted for B55.
[0094] Tumour-induced body fluids (e.g. pleural effusion, ascites,
seroma or wound drainage fluid) are diluted 1/10 with phosphate
buffered saline (PBS) and filtered to 0.45 .mu.m.
[0095] Diluted body fluids are incubated with the anti-MUC1
sepharose beads (25 ml diluted fluid to 1 ml packed volume of
beads) overnight at 4.degree. C. with rolling ("batch" method) or
re-circulated overnight through a packed column containing
anti-MUC1 sepharose beads ("column" method).
"Batch" Method:
[0096] Beads are packed by centrifugation and the supernatant
removed;
[0097] Beads re-suspended in 5-10 ml PBS and rolled for 10 minutes
then packed by centrifugation and the supernatant removed; repeat 5
times (or until A.sub.280 nm.about.0)
[0098] Beads re-suspended in 5 ml 100 mM DEA pH 11, and rolled at
room temperature for 10 minutes;
[0099] Beads packed by centrifugation and the supernatant removed,
pH adjusted to 7 by the addition of pH 7 Tris buffer, dialyzed
against PBS for 24 hours minimum (100 DEA fraction);
[0100] Beads re-suspended in 5 ml PBS and rolled for 10 minutes
then packed by centrifugation and the supernatant removed, pH
adjusted to 7 by the addition of pH 7 Tris buffer, dialyzed against
PBS for 24 hours minimum (post-DEA fraction);
[0101] MUC1 content of each fraction confirmed by ELISA using, for
instance, the monoclonal anti-MUC1 antibody C595 (available from
Cancer Research Campaign Laboratories, UK) (see example 5 for
details) or B55; prior to pooling of the two fractions and storage
at -20.degree. C.
"Column" Method:
[0102] Column washed with 5 column volumes of PBS, or until eluate
reads .about.0 at A.sub.280 nm;
[0103] 1 column volume of 100 mM DEA pH 11 applied, followed by 5
column volumes of PBS;
[0104] Eluate fractions (2 ml) collected from the time of DEA
application through the application of PBS;
[0105] Fractions dialyzed overnight against PBS;
[0106] Fractions assayed for MUC1 content by ELISA using, for
instance, the monoclonal anti-MUC1 antibody C595 or B55, prior to
pooling MUC1 positive fractions and storage at -20.degree. C.
[0107] In order to remove contaminating immunoglobulins, MUC1
pooled fractions are incubated with dithiothreitol (DTT) to 50 mM
for 30 minutes, then iodoacetamide (to 75 mM) before being
subjected to gel filtration on an 5300 column.
[0108] Resulting fractions (5 ml) are assayed for MUC1 and human
immunoglobulin (Ig) content by ELISA.
[0109] MUC1 containing fractions (uncontaminated with human Ig) are
pooled and stored at -20.degree. C.
EXAMPLE 2a
General Protocol for Purification of MUC16 Antigen (Previously
Known as CA125)
[0110] One volume (e.g. 50 ml) of saturated ammonium sulphate was
added to one volume (e.g. 50 ml) of tumour-induced body fluid (e.g.
pleural effusion, ascites, seroma or wound drainage fluid) and
incubated overnight at 4.degree. C.
[0111] The resultant precipitate is collected by centrifugation
(3500 rpm for 30 min in a standard bench top centrifuge) and
resuspended in 1/2 volume PBS.
[0112] This resuspension is subjected to gel filtration
chromatography through an S300 column (2.5.times.100 cm) using PBS
as the eluting buffer.
[0113] Fractions (5 or 10 ml) are collected and assayed by ELISA
for MUC16, using for instance anti-CA125 from ICN or the anti-MUC16
antibody VK8 (Memorial Sloane Kettering, N.Y.), prior to pooling
MUC16 positive fractions and storage at -20.degree. C.
[0114] In order to remove contaminating immunoglobulins, MUC16
pools are incubated with NaSCN (to 1.5 M) for 10 minutes, DTT (to
50 mM) for 30 minutes, then iodoacetamide (to 75 mM) for 30 minutes
before being subjected to gel filtration on, for instance, an S300
or a SUPERDEX.TM. 75 column.
[0115] Resulting fractions (5 ml) are assayed for MUC16 and human
immunoglobulin (Ig) content by ELISA.
[0116] MUC16 containing fractions (uncontaminated with human Ig)
are pooled and stored at -20.degree. C.
EXAMPLE 2b
Post Ig Disruption Gel Filtration Chromatography
[0117] For a sample prepared in the manner described above,
fractions from a post-Ig disruption gel filtration were assayed for
MUC16 using anti-MUC16 antibody VK8 and for human Ig using an
anti-human Ig. The results are shown in FIG. 1. As is clearly
demonstrated, two substantially immunoglobulin free MUC16 peaks are
eluted.
EXAMPLE 3
Purification of c-myc Antigen
[0118] Methodology as per purification of MUC1 (Example 1), except
that:
[0119] Monoclonal anti-c-myc antibody 9E10 (ATCC) is used (or
equivalent anti-c-myc antibody).
[0120] Gel filtration is performed on a SUPERDEX.TM. 75 column.
Electrophoresis and Western Blotting
[0121] Purity of MUC1, MUC16 and c-myc fractions are assessed by
denaturing polyacrylamide gel electrophoresis and Western blotting,
performed according to standard protocols using BIO-RAD.TM. MINI
PROTEAN III.TM. gel electrophoresis assembly system and BIO-RAD.TM.
DRYBLOT.TM. protein transfer assembly system.
[0122] Protein patterns were revealed on gels for c-myc by silver
staining (FIG. 2). Western blots of c-myc were immuno-probed using
monoclonal antibodies 9E10 (FIG. 3). In each case, c-myc as well as
immunoglobulin heavy and light chains are identified.
EXAMPLE 4
Standard Auto-Antibody Assay
[0123] Tumour antigen (e.g. MUC1, MUC16 or c-myc prepared according
to Examples 1-3) diluted appropriately in PBS is plated out at 50
.mu.l per well in a standard 96 well microtiter plate and left to
air dry overnight;
[0124] Plate washed once with PBS/TWEEN.TM. washing buffer to
remove residual salt crystals;
[0125] Plate blocked for 60 minutes with 0.1% casein or 1% BSA in
PBS;
[0126] Plate washed 3 times with PBS/TWEEN.TM. washing buffer;
[0127] Serum (diluted 1/100 in PBS/0.1% casein) plated out in
triplicate (50 .mu.l per well), also monoclonal antibody
controls;
[0128] Incubate for 60 minutes at room temperature with
shaking;
[0129] Wash plate 4 times with PBS/TWEEN.TM. washing buffer;
[0130] Add horseradish peroxidase (HRP)-conjugated anti-Ig antibody
(Dako) to each well (50 .mu.l per well) at 1/8000 dilution for
anti-human and 1/1000 for anti-mouse;
[0131] Incubate for 60 minutes at room temperature with
shaking;
[0132] Wash plate 4 times with PBS/TWEEN.TM. washing buffer;
[0133] Add 50 .mu.l TMB (tetramethylbenzadine) per well and read
kinetically over a 10 minute period at A.sub.650 nm.
Experimental Data
[0134] Using the method as described in Example 4,
cancer-associated autoantibodies to MUC1 and MUC16 were measured in
a variety of sera using MUC1 and MUC16 isolated from the various
sources as described herein. Results generated are shown in FIGS. 4
to 6.
[0135] FIG. 4 shows a comparison of patient serum auto-antibody
reactivity against MUC1 isolated from various body fluids: urine
(from "normal" individuals), pleural effusion from a cancer patient
and serum from advanced breast cancer patients (ABC serum). The
patient serum tested was from either individuals with no evidence
of breast cancer themselves, but with a family history of breast
cancer (i.e. one or more relatives who had breast cancer at a young
age), or individuals with primary breast cancer.
[0136] Standard auto-antibody ELISAs were performed as described
above, utilizing MUC1 isolated from urine (normal), pleural
effusion or ABC serum as antigen. Data was normalized to an
internal control reaction using the DF3 anti-MUC1 monoclonal
antibody (as opposed to a serum sample) against each of the MUC1
antigens.
[0137] As can be seen from the Figure, MUC1 derived from normal
urine (nMUC1) was consistently lower in its reactivity than MUC1
derived from either pleural effusion (PE) or ABC serum.
Furthermore, MUC1 derived from PE was of similar reactivity to
cancer-associated MUC1 autoantibodies as MUC1 isolated from the
serum of patients with ABC and therefore of equal diagnostic
value.
[0138] FIG. 5 shows the results of an identical exercise to FIG. 4
except that all serum samples tested were for normal individuals
(no breast cancer or family history of breast cancer). As can be
seen, there is no significant difference in the reactivity of the
serum to the three different antigens.
[0139] FIG. 6 shows reactivity of MUC16 cancer-associated
autoantibodies from serum of patients with ovarian masses
(pre-operative) against MUC16 (CA125) isolated from the serum of
normal individuals and from ascites fluid in a patient with breast
cancer. Antigens were prepared as in Example 2 and autoantibodies
detected using ELISA assay as described in Example 4.
[0140] As can be seen, greatly enhanced reactivity of the
cancer-associated MUC16 autoantibodies is seen with the MUC16
antigen from ascites fluid as compared to the "normal" MUC16. This
experimental result therefore confirms the usefulness of ascites
fluid as an antigen source for detection of cancer-associated
autoantibodies.
EXAMPLE 5
Measurement of Cancer-Associated MUC1 Levels in Ascites Fluid,
Pleural Effusion, Seroma and Wound Drainage Fluid
[0141] MUC1 levels found in the serum of a patient with cancer were
compared with the levels found in ascites fluid, pleural effusion,
wound drainage fluid or seroma, in each case in the same patient
from whom the serum sample was taken. MUC1 in the samples was
quantified according to the following protocol:
Capture MUC1 ELISA Protocol
[0142] Aliquot 50 .mu.l per well antibody solution into triplicate
wells of a microtitre plate (usually 1 .mu.g ml.sup.-1 C595 (IgG)
and appropriate negative control) and incubate at room temperature
(RT) with shaking for 1 hour for the protein to adsorb to the
plate.
[0143] Wash the plate 4 times with PBS/TWEEN.TM. washing buffer
using 250 .mu.l per well.
[0144] Block the plate using 1% BSA 100 .mu.l per well and incubate
at RT with shaking for 1 hr.
[0145] Wash the plate 4 times with PBS/TWEEN.TM. washing buffer
using 250 .mu.l per well.
[0146] Apply 50 .mu.l per well of fluid being tested, diluted 1/10
in PBS and incubate at RT with shaking for 1 hr.
[0147] Wash the plate 4 times with PBS/TWEEN.TM. washing buffer
using 250 .mu.l per well.
[0148] Add 50 .mu.l per well biotinylated C595 (1 .mu.g/ml) and
incubate at RT with shaking for 1 hr.
[0149] Wash the plate 4 times with PBS/TWEEN.TM. washing buffer
using 250 .mu.l per well.
[0150] Add 50 .mu.l per well extra-avidin peroxidase at 1/1000
dilution and incubate at RT with shaking for 1 hr.
[0151] Wash the plate 4 times with PBS/TWEEN.TM. washing buffer
using 250 .mu.l per well.
[0152] Add 50 .mu.l per well TMB substrate and read kinetically at
650.sub.nm for 10 minutes.
[0153] The results are shown in FIGS. 7 and 8.
[0154] As will be readily apparent from the data serum levels of
the cancer-associated MUC1 antigen are significantly lower than the
level found in either ascites fluid, pleural effusion, seroma or
wound drainage fluid. Accordingly, there are substantial benefits
to be gained in terms of yield in recovering tumour marker antigen
from those body cavity fluids.
EXAMPLE 6
Reactivity of Human Anti-MUC1 Antibodies Purified Against
Cancer-Associated MUC1 from Seroma
[0155] Human antibodies from seroma from patient M were purified by
immunoaffinity chromatography against MUC1 derived from seroma
fluid from the same cancer patient M. Purified antibodies were then
tested against BSA conjugated protein core peptide to MUC1 and MUC1
derived from:--patient M's urine taken two years prior to cancer
diagnosis; patient M's seroma taken after cancer diagnosis. The
antibody purification from seroma was carried out according to the
following protocol:
Human Anti-MUC1 Antibody Purification
[0156] Purification of human anti-MUC1 auto-antibodies was by
affinity chromatography.
[0157] Seroma fluid, diluted 10 fold in PBS pH 7.6, was applied at
0.5 ml/min by overnight re-circulation at 4.degree. C., to an
affinity matrix in column format, consisting of CNBr sepharose
(Pharmacia) coupled (following the manufacturer's instructions) to
Pt-MUC1.
[0158] After seroma fluid application, the column was washed with
15 ml of PBS (ensuring return of A.sub.280 nm reading to zero)
prior to elution of antibody using 10 ml of 3M NaSCN, at 1
m/min.
[0159] Fractions of 1 ml were collected throughout, desalted by
dialysis against PBS and tested by ELISA for the presence of
antibody.
[0160] Positive fractions were pooled, purity of antibody verified
(by PAGE) and antibody concentration determined.
[0161] Assay of the purified antibodies against the three MUC1
antigens identified above was carried out according to the
following protocol:
MUC1 ELISA Protocol
[0162] Aliquot 50 .mu.l per well of the MUC1 antigen solution into
triplicate wells of a microtitre plate and dry down at RT
overnight.
[0163] Wash the plate 2 times with PBS/TWEEN.TM. washing buffer
using 250 .mu.l per well.
[0164] Block the plate with 1% BSA using 100 .mu.l per well and
incubate at RT with shaking for 1 hr.
[0165] Wash the plate 2 times with PBS/TWEEN.TM. washing buffer
using 250 .mu.l per well.
[0166] Add 50 .mu.l per well purified antibody solution at 1
.mu.g/ml and incubate at RT with shaking for 1 hr.
[0167] Wash the plate 4 times with PBS/TWEEN.TM. washing buffer
using 250 .mu.l per well.
[0168] Add 50 .mu.l per well .alpha.-human Ig HRP (DAKO), freshly
diluted as per manufacturer's instructions, and incubate at RT with
shaking for 1 hr.
[0169] Wash the plate 4 times with PBS/TWEEN.TM. washing buffer
using 250 .mu.l per well.
[0170] Add 50 .mu.l per well TMB substrate and read kinetically at
650.sub.nm for 10 minutes.
[0171] The results are shown in FIG. 9.
[0172] Reactivity of the antibodies against MUC1 peptide was
negligible. Reactivity of antibodies against normal MUC1 was
considerably lower than that seen towards patient M's seroma
derived MUC1. It can be inferred from this result that normal MUC1
molecule is substantially different with regard to its immune
recognition, to that found in seroma fluid from an individual with
cancer.
EXAMPLE 7
Serum Reactivity Against MUC1 Purified from Pooled Ascitic Fluid
and Pleural Effusions
[0173] MUC1 was purified from pooled ascitic fluid and from pooled
pleural effusion from patients with advanced breast cancer using
the protocol described in Example 1 and its reactivity against
serum from patients with primary breast cancer measured as
described in Example 4. The antigen from the pooled fluids was
compared in each case with antigen isolated from 3 individual
samples of ascitic fluid or pleural effusion respectively from
patients with ABC. The results are shown in FIGS. 10 and 11.
[0174] In the case of both ascitic fluid and pleural effusion the
reactivity of the MUC1 from pooled fluid is as good as that
isolated from individual samples. Furthermore, while there is great
scope for variability of reactivity using samples from individuals,
pooled samples provide greater consistency of product so that one
would not expect the reactivity to significantly vary between
batches from pooled samples.
EXAMPLE 8
Calibration Curve Using MUC1
[0175] Serial dilutions of MUC1 which had been isolated from
pleural effusion were prepared. Their MUC1 concentrations were
measured by the method as shown in example 4 except that no human
sera were used. Detection was by mouse B55 antibody followed by
Dako anti-mouse HRP using an end-point rather than a kinetic
reading.
[0176] The results are shown in FIG. 12 and confirm the utility of
the tumour marker proteins prepared in accordance with the
invention as a calibration material.
Sources of Antibodies to Tumour Marker Proteins
[0177] The following lists sources of antibodies which may be used
in the purification of tumour marker proteins by affinity
chromatography. Affinity chromatography may be performed following
the general methodology described in Example 1 (in relation to
MUC1), with appropriate modification. It will be appreciated that
other antibodies specific for the relevant marker protein may also
be used.
Carcinoembryonic Antigen (CEA):
[0178] 1116NS-3d, ATCC number CRL-8019, B lymphocyte hybridoma
producing monoclonal antibody against CEA; T84.66A3.1A.1F2, ATCC
number HB-8747, B lymphocyte hybridoma producing monoclonal
antibody against CEA.
P53:
[0179] Rabbit anti-human p53 polyclonal, commercially available
from Serotec Ltd, Kidlington, Oxford OX5 1JE, United Kingdom.
[0180] Monoclonal anti-p53, clone BP53-12, commercially available
from Sigma. CA19-9:
[0181] Mouse anti-human CA19-9 monoclonal, type clone 1116-NS-19-9,
IgG1, commercially available from Serotec Ltd, Kidlington, Oxford
OX5 1JE, United Kingdom.
H-ras p21:
[0182] Rabbit polyclonal IgG, commercially available from Santa
Cruz Biotechnology, Inc., Santa Cruz, Calif., USA.
BRCA1:
[0183] Rabbit polyclonal IgG, commercially available from Santa
Cruz Biotechnology, Inc., Santa Cruz, Calif., USA.
BRCA2:
[0184] Goat polyclonal IgG, commercially available from Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif., USA.
APC:
[0185] Rabbit polyclonal IgG, commercially available from Santa
Cruz Biotechnology, Inc., Santa Cruz, Calif., USA.
PSA:
[0186] Mouse monoclonal IgG, commercially available from Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif., USA.
* * * * *