U.S. patent application number 13/438344 was filed with the patent office on 2013-04-11 for tumour markers.
This patent application is currently assigned to Onclmmune Limited. The applicant listed for this patent is Catherine R.L. Graves, Frances M. Price, John F.R. Robertson. Invention is credited to Catherine R.L. Graves, Michael R. Price, John F.R. Robertson.
Application Number | 20130090251 13/438344 |
Document ID | / |
Family ID | 10831824 |
Filed Date | 2013-04-11 |
United States Patent
Application |
20130090251 |
Kind Code |
A1 |
Robertson; John F.R. ; et
al. |
April 11, 2013 |
Tumour Markers
Abstract
A method of determining the immune response of a mammal to
circulating tumour marker proteins is described in which a sample
of bodily fluid, for example plasma or serum, is contacted with a
panel of two or more distinct tumour marker antigen. The presence
of complexes between the tumour marker antigens and any
autoantibodies to the antigens present in the sample are detected
and provide an indication of an immune response to a circulating
tumour marker protein. The method is useful for the diagnosis of
cancer, particularly for identifying new or recurrent cancer in an
otherwise assymptomatic patient.
Inventors: |
Robertson; John F.R.;
(Nottingham, GB) ; Graves; Catherine R.L.;
(Nottingham, GB) ; Price; Michael R.; (Nottingham,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Robertson; John F.R.
Graves; Catherine R.L.
Price; Frances M. |
Nottingham
Nottingham
Nottingham |
|
GB
GB
GB |
|
|
Assignee: |
Onclmmune Limited
Derby
GB
|
Family ID: |
10831824 |
Appl. No.: |
13/438344 |
Filed: |
April 3, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13349348 |
Jan 12, 2012 |
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13438344 |
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11953237 |
Dec 10, 2007 |
8114604 |
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13349348 |
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09700092 |
May 16, 2001 |
7402403 |
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PCT/GB99/01479 |
May 11, 1999 |
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11953237 |
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Current U.S.
Class: |
506/9 |
Current CPC
Class: |
C12Q 1/6886 20130101;
Y10S 436/813 20130101; C07K 14/4748 20130101; G01N 33/6854
20130101; G01N 2800/52 20130101; G01N 33/574 20130101 |
Class at
Publication: |
506/9 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 1998 |
GB |
9810040.7 |
Claims
1-19. (canceled)
20. A method for the identification of those individuals which are
at increased risk of developing cancer in a population of
asymptomatic individuals comprising: (a) contacting a sample of
bodily fluids from a mammal with a panel of two or more distinct
tumour marker antigens; (b) determining the presence or absence of
complexes of the tumour marker antigens bound to autoantibodies
present in the sample of bodily fluids, the autoantibodies being
immunologically specific to the tumour marker proteins; wherein the
presence of the complexes is indicative of an increased risk of
developing cancer.
21. (canceled)
22. A method for the determination of the tumour marker profile of
an individual suffering from cancer comprising: (a) contacting a
sample of bodily fluids from the individual with a panel of two or
more distinct tumour marker antigens; (b) determining the presence
or absence of complexes of the tumour marker antigens bound to
autoantibodies present in the sample of bodily fluids, the
autoantibodies being immunologically specific to the tumour marker
proteins; wherein the presence of the complexes provides the tumour
marker profile of the individual.
23. (canceled)
24. A method for predicting the response of an individual with
cancer to anti-cancer treatment comprising: (a) contacting a sample
of bodily fluids from a mammal with a panel of two or more distinct
tumour marker antigens; (b) determining the presence or absence of
complexes of the tumour marker antigens bound to autoantibodies
present in the sample of bodily fluids, the autoantibodies being
immunologically specific to the tumour marker proteins; wherein the
presence of the complexes correlates with the response to the
anti-cancer treatment.
25.-72. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 13/349,348, which is a continuation of
U.S. patent application Ser. No. 11/953,237, now U.S. Pat. No.
8,114,604, which is a continuation of U.S. patent application Ser.
No. 09/700,092, filed May 16, 2001, which is a national stage
filing under 35 U.S.C. .sctn.371 of PCT International Application
PCT/GB99/01479, filed May 11, 1999, which claims priority to Great
Britain Application No. 9810040.7, filed May 11, 1998, all of which
are incorporated herein by reference.
[0002] The invention relates to methods of detecting or
quantitatively measuring the immune response of a mammal to
circulating tumour markers or tumour markers expressed on the
surface of tumour cells, also to tumour marker antigens for use in
these methods, to kits for performing the methods and to the use of
these methods in the detection of cancer, in monitoring the
progress of cancer, in detecting recurrent disease in cancer
patients who have previously undergone anti-cancer treatment and in
predicting the response of a cancer patient to a particular course
of treatment.
[0003] 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 the 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. Alternatively,
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.
[0004] 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 disease progression, for example in the screening of
asymptomatic patients. Thus, in the search for tumour markers
present in bodily fluid that are of use earlier in the disease
process the present inventors have sought to identify markers which
do not depend on tumour bulk per se.
[0005] 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 recognised
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 recognises 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.
[0006] As an alternative to the direct measurement or detection of
tumour marker protein in bodily fluids, assays could be developed
to measure the immune response of the individual to the presence of
tumour marker protein in terms of autoantibody production. Such
assays would 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 to
identify individuals "at risk" of developing disease.
[0007] 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).
[0008] In most cancers resulting from a progressive accumulation of
genetic alterations, such as breast cancer, the presence of tumour
markers in bodily fluids reflects the development and progression
of disease but no single marker on its own summates all clinically
important parameters. For example, the characteristics of a marker
useful for diagnosis of cancer may be quite different from markers
which convey information about prognosis. Furthermore, in each
clinical situation (i.e. diagnosis or prognosis) different markers
may be required when dealing with primary cancer and secondary
(metastatic) cancer and a different marker again may be required to
provide a method of measuring the effectiveness of a particular
course of treatment. Different clinical situations therefore
require different biological markers and, as has been observed with
p53, not all patients express the same set of tumour marker
proteins. It is therefore difficult to envisage any one single
tumour marker being universally applicable to all patients in all
stages of disease.
[0009] It is an object of the present invention to provide an
improved assay system for the detection of bodily fluids-borne
tumour markers which is more generally useful in all patients and
in a variety of different clinical situations.
[0010] Accordingly, in a first aspect the invention provides a
method of detecting the immune response of a mammal to circulating
tumour marker proteins or tumour cells expressing said tumour
marker proteins, which method comprises steps of: [0011] (a)
contacting a sample of bodily fluids from said mammal with a panel
of two or more distinct tumour marker antigens; [0012] (b)
determining the presence or absence of complexes of said tumour
marker antigens bound to autoantibodies present in said sample of
bodily fluids, said autoantibodies being immunologically specific
to said tumour marker proteins. whereby the presence of said
complexes is indicative of the immune response to circulating
tumour marker proteins or tumour cells expressing said tumour
marker proteins.
[0013] The method of the invention, which may be hereinafter
referred to as a `panel assay`, utilises a panel of two or more
tumour marker antigens to monitor the overall immune response of an
individual to a tumour or other carcinogenic/neoplastic change. The
method thus provides essentially a `profile` of the immune response
for that individual, indicating which tumour markers elicit an
immune response resulting in autoantibody production. The method of
the invention is preferred for the detection of an immune response
resulting in the production of circulating free autoantibodies.
[0014] Because the assay method of the invention performed on a
sample of bodily fluids taken from the patient it is essentially
non-invasive and can be repeated as often as is thought necessary
to build up a profile of the patient's immune response throughout
the course of disease. As used herein the term `bodily fluids`
includes plasma, serum, whole blood, urine, sweat, lymph, faeces,
cerebrospinal fluid or nipple aspirate. The type of bodily fluid
used may vary depending upon the type of cancer involved and the
use that the assay is being put to. In general, it is preferred to
perform the method on samples of serum or plasma.
[0015] As will be illustrated in the Examples given below, the use
of a panel of two or more tumour marker antigens to monitor
autoantibody production is more sensitive than the use of single
markers and gives a much lower frequency of false negative results.
The actual steps of detecting autoantibodies in a sample of bodily
fluids may be performed in accordance with immunological assay
techniques known per se in the art. Examples of suitable techniques
include ELISA, radioimmunoassays and the like. In general terms,
such assays use an antigen which may be immobilised on a solid
support. A sample to be tested is brought into contact with the
antigen and if autoantibodies specific to the tumour marker protein
are present in the sample they will immunologically react with the
antigen to form autoantibody-antigen complexes which may then be
detected or quantitatively measured. Detection of
autoantibody-antigen complexes is preferably carried out using a
secondary anti-human immunoglobulin antibody, typically anti-IgG or
anti-human IgM, which recognise general features common to all
human IgGs or IgMs, respectively. The secondary antibody is usually
conjugated to an enzyme such as, for example, horseradish
peroxidase (HRP) so that detection of
autoantibody/antigen/secondary antibody complexes is achieved by
the addition of an enzyme substrate and subsequent colorimetric,
chemiluminescent or fluorescent detection of the enzymatic reaction
products.
[0016] The panel assay of the invention uses a panel of tumour
marker-related antigens. The panel may be tailored to detect a
particular cancer, or a cancer at a particular stage of
development. The tumour marker antigens may be wild type or mutant
tumour marker proteins isolated from samples of biological fluid
from normal individuals or from cancer patients or from cell lines
expressing the tumour marker protein or they may be full length
recombinant tumour marker proteins, viral oncogenic forms of tumour
marker proteins or antigenic fragments of any of the aforementioned
proteins. The term `antigenic fragment` as used herein means a
fragment which is capable of eliciting an immune response.
[0017] The panel assay may be performed in a multi-well format in
which each one of the two or more antigens is placed in a separate
well of a multi-well assay plate or, alternatively, in a single-pot
format in which the entire panel of antigens is placed in a single
container. The panel assay may be performed in a qualitative format
in which the objective is simply detection of the presence or
absence of autoantibodies or in a quantitative format which
provides a quantitative measurement of the amount of autoantibodies
present in a sample.
[0018] Preferred markers for inclusion into the panel of tumour
marker antigens include 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) Mel. Cell. Biol. 5: 1887-1893) and ras (or Ras)
(Capella, G. et al. (1991) Environ Health Perspectives. 93:
125-131), including the viral oncogenic forms of ras which can be
used as antigens to detect anti-ras autoantibodies, 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), CAl25 (Nouwen, E. J. et al. (1990) Differentiation. 45:
192-8) and PSA (Rosenberg, R. S. et al. (1998) Biochem Biophys Res
Commun. 248: 935-939). As aforementioned, the assays can be formed
using tumour marker antigens which are forms of these proteins
isolated from human bodily fluids or from cultured cells or
antigenic fragments thereof or full length or truncated recombinant
proteins or antigenic fragments thereof.
[0019] Preferably the tumour marker antigens are labelled with
biotin so that they can easily be attached to a solid support, such
as a multi-well assay plate, by means of the biotin/avidin or
biotin/streptavidin interaction. Tumour marker antigens labelled
with biotin may be referred to herein as `biotinylated` proteins.
To facilitate the production of biotinylated tumour marker antigens
for use in the assay methods of the invention, cDNAs encoding a
full length recombinant tumour marker protein, a truncated version
thereof or an antigenic fragment thereof may be expressed as a
fusion protein labelled with a protein or polypeptide tag to which
the biotin co-factor may be attached via an enzymatic reaction. A
useful system for the expression of biotinylated fusion proteins is
the PinPoint.TM. system supplied by Promega Corporation, Madison
Wis., USA. The present inventors have surprisingly found that with
the use of biotinylated tumour marker antigens as antigens they are
able to detect autoantibodies in a much higher percentage of
patients than is observed using non-biotinylated antigen.
[0020] The assay method of the invention may be employed in a
variety of different clinical situations such as, for example, in
the detection of primary or secondary (metastatic) cancer, in
screening for early neoplastic or early carcinogenic change in
asymptomatic patients or identification of individuals `at risk` of
developing cancer (particularly breast cancer, bladder cancer,
colorectal cancer or prostate cancer) in a population or
asymptomatic individuals, in the detection of recurrent disease in
a patient previously diagnosed as carrying tumour cells who has
undergone treatment to reduce the number of tumour cells or in
predicting the response of an individual with cancer to a course of
anti-cancer treatment.
[0021] The assay method of the invention is suitable for detection
of many different types of cancer, of which examples are breast,
bladder, colorectal, prostate and ovarian. The assay of the
invention may complement existing methods of screening and
surveillance. For example in the case of primary breast cancer it
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 method of the invention is 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.
[0022] As aforesaid the panel of tumour marker antigens may be
tailored having regard to the particular application. A panel of 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 MUC 1 and/or c-myc and/or BRCA1 and/or
BRCA2 and/or PSA whereas bladder cancer the panel might optionally
include MUC 1 and/or c-myc, for colorectal cancer ras and/or APC,
for prostate cancer PSA and/or BRCA 1 or for ovarian cancer BRCA1
and/or CAl25. 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:
[0023] p53 and MUC 1 with optional c-erbB2 and/or c-myc, and/or
BRCA1 and/or BRCA2 and/or PSA; [0024] p53 and c-myc with optional
c-erbB2 and/or MUC1 and/or BRCA1 and/or BRCA2 and/or PSA; [0025]
p53 and BRCA1 with optional c-erB2 and/or MUC 1 and/or c-myc and/or
BRCA2 and/or PSA; [0026] p53 and BRCA2 with optional c-erbB2 and/or
MUC 1 and/or c-myc and/or BRCA1 and/or PSA; [0027] c-erbB2 and MUC
1 with optional p53 and/or c-myc, and/or BRCA1 and/or BRCA2 and/or
PSA; [0028] c-erbB2 and c-myc with optional p53 and/or MUC1 and/or
BRCA1 and/or BRCA2 and/or PSA; [0029] c-erbB2 and BRCA1 with
optional p53 and/or MUC 1 and/or c-myc and/or BRCA2 and/or PSA;
[0030] c-erbB2 and BRCA2 with optional p53 and/or MUC 1 and/or
c-myc and/or BRCA1 and/or PSA;
[0031] In the case of colorectal cancer suitable panels could be
selected from the following: [0032] 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
[0033] In the case of prostate cancer suitable panels could be
selected from the following: [0034] p53 and PSA with optional BRCA1
and/or c-erbB2; c-erbB2 and PSA with optional p53 and/or BRCA1.
[0035] In the case of ovarian cancer suitable panels could be
selected from the following: [0036] p53 and CAl25 with optional
c-erbB2 and/or BRCA1; c-erbB2 and CAl25 with optional p53 and/or
BRCA1.
[0037] In a second aspect the invention provides a method of
determining the immune response of a patient to two or more
circulating tumour marker proteins or to tumour cells expressing
said tumour marker proteins and identifying which one of said two
or more tumour marker proteins elicits the strongest immune
response in the patient, the method comprising contacting a sample
of bodily fluids from said patient with a panel of two or more
distinct tumour marker antigens, measuring the amount of complexes
formed by binding of each of said tumour marker antigens to
autoantibodies present in the sample of bodily fluids, said
autoantibodies being immunologically specific to said tumour marker
proteins and using the measurement obtained as an indicator of the
relative strength of the immune response to each tumour marker
protein and thereby identifying which one of said two or more
tumour marker proteins elicits the strongest immune response in the
patient.
[0038] The assay described above, which may be hereinafter referred
to as a `selection assay` is useful in the selection of a course of
vaccine treatment wherein the single tumour marker protein
identified as eliciting the strongest immune response or a
combination of markers eliciting strong immune response is/are used
as the basis of an anti-cancer vaccine treatment.
[0039] Preferred tumour marker antigens for use in the selection
assay are any of the tumour marker antigens mentioned above and
preferably the antigens are labelled with biotin. The actual steps
of detecting autoantibodies in a sample of bodily fluids may be
performed in accordance with known immunological assay techniques,
as described above for the panel assay.
[0040] The invention also provides methods for the detection or
quantitative measurement of the immune response of a mammal to a
circulating tumour marker protein or tumour cells expressing the
tumour marker protein wherein the tumour marker protein is MUC1,
c-erbB2, Ras, c-myc, BRCA1, BRCA2, PSA, APC, CAl25 or p53, the
method comprising the steps of contacting a sample of bodily fluids
from the mammal with the tumour marker antigen and determining the
presence or absence of complexes of the tumour marker antigen bound
to autoantibodies immunologically specific to the tumour marker
protein or antigenic fragment thereof, whereby the presence of said
complexes is indicative of the immune response to said circulating
tumour marker protein or tumour cells expressing the tumour marker
protein.
[0041] The assays described above, which may be hereinafter
referred to as `single marker assays`, use a single type of tumour
marker as antigen rather than using a panel of two or more tumour
markers. The single marker assays may be used in any clinical
situation, for example, screening for early neoplastic or
carcinogenic change in asymptomatic patients, identification of
individuals `at risk` of developing cancer, early diagnosis and
early detection of recurrence in a patient previously diagnosed as
carrying tumour cells which patient has undergone treatment to
reduce the number of said tumour cells or in predicting the
response of a patient to a course of anti-cancer treatment,
including surgery, radiotherapy, immune therapy, vaccination
etc.
[0042] The single marker assays are particularly useful in
situations where the tumour marker eliciting the strongest immune
response in a given patient has been previously identified,
possibly using the selection assay described above. For example, in
a situation in which an initial selection assay has been performed
to establish which tumour marker elicits the strongest immune
response in a given patient, subsequent follow-up, detection of
recurrence or monitoring of treatment may be carried out using a
single marker assay to only detect or measure autoantibodies to
that tumour marker previously identified as eliciting a strong
immune response in that patient.
[0043] The actual steps of detecting autoantibodies in a sample of
bodily fluids may be performed in accordance with known
immunological assay techniques, as described above for the panel
assay. Preferably the tumour marker protein used as antigen is
labelled with biotin so that it may be easily attached to a solid
support by means of the biotin/avidin or biotin/streptavidin
interaction.
[0044] In a further aspect, the present invention provides a
preparation comprising a human MUC1 protein which MUC1 protein
manifests all the antigenic characteristics of a MUC1 protein
obtainable from the bodily fluids of a patient with advanced breast
cancer.
[0045] Preferably the MUC1 protein exhibits altered affinity for
the antibodies B55, C595, BC4W154, DF3, B27.29, 115D8, 27.1, SM3,
Ma552, HMPV and BC2 compared to MUC1 protein isolated from normal
human urine. Most preferably the MUC1 protein is isolated from the
serum of one or more human patients with advanced breast cancer.
This can be accomplished using the protocol given in the Examples
listed herein.
[0046] As will be described in detail in Example 2, the present
inventors have found immunological differences between MUC1
isolated from normal individuals and MUC1 isolated from patients
with advanced breast cancer. Possibly as a result of these
differences, the inventors have found that the MUC1 protein
isolated from serum of patients with advanced breast cancer
(hereinafter referred to as ABC MUC1) is more sensitive when used
as antigen in an assay to detect autoantibodies specific to MUC1
than either MUC1 isolated from urine of normal individuals,
synthetic MUC1 or MUC1 isolated from a range of different cultured
cells. MUC1 isolated from the serum of patients with advanced
breast cancer is therefore preferred for use as antigen in the
panel assay method and the single marker assay methods described
herein.
[0047] MUC1 has recently attracted interest as a target for
immunotherapy of adenocarcinomas and several Phase I clinical
trials involving different MUC1 vaccine substrates, adjuvants and
carrier proteins have been carried out (Goydos, J. S. et al. (1996)
J Surgical Res. 63: 298-304; Xing, P. X. et al. (1995) Int. J.
Oncol. 6: 1283-1289; Reddish, M. A. et al. (1996) Cancer Immunol.
Immunother. 42: 303-309; Graham, R. A. et al. (1996) Cancer
Immunol. Immunother. 42: 71-80). Methods for the detection of
anti-MUC1 autoantibodies using MUC1 isolated from the serum of
patients with advanced breast cancer as antigen will be of
particular use in monitoring the success of MUC1 vaccine therapy.
In this case the aim of the assay will be to detect anti-MUC1
antibodies produced in response to the vaccine rather than
autoantibodies i.e. antibodies produced in response to an exogenous
antigen introduced into the body by vaccination. Methods for the
detection of autoantibodies directed to other tumour markers would
also be of use in monitoring the success of vaccine therapy using
the relevant tumour marker. For example, following vaccination with
a p53 antigenic preparation, the presence of anti-p53 antibodies
could be monitored using the assay based on the use of biotinylated
p53 antigen described in the examples given below. Moreover, the
panel assay method could also be used in monitoring the success of
vaccine therapy, for example, in a situation where an individual
has been vaccinated with an antigenic preparation designed to
elicit antibodies to two or more different tumour markers.
[0048] In a still further aspect the invention provides a method of
detecting recurrent disease in a patient previously diagnosed as
carrying tumour cells, which patient has undergone treatment to
reduce the number of said tumour cells, which method comprises
steps of contacting a sample of bodily fluids from the patient with
MUC1 protein or an antigenic fragment thereof, determining the
presence or absence of complexes of said MUC1 protein or antigenic
fragment thereof bound to autoantibodies present in said sample of
bodily fluids, said autoantibodies being immunologically specific
to MUC1, whereby the presence of said complexes indicates the
presence of recurrent disease in said patient.
[0049] The method described above may be repeated on a number of
occasions to provide continued monitoring for recurrence of
disease. The method is particularly preferred for the monitoring of
patients previously diagnosed with primary breast cancer,
colorectal cancer, prostate cancer or bladder cancer, which
patients have undergone treatment (e.g. surgery) to remove or
reduce the size of their tumour. In this instance, the presence of
anti-MUC1 autoantibodies in the patient's serum after treatment may
be indicative of recurrence of disease.
[0050] Also provided by the invention are assay kits suitable for
performing the methods for the detection of autoantibodies
described herein. Such kits include, at least, samples of the
tumour marker antigens to be used as antigen in the assay and means
for contacting the sample to be tested with a sample of the
antigen.
[0051] The contents of all documents, articles and references cited
herein are incorporated herein by reference.
[0052] The present invention will be further understood with
reference to the following Examples and the accompanying Figures in
which:
[0053] FIG. 1: shows the results of assays for autoantibodies to
MUC1, p53 and c-erbB2 in samples of serum taken from 21 patients
diagnosed with primary breast cancer. Panel A: anti-p53
autoantibodies; Panel B: anti-c-erbB2 autoantibodies and Panel C:
anti-MUC1 autoantibodies. In each case, the dotted line represents
the cut-off value for normality.
[0054] FIG. 2: shows reactivity profiles of MUC1 protein isolated
from normal human urine (panel A), ABC MUC1 isolated from the serum
of patients with advanced breast cancer (panel B) or MUC1 isolated
from the human breast cancer cell line ZR75-1 (panel C) with
various monoclonal anti-MUC1 antibodies.
[0055] FIG. 3: shows continuous monitoring for recurrent disease in
three post-operative breast cancer patients. Quantitative assays
for anti-MUC1, anti-cerbB2 and anti-p53 autoantibodies and for the
tumour marker CA15-3 (TM) were performed on samples of serum taken
at two or three monthly intervals post-surgery.
[0056] FIG. 4: shows the range of autoantibody levels found in
assays for autoantibodies to c-erbB2, c-myc, MUC1 and p53 in normal
individuals and patients with early primary breast cancer
(PBC).
[0057] FIG. 5: summarises the detection rate for primary breast
cancer in an analysis of autoantibody levels in a series of healthy
controls and patients with primary breast cancer, PBC subdivided by
Stage 1--i.e. lymph node negative and Stage 2--i.e. lymph node
positive and patients with metastatic cancer at 100%
confidence.
[0058] FIG. 6: summarises the detection rate for primary breast
cancer in an analysis of autoantibody levels in a series of healthy
controls and patients with PBC subdivided by Stage 1--i.e. lymph
node negative and Stage 2--i.e. lymph node positive and patients
with metastatic cancer at 95% confidence.
[0059] FIG. 7: shows the sensitivity for primary breast cancer in
an analysis of autoantibody levels in a series of healthy controls
and patients with Stage 1 or Stage 2 primary breast cancer at 95%
confidence.
[0060] FIG. 8: shows the levels of autoantibodies to MUC1, p53 and
c-erbB2 in the serum of three patients previously diagnosed with
breast cancer measured sequentially during follow-up until the
patient manifested recurrent disease.
[0061] FIG. 9: shows the autoantibody levels in further samples
from the second patient in FIG. 10 (AEC at 36 months) taken up to
recurrence and during treatment for recurrence. Sequential
measurements of established tumour markers reflecting tumour bulk
(e.g. CA15-3 and CEA) were within the normal range throughout this
period (data not shown).
[0062] FIG. 10: shows follow-up autoantibody levels in
post-operative serum samples from two patients, one who did not
develop recurrent disease (no REC) and the other who did (REC at 36
months).
[0063] FIG. 11: summarises the detection rates in an analysis of
autoantibody levels (p53, MUC1, c-erbB2 and c-myc) in samples of
serum taken from patients with urologically benign disorders and
various stages of bladder cancer.
[0064] * indicates patients which were benign with respect to
urology (i.e. did not have a urological malignancy), but six cases
(all with positive autoantibody status) had evidence of other
malignancies.
[0065] ** Other malignancies were:--lung cancer, skin cancer,
adenocarcinoma of unknown primary. Evidence of other neoplasia
consisted of:--pleural effusion, ovarian cysts, colon polyps.
[0066] FIG. 12: summarises the detection rate for colorectal cancer
in an analysis of autoantibody levels in the serum of healthy
controls, patients with colonic polyps and patients with colorectal
cancer at 100% confidence compared to a pre-defined group of
healthy controls.
[0067] FIG. 13: summarises the detection rate for colorectal cancer
in an analysis of autoantibody levels serum of healthy controls,
patients with colonic polyps and patients with colorectal cancer at
at 95% confidence compared to a pre-defined group of healthy
controls.
[0068] FIG. 14: summarises the detection rate in an analysis of
autoantibody levels in the serum of healthy controls, patients with
primary breast cancer and asymptomatic women known to be BRCA1
mutant carriers at 100% confidence compared to a pre-defined group
of healthy controls.
[0069] FIG. 15: summarises the detection rate for prostate cancer
in an analysis of autoantibody levels in the serum of healthy
controls and patients with prostate cancer at 95% confidence
compared to a pre-defined group of healthy controls.
EXAMPLES
Example 1
Isolation of ABC MUC1 from Advanced Breast Cancer Patients
Method
[0070] ABC MUC1 was purified from pooled sera taken from 20
patients with advanced breast cancer using immunoaffinity
chromatography as follows:
[0071] The mouse monoclonal anti-MUC1 antibody B55 (also known as
NCRC 11 and described by Ellis et al. (1984) Histopathology. 8:
501-516 and in International patent application No. WO 89/01153)
was conjugated to CNBrsepharose beads. Pooled sera from patients
diagnosed with advanced breast cancer was diluted 1/10 in phosphate
buffered saline (PBS) and then incubated with the antibody
conjugated sepharose beads (25 ml diluted sera to 1 ml packed
volume of beads) overnight at 4.degree. C. with rolling. The beads
were then packed by centrifugation and the supernatant removed. In
order to wash away unbound serum components the beads were
resuspended in PBS, rolled for 10 minutes, packed by centrifugation
and the supernatant removed. This washing sequence was repeated 5
times (or until A280 nm of the supernatant was -0). The washed
beads were then resuspended in 0.25M glycine pH 2.5, rolled at room
temperature for 10 minutes, packed by centrifugation and the
supernatant removed. This supernatant was adjusted to pH 7 by the
addition of Tris and stored at 4.degree. C. labelled `glycine
fraction`. The beads were then resuspended in 1 ml 25 mM
diethylamine (DEA) pH11, rolled at room temperature for 10 minutes,
packed by centrifugation and the supernatant removed. This
supernatant was again adjusted to pH 7 by the addition of Tris and
stored at 4.degree. C. labelled `25 DEA fraction`. The beads were
finally resuspended in 1 ml 100 mM DEA pH11, rolled at room
temperature for 10 minutes, packed by centrifugation and the
supernatant removed. The final supernatant was again adjusted to pH
7 by the addition of Tris and stored at 4.degree. C. labelled `100
DEA fraction`. The MUC1 content of the three fractions (glycine
fraction, 25 DEA fraction and 100 DEA fraction) was confirmed by
ELISA using the mouse monoclonal anti-MUC1 antibody C595
(commercially available from Serotec).
Example 2
Immunological Characterisation of ABC MUC1 Isolated from the Serum
of Patients with Advanced Breast Cancer
[0072] ABC MUC1 isolated from the serum at least 20 patients with
advanced breast cancer according to the procedure described in
Example 1 can be distinguished from MUC1 isolated from the urine of
normal human subjects (normal human urinary MUC1) on the basis of
altered affinity for the following mouse monoclonal anti-MUC1
antibodies:
TABLE-US-00001 B55 (NCRC 11) C595 BC4W154 Obtainable from
Hybritech, Inc DF3 Obtainable from Centocor B27.29 Obtainable from
Biomira, Inc 115D8 Obtainable from Centocor 27.1 Obtainable from
Austin Research Institute SM3 Obtainable from the Imperial Cancer
Research Fund Ma552 Obtainable from CanAg HMPV Obtainable from
Austin Research Institute BC2 Obtainable from Austin Research
Institute
[0073] Normal urinary MUC1 is available from Dr M. R. Price, Cancer
Research Laboratories, The University of Nottingham, University
Park, Nottingham. NG7 2RD, United Kingdom.
[0074] The affinity of each of the above antibodies for ABC MUC1,
normal human urinary MUC1 and also MUC1 protein purified from the
human breast cancer cell line ZR75-1 (purified from a tissue
culture supernatant by gel filtration) was measured by performing
colorimetric ELISA assays using each of the different antibodies
and secondary anti-immunoglobulin antibodies conjugated to HRP.
Following addition of the colorimetric substrate (TMB),
measurements were taken of OD at 650 nm. The results of the ELISA
assays are presented graphically in FIG. 2. Values of Kd for the
binding of several of these antibodies to ABC MUC1 and normal human
urinary MUC1 are summarised in Table 1:
TABLE-US-00002 TABLE 1 Kd values for binding of monoclonal
antibodies to ABC MUC1 and normal human urinary MUC1. Monoclonal Kd
vs ABC MUC1 Kd vs urinary MUGS BC4W154 2.4 .times. 10.sup.-7 1.7
.times. 10.sup.-9 115D8 .sup. 1 .times. 10.sup.-8 3.38 .times.
10.sup.-8 C595 2.4 .times. 10.sup.-8 2.5 .times. 10.sup.-8
Example 3
Cloning of Biotinylated p53
Method
[0075] Commercially available cDNA for p53 (E. coli clone pBH53,
deposited in the American Type Culture Collection under accession
number 79110) was cloned into the PinPoint.TM. plasmid vector
(Promega Corporation, Madison Wis., USA) using standard molecular
biology techniques. The PinPoint.TM. vector is designed to
facilitate the production of fusion proteins comprising a
biotinylation domain (consisting of a fragment of a biotin
carboxylase carrier protein) fused N-terminally to the target
protein of interest. Care was therefore taken during the cloning
procedure to ensure that the reading frame of p53 was maintained in
the fusion protein. Procedures for cloning in PinPoint.TM. vectors
are described in detail in the Promega Protocols and Applications
Guide obtainable from Promega Corporation, Madison Wis., USA.
[0076] Fusion proteins expressed from the PinPoint.TM. vector in E.
coli are biotinylated by an enzyme system of the E. coli host cells
and may therefore be purified or bound to an assay plate using
conventional avidin or streptavidin technology. For example,
procedures for purification of the fusion protein using avidin
covalently attached to a polymethacrylate resin are described in
the Promega Protocols and Applications Guide obtainable from
Promega Corporation, Madison Wis., USA.
Example 4
Cloning of c-erbB2
Method
[0077] Full-length cDNA encoding c-erbB2 was cloned from the human
breast cancer cell line ZR75-1, which can be induced to up-regulate
c-erbB2 expression by treatment with the anti-cancer drug
tamoxifen.
[0078] Two T25 flasks of sub-confluent ZR75-1 cells (available from
the American Type Culture Collection and from the European
Collection of Cell Cultures, deposit number ATCC CRL1500) grown in
RPMI plus 10% foetal calf serum were induced to express c-erbB2 by
4 day stimulation with tamoxifen at 7.5 pM (see Warri et al. (1996)
Eur. J. Cancer. 32A: 134-140). The cells were then harvested using
trypsin/EDTA and washed three times with PBS.
[0079] mRNA was extracted from the cell pellet using a Dynabead
mRNA purification kit according to the manufacturer's recommended
protocol. The mRNA was then used as a template for first strand
cDNA synthesis using the Pharmacia Ready-To-Go.TM. T primed first
strand cDNA synthesis kit. cDNA/mRNA was then blunt end ligated
into the EcoRV site of the PinPoint.TM. vector. The ligation
products were then transformed into Top 10 F E. coli cells
(Invitrogen) following the manufacturer's supplied protocol and the
transformed cells grown overnight on LB agar plates containing
ampicillin. Colonies of the transformed E. coli were copied onto
nitrocellulose filter and then grown for 2 hours on LB agar
containing ampicillin and IPTG (1 mM). The colonies on the
nitrocellulose filter were fixed and lysed (15 minutes in the
presence of chloroform vapour followed by 18 hours in 100 mM
Tris/HCL pH 7.8; 150 mM NaCl; 5 mM MgCl2; 1.5% BSA; 1 .mu.g/ml
DNase 1; 40 .mu.g/ml lysozyme).
[0080] Screening for colonies expressing anti-c-erbB2 reactive
protein was carried out as follows: [0081] 1. Wash nitrocellulose
filter three times in TNT (10 mM Tris/HCl pH 8; 150 mM NaCl; 0.05%
Tween 20) then block for 60 minutes in TNT+5% dried milk protein.
[0082] 2. Incubate nitrocellulose filter for 2 hours at room
temperature with mouse anti-c-erbB2 antibody (Ab-3 from Oncogene
Research Products, Calbiochem). [0083] 3. Wash the filter three
times in TNT then incubate overnight at 4.degree. C. with
anti-mouse HRP conjugate. [0084] 4. Wash filter three times in TNT,
twice in TN (10 mM Tris/HCl pH 8; 150 mM NaCl) then visualise
colonies expressing anti-c-erbB2 reactive protein using
chloronaphthol (6 mg chloronaphthol in TN+6 .mu.l 30%
H.sub.2O.sub.2). [0085] 5. After development (approximately 20
minutes treatment with chloronaphthol as described in step 4) wash
filter with water and allow to air dry.
[0086] Colonies identified as positive for c-erbB2 expression were
picked and grown up overnight in liquid culture of LB+ampicillin
and small amounts of plasmid DNA and protein were prepared from the
culture for analysis. Plasmids containing a c-erbB2 cDNA insert
were identified using restriction enzyme digestion and PCR using a
primer pair specific to the published c-erbB2 cDNA sequence,
described by Yazici, H. et al., (1996) Cancer Lett. 107: 235-239.
DNA sequence analysis could then be used to confirm 1) the presence
of a c-erbB2 insert and 2) that the reading frame of c-erbB2 is
maintained in the resultant biotinylated fusion protein. Protein
samples prepared from E. coli cultures carrying a plasmid with a
c-erbB2 insert were analysed by SDS-PAGE and western blotting to
ensure that the correct protein was being expressed.
Example 5
Detection of the Immune Response of Patients with Primary Breast
Cancer Using a Panel Assay
Methods:
(A) Preparation of Biotinylated Antigen
[0087] E. coli transformed with the appropriate PinPoint.TM.
plasmid expressing biotinylated antigen were grown in a 5 ml
overnight culture (LB+amp+biotin) and the overnight culture used to
inoculate a 150 ml culture. The 150 ml culture was grown to OD
0.4-0.6 then expression of the fusion protein was induced by the
addition of IPTG to a final concentration of 1 mM and the induced
culture incubated at 25.degree. C. The bacterial cells were
harvested by centrifugation and then lysed by gentle sonication in
a Tris/EDTA buffer containing the protease inhibitor PMSF. Cellular
debris was removed by centrifugation at .about.50,000 g and the
resultant particle-free supernatant assayed by avidin ELISA to
confirm the presence of biotinylated protein.
(B) c-erbB2/p53 Autoantibody Assay Method [0088] 1. Standard 96
well microtitre assay plates were coated with avidin, using 50
.mu.l of a 1 .mu.g/ml solution per well, and allowed to air dry
overnight. The plates were then washed once with PBS/Tween to
remove residual salt crystals, blocked for 60 minutes with a
solution of 2% (w/v) PVP (polyvinylpyrolidone 360) in PBS and
washed three times using PBS/Tween. [0089] 2. Particle free
supernatant containing the appropriate biotinylated antigen
(prepared as described in section (1) above) was plated out at 50
.mu.l per avidin-coated well and then incubated for 60 minutes at
room temperature with shaking to allow the biotin/avidin binding
reaction to take place. The plates were then washed four times with
PBS/Tween. [0090] 3. Serum samples to be tested for the presence of
autoantibodies (diluted 1/50 and 1/100 in PBS) were plated out in
triplicate (50 .mu.l per well) and then incubated for 60 minutes
with shaking to allow formation of any autoantibody/antigen
complexes. Plates were then washed four times with PBS/Tween to
remove unbound serum components. [0091] 4. 50 .mu.l of ARP
conjugated anti-human IgG/IgM antibody (obtained from Dako and used
at a dilution recommended by the manufacturer) was added to each
well and incubated for 60 minutes at room temperature with shaking.
The plates were then washed again four times with PBS/Tween. [0092]
5. 50 .mu.l of TMB was added to each well and measurements of OD at
650 nm for each well of the assay plate were taken kinetically over
a period of 10 minutes.
[0093] For each antigen, control assays were performed following
the procedure described above but using a sample of protein induced
from E. coli transformed with a control PinPoint.TM. vector
containing an out-offrame cDNA instead of the particle free
supernatant containing biotinylated antigen. As it will be apparent
to persons skilled in the art, the above methodology can be adapted
for use in the detection of autoantibodies of any specificity with
use of an appropriate biotinylated antigen.
(C) MUC1 Autoantibody Assay
[0094] 1. ABC MUC1 isolated from the serum of patients with
advanced breast cancer according to the method of Example 1 (all
three fractions pooled) was diluted appropriately in PBS, plated
out on a 96 well microtitre assay plate at 50 .mu.l per well and
left to dry overnight. The plate was then washed once with
PBS/Tween to remove residual salt crystals, blocked for 60 minutes
using a solution of 2% (w/v) PVP in PBS and washed three times with
PBS/Tween. [0095] 2. Serum samples to be tested for the presence of
autoantibodies (diluted 1/50 and 1/100 in PBS) were plated out in
triplicate, adding 50 .mu.l per well, and incubated for 60 minutes
at room temperature with shaking. The plate was then washed four
times with PBS/Tween. [0096] 3. 50 .mu.l of HRP conjugated
anti-human IgG/IgM antibody (obtained from Dako and used at a
dilution recommended by the manufacturer) was added to each well
and incubated for 60 minutes at room temperature with shaking. The
plates were then washed again four times with PBS/Tween. [0097] 4.
50 .mu.l of TMB was added to each well and measurements of OD at
650 nm for each well of the assay plate were taken kinetically over
a period of 10 minutes.
Results
[0097] [0098] Pre-operative blood samples taken from 21 patients
diagnosed with primary breast cancer were assayed for the presence
of autoantibodies against MUC1, p53 and c-erbB2. The results of
these assays are shown in FIG. 1 and summarised in Table 2.
TABLE-US-00003 [0098] TABLE 2 anti- anti- Predic- c- Predic- anti-
Predic- Sample p53 tion erbB2 tion MUC I tion Combined 1 + cancer -
normal + cancer CANCER 2 +/- ? +/-. ? +/- ? cancer 3 + cancer +/- ?
+ cancer CANCER 4 + cancer + cancer + cancer CANCER 5 + cancer +
cancer +/- ? CANCER 6 - normal + cancer +/- ? cancer 7 + cancer +
cancer + cancer CANCER 8 +/- ? + cancer +/- ? CANCER 9 + cancer +
cancer + cancer CANCER 10 + cancer + cancer - normal CANCER 11 +/-
? + cancer + cancer CANCER 12 - normal + cancer - normal cancer 13
+ cancer - normal + cancer CANCER 14 +/- ? + cancer + cancer CANCER
15 + cancer - normal + cancer CANCER 16 - normal - normal +/- ? ?
17 +/- ? - normal + cancer cancer 18 + cancer + cancer + cancer
CANCER 19 + cancer + cancer + cancer CANCER 20 + cancer - normal +
cancer CANCER 21 + cancer +/- ? - normal cancer
[0099] FIG. 1 shows the results of the assays for autoantibodies
specific to MUC1, c-erbB2 and p53. For each set of data the dotted
line represents the cut-off value for normality. For the purposes
of this study the normal control patients were women who clinically
and/or mammographically had no evidence of breast cancer at the
time of taking the serum sample. In order to establish the cut-off
value for normality, control assays were performed on a total of 30
normal patients. Values below the dotted line fall within the
normal control range and were scored as negative (-) in Table 2
whereas values above the dotted line were scored as positive (+).
Values which were difficult to score as negative or positive with a
reasonable degree of certainty were scored +/-. Patients scoring
positive in at least two of the assays were identified as strongly
positive for breast cancer (indicated "CANCER" in Table 2);
patients scoring positive in at least one of the assays were
identified as probable for breast cancer (indicated "cancer" in
Table 2).
[0100] The results presented illustrate the predictive value of the
three autoantibody assays both when used individually and when used
as a panel. The use of a single assay to predict breast cancer gave
approximately 40% of the results as a false negatives. However, by
combining the results from all three assays only one patient
appeared as a false negative (<5%), 71% of patients were scored
as strongly positive for breast cancer (i.e. positive in at least
two assays) and 23% of patients were scored as probable for breast
cancer (i.e. positive in at least one assay). The results also show
that a group of patients which have all been diagnosed with primary
breast cancer have different serological profiles in terms of the
immune response to their cancer. Thus, no single one of the three
autoantibody assays would be useful in all primary breast cancer
patients.
Example 6
Cloning of a Ras Antigen
Method
[0101] cDNA encoding a mutant oncogenic form of ras (designated
K-ras) was cloned from the cell line KNRK (Rat kidney, Kirsten MSV
transformed, see Aaronson, S. A. and Weaver, C. A. (1971) J. Gen.
Virol. 13: 245-252; ATCC accession number CRL 1569). mRNA was
extracted from the cell pellet using a Dynabead mRNA purification
kit according to the manufacturer's recommended protocol cDNA
synthesis, cloning into the EcoRV site of the PinPoint.TM. vector
and transformation of E. coli was carried out as described in
Example 4. Clones expressing ras were then identified by expression
screening using the anti-ras antibody F234-4.2 from Calbiochem.
Example 7
Cloning of c-myc
Method
[0102] cDNA encoding human c-myc was cloned from the breast cancer
cell line T47-D (European Collection of Animal Cell Cultures
accession number 85102201). mRNA was extracted from the cell pellet
using a Dynabead mRNA purification kit according to the
manufacturer's recommended protocol. cDNA synthesis, cloning into
the EcoRV site of the PinPoint.TM. vector and transformation of E.
coli was carried out as described in Example 4. Clones expressing
c-myc were then identified by expression screening using the
anti-cmyc antibody 4111.1 from Unilever.
Example 8
Assay for ras and c-myc Autoantibodies
[0103] Biotinylated c-myc and ras antigens were prepared from E.
coli transformed with the appropriate PinPoint.TM. plasmid vector
expressing biotinylated c-myc or biotinylated ras, as described in
Example (5), part (A). The assays for c-myc and ras autoantibodies
were then performed according to the protocol described in Example
(5), part (B).
Example 9
Method of Detecting Recurrent Disease in a Patient Previously
Diagnosed as Carrying Tumour Cells
[0104] A group of nine patients previously diagnosed with primary
breast cancer were selected. Pre-operative serum samples were taken
from each of these patients prior to surgery for the removal of the
primary breast cancer. Follow-up serum samples were then taken
postoperatively at 2 or 3 monthly intervals and during the same
period of time the patients were assessed clinically for signs of
recurrent disease. None of the patients received any post-operative
therapy until recurrence was diagnosed clinically. The preoperative
and post-operative serum samples from each of the patients were
assayed for the presence of autoantibodies to MUC1, c-erbB2 and
p53, using the assay methods described above under Example 5, and
also for the presence of the commonly used serum tumour marker
protein CA15-3. The results of these assays are summarised in Table
3 and results for three of the nine patients are presented
graphically in FIG. 3. Clinical signs of recurrent disease were
scored as follows:
LN recurrent disease in the lymph nodes LR local recurrence METS
distant metastases present
Results
[0105] In each of the patients at least one class of autoantibody
was observed to remain above normal level. This suggests continued
presence of the tumour marker (immunogen) and hence continued
presence of tumour. Serum levels of the tumour marker protein
CA15-3 were not found to be predictive of recurrent disease.
TABLE-US-00004 TABLE 3 Date of Sample CA Anti- Anti Anti first DFI
Patient date 15-3 p53 Prediction c-erbB2 Prediction MUCI Prediction
Predicted Recurrence recurrence (months) 0001 December 1988 11 - +
Cancer + Cancer CANCER - March 1987 12 - + Cancer + Cancer CANCER -
May 1987 13 - + Cancer + Cancer CANCER - August 1987 22 +/- ? +
Cancer + Cancer CANCER - November 1987 56 +/- ? + Cancer + Cancer
CANCER METS December 1987 79 +/- ? + Cancer + Cancer CANCER METS 11
0002 January 1987 16 - + Cancer +/- ? Cancer - May 1987 8 - +
Cancer +/- ? Cancer - August 1987 10 - + Cancer + Cancer CANCER -
November 1987 12 +/- ? + Cancer + Cancer CANCER - February 1988 16
- + Cancer + Cancer CANCER - February 23 1989 0003 February 1987 10
- + Cancer - Cancer - May 1987 7 + Cancer + Cancer - CANCER -
August 1987 8 + Cancer + Cancer - CANCER - November 1987 12 +
Cancer + Cancer - CANCER - February 1988 12 + Cancer + Cancer -
CANCER - May 1988 11 - + Cancer - Cancer - December 34 1989 0004
February 1987 8 + Cancer ++ Cancer - CANCER - April 1987 + Cancer +
Cancer - CANCER - June 1987 4 + Cancer + Cancer - CANCER - December
1987 0.4 + Cancer ++ Cancer - CANCER - March 1988 7 ++ Cancer ++
Cancer - CANCER - February 71 1993 0005 March 1987 16 +/- ? +
Cancer - Cancer - June 1987 13 +/- ? + Cancer - Cancer - September
1987 14 + Cancer + Cancer +/- ? CANCER - December 1987 17 +/- ? +
Cancer +/- ? CANCER - March 1988 16 - May 1988 LN 15 0006 May 1987
12 - + Cancer + Cancer CANCER - July 1987 15 - + Cancer + Cancer
CANCER - September 1987 9 +/- ? + Cancer +/- ? Cancer LR 4 November
1987 12 - + Cancer +/- ? Cancer - March 1988 15 - +/- ? - - May
1988 13 - +/- ? - - November 000& June 1987 26 + Cancer ++
Cancer - CANCER - August 1987 28 + Cancer + Cancer CANCER - October
1987 42 + Cancer + Cancer - CANCER - December 1987 105 + Cancer ++
Cancer + Cancer CANCER METS December 6 0008 June 1987 48 + Cancer +
Cancer + Cancer CANCER - August 1987 30 + Cancer + Cancer + Cancer
CANCER - October 1987 17 + Cancer + Cancer + Cancer CANCER -
January 1988 14 + Cancer + Cancer + Cancer CANCER - May 1988 22 +
Cancer + Cancer +/- ? CANCER LR May 1988 11 0009 May 1987 17 - +/-
? - - August 1987 17 - + Cancer - Cancer - November 1987 18 - +
Cancer - Cancer LR 6 January 1988 31 - + Cancer +/- ? Cancer METS
8
Example 10
Retrospective Analysis of a Well Characterised Series of Healthy
Controls and Patients with Early Breast Cancer
[0106] The above-described methods for detecting autoantibodies to
MUC1, p53, c-erbB2 and c-myc were used to carry out a retrospective
study on a large number of early (stage 1 and 2) breast cancer sera
as well as a large number of control serum samples from individuals
with no evidence of malignancy (control group). The serum samples
from patients were all taken within a 4 week pre-operative period.
At the same time, the serum samples were assayed for the presence
of circulating antigen (MUC1 and c-erbB2) using conventional tumour
marker kits (used normally in advanced disease only). This would
allow an assessment of whether the autoantibody assays are more
sensitive than the conventional antigen assays. As used herein, the
terms early or primary breast cancer means that the primary tumour
has a diameter of less than 5 cm. Stage 1 early breast cancer is
defined as lymph node negative; Stage 2 early breast cancer is
defined as lymph node positive.
[0107] In total, pre-operative serum samples from 200 patients
diagnosed with primary breast cancer and 100 normal control samples
were assayed for autoantibodies against MUC1, p53, c-erbB2 and
c-myc. The results are summarised in Tables 4-7 and FIGS. 4-7.
[0108] FIG. 4 depicts the range of autoantibody levels found for
each assay in normal individuals and patients with early breast
cancer. It is apparent that cancer patients have a considerably
higher level of circulating autoantibodies to these markers than do
normal individuals. Using the range for the normal individuals it
is possible to set a `cut-off` above which no normal values should
lie. Therefore, samples with autoantibody levels above this cut-off
can be deemed to be positive for cancer. Cut-off points determined
in this manner were used to score the results of the retrospective
study in early breast cancer patients.
[0109] The results presented in Tables 4-7 and FIGS. 5-7
demonstrate the predictive value of the four autoantibody assays
both individually and when used in combination as a panel of
assays. Table 4 indicates the increased sensitivity of combining
the results of a number of assays. By using one assay on its own,
less than 50% of cancers are detected, however the power of
detection increases as more assays are added to the panel until the
combination of all four assays allows 82% of primary cancers to be
detected. FIG. 7 shows the percentage of samples which are positive
in 0 out of 4 assays up to 4 out of 4 assays. This provides good
evidence that the panel assay is more powerful in the detection of
cancer than any one single marker assay since not all patients with
cancer have raised autoantibodies to all markers.
[0110] Tables 5-7 summarise the detection rates in stage 1, stage 2
and in early breast cancer (i.e. stage 1 and 2) for various
combinations of autoantibody assays. The use of a single
autoantibody assay to predict breast cancer gives approximately
60-70% of the results as false negatives in the stage 1 group; and
50-60% in stage 2. However, by combining the results from all four
assays, 76% of stage 1 and 89% of stage 2 cancers were positive in
one or more assay. The overall detection rate for early breast
cancer (i.e. both stage 1 and stage 2 cancers) using this system
was 82%. In both stage 1 and stage 2 cancer, assaying for
autoantibodies to MUC1 appeared to add predictive power to any
combination of assays.
[0111] The results for this study were obtained using a 100%
confidence limit, in other words for a result to be deemed positive
it had to fall above the cut-off for readings in the normal range.
This normal range was previously evaluated from a large number of
normal individuals and then confirmed using the control group of
100 normal individuals mentioned above. Therefore, within the
normal control group, none of the samples were found to be
positive, meaning that the sensitivity of the panel of autoantibody
assays was 100% for the detection of early breast cancer (FIG.
5).
[0112] FIGS. 6 and 7 demonstrate the detection rates which are
achievable if specificity is reduced from a 100% confidence level
(no false positives) to a 95% confidence level, where some degree
of false positive detection is expected. In this case, the cut-off
point is defined as the mean value plus twice the standard
deviation of the normal sample range. Using this cut-off point,
approximately 5% of the normal samples were determined to be
positive for cancer (i.e. false positives); whilst detection of
primary cancer increased to approximately 94% (i.e. 6% false
negatives). Again, the greatest percentage of the sample group were
positive in only 1 out of the 4 assays, however, the percentage of
samples that were positive in all 4 assays increased
considerably.
[0113] Since the above study was carried out retrospectively,
clinical data was available regarding the initial diagnosis as well
as clinical data regarding the post-operative outcome (i.e.
follow-up data). This allowed analysis of the prognostic value of
the data obtained from the autoantibody assays. Table B shows the
correlations between serum levels of autoantibodies to MUC1, p53,
c-erbB2 and c-myc and a number of clinical factors. For instance,
the presence of autoantibodies to any of the 4 tumour associated
proteins (MUC1, p53, c-erbB2 or c-myc) appears to correlate with
the development of a recurrence. In other words, those patients who
had autoantibodies were more likely to go on to develop a
recurrence of their disease. In the case of autoantibodies to MUC1,
c-myc and c-erbB2, this was most likely to be distant metastases,
only autoantibodies to p53 were not associated with the later
development of distant metastases with any statistical
significance. In fact, the presence of autoantibodies to p53 was
the weakest indicator of a later recurrence of disease;
furthermore, p53 autoantibodies correlated with disease free
interval.
[0114] Table 9 presents an analysis of whether the degree of
autoantibody positivity may be of value in the prediction of which
stage 1 tumour will go on to develop a recurrence. At the present
time, there is little to indicate at the time of diagnosis whether
a patient with a stage 1 tumour (i.e. no evidence of spread of
tumour to the lymphatic system) will go on to develop recurrent
disease. As can be seen in Table 9, of those patients with stage 1
tumours from the sample group that went on to develop recurrent
disease, 71% were positive in two or more autoantibody assays. Of
the patients with stage 1 tumours that have not yet recurred, only
30% were positive in two or more autoantibody assays.
TABLE-US-00005 TABLE 4 Sensitivity of autoantibody assays in the
detection of early breast cancer. % PBC positive Single marker
assay 35-47 Two marker assay 51-60 Three marker assay 63-76 Four
marker assay 82
TABLE-US-00006 TABLE 5 Sensitivity of autoantibody panel assays in
the detection of stage 1 breast cancer. p53 c-erbB2 c-myc MUC1 p53
38 48 58 59 c-erbB2 31 50 51 c-myc 41 55 MUC1 38 p53/c-erbB2 61 66
p53/c-myc 73 c-erbB2/c-myc 65 p53/c-erbB2/c-myc 76
TABLE-US-00007 TABLE 6 Sensitivity of autoantibody panel assays in
the detection of stage 2 breast cancer. p53 c-erbB2 c-myc MUC1 p53
40 56 55 73 c-erbB2 42 56 73 c-myc 33 69 MUC1 56 p53/c-erbB2 65 84
p53/c-myc 80 c-erbB2/c-myc 84 p53/c-erbB2/c-myc 89
TABLE-US-00008 TABLE 7 Sensitivity of autoantibody panel assays in
the detection of primary breast cancer. p53 c-erbB2 c-myc MUC1 p53
38 51 57 64 c-erbB2 35 53 59 c-myc 37 60 MUC1 47 p53/c-erbB2 63 73
p53/c-myc 76 c-erbB2/c-myc 72 p53/c-erbB2/c-myc 82
TABLE-US-00009 TABLE 8 Correlations between serum autoantibody
level and various clinical factors. FACTOR MUC1 p53 c-erbB2 c-myc
recurrence 1 4 local recurrence 1 2 1 2 1 2 1 4 distant metastases
X stage X X X X grade X X X X family history X X X X disease free
interval X X X age X X X X menopausal status X X X X Key: Good
correlation 1 2 Moderate correlation 1 4 Weak correlation X No
correlation
TABLE-US-00010 TABLE 9 Analysis of the degree of positivity in
autoantibody assays for recurrent and non-recurrent stage 1 breast
cancer tumours. Negative-no +ve auto- +ve auto- autoantibodies
antibodies to antibodies to detected one marker 2-4 markers
Recurrent 12% 17% 71% Non-recurrent 22% 48% 30%
Example 11
Detection of Autoantibodies in Sequential Serum Samples-Application
to the Monitoring of Disease Progression
[0115] This study was carried out in order to assess whether
autoantibody assays could be useful in the earlier detection of
recurrent disease.
[0116] Levels of autoantibodies to MUC1, p53 and c-erbB2 in the
serum of patients previously diagnosed with breast cancer were
measured sequentially during follow-up until the patient manifested
recurrent disease. The results are summarised in FIGS. 8-10. All
three patients went on to develop recurrent disease. In all three
patients, autoantibody levels were indicative of the presence of
cancer. However, there is no evidence from this group that
autoantibody levels decrease after removal of the primary tumour.
FIG. 10 shows the levels of autoantibodies post-operatively of a
patient with non-recurrent disease and a patient with recurrent
disease. Autoantibody levels in the patient with non-recurrent
disease remained below the cut-off point during the period of
sample collection (48 months). In the second patient, whose disease
recurred at 36 months, autoantibody levels are seen to be steadily
rising towards the cut-off point, with c-erbB2 autoantibodies
rising above cut-off. Furthermore, as can be seen in FIG. 9, when
further sequential samples are added to the analysis, 3 out of the
4 assays become positive for cancer and these levels then decrease
again once treatment of the recurrence is underway. This data
supports the utility of autoantibody assays in the earlier
detection of recurrent disease.
Example 12
Analysis of a Series of Patients with Bladder Cancer and Benign
Urological Disorders
[0117] Serum samples were collected from a group of 80 patients
with bladder cancer/benign urological disorders and analysed for
the presence of autoantibodies to MUC1, p53, c-erbB2 and c-myc
using the assay methods described above.
[0118] The data summarised in Table 10 shows that single assay
sensitivities for bladder cancer detection range from 15-500 (as
opposed to 35-47% for breast cancer). The detection sensitivity
using all 4 assays was 80%, similar to that found for early breast
cancer.
[0119] FIG. 11 shows the break down of detection rates between
urologically benign disorders (`benign`) and the three stages of
bladder cancer. Upon further investigation of the relevant clinical
data it became apparent that 6 of the patients in the `benign`
group had evidence of other malignancies. These other malignancies
were lung cancer, skin cancer and adenocarcinoma. Evidence of other
malignancies were: pleural effusion, ovarian cysts and colon
polyps. Serum samples from all 6 of these patients had been scored
as positive for cancer using the panel of autoantibody assays,
illustrating the general application of the panel assay to the
detection of cancers. Furthermore, it is known that some patients
with stage PT1/2 and PT3/4 disease had previously received systemic
therapy.
TABLE-US-00011 TABLE 10 Sensitivity of autoantibody assays in the
detection of bladder cancer. % positive Single marker assay 15-50
Two marker assay 28-73 Three marker assay 46-76 Four marker assay
80
TABLE-US-00012 TABLE 11 Sensitivity of autoantibody panel assays in
the detection of bladder cancer. p53 c-erbB2 c-myc MUC1 p53 50 73
73 73 c-erbB2 17 28 36 c-myc 15 35 MUC1 24 p53/c-erbB2 76 76
p53/c-myc 75 c-erbB2/c-myc 46 p53/c-erbB2/c-myc 80
Example 13
Sensitivity of Autoantibody Assay in Diagnosis of Colorectal
Cancer
[0120] An autoantibody assay as previously described was carried
out on serum samples from patients with colorectal cancer using the
tumour antigens c-myc, p53, c-erbB2 and K-ras individually and as a
panel. The results are shown in FIGS. 12 and 13. As has been
demonstrated previously increased sensitivity is shown when a panel
of antigens is used.
Example 14
Use of BRCA1 in Panel Assay for Detection of Breast Cancer
[0121] A BRCA1 antigen suitable for use in the detection of
anti-BRCA1 autoantibodies was cloned from the breast cancer cell
line MCF7 using an RT-PCR strategy. Briefly, mRNA isolated from
MCF7 cells was reverse transcribed to give first-strand cDNA. These
cDNA was used as a template for PCR using a primer pair designed to
amplify a product covering the first 1500 base pairs of the BRCA1
cDNA but including a known mis-match mutation that leads to an
early stop codon and therefore the production of truncated protein.
Different sites for restriction enzyme digestion were also
incorporated into the forward and reverse PCR primers to facilitate
the cloning of the PCR product. The PCR primers were as
follows:
TABLE-US-00013 (SEQ. ID. NO. 1) 5'-GAC AGG ATC CGG ATG GAT TTA TCT
GCT CTT CGC GTT G (SEQ. ID. NO. 2) 5'-GCG GCC GCC CTC ATG TAG GTC
TCC TTT TAC GC
[0122] The PCR product obtained using these primers was then cloned
into the PinPoint.TM. vector and used to transform E. coli Top 10 F
cells, as described hereinbefore. Clones expressing the fusion
protein of truncated BRCA1 antigen fused in-frame to the N-terminal
biotinylation domain were then identified by expression screening,
according to the procedure described in Example 4, using the
antibody MAB4132 from Chemicon.
[0123] Biotinylated truncated BRCA1 antigen is then prepared from
E. coli transformed with the appropriate PinPoint.TM.plasmid vector
expressing the fusion protein, as described in Example (5), part
(A). The assay for BRCA1 autoantibodies is then performed according
to the protocol described in Example (5), part (B).
[0124] FIG. 14 shows the results of a study in which the
above-described assays for autoantibodies to cmyc, p53, c-erbB2,
MUC1 and BRCA1 were performed individually, as a panel and as a
panel without BRCA1 to detect autoantibodies in samples of serum
taken from normal individuals, patients diagnosed with primary
breast cancer and BRCA1 mutation carriers. As demonstrated
previously, increased sensitivity is shown when a panel of markers
is used.
Example 15
Use of Autoantibody Panel Assay for Detecting Prostate Cancer,
Incorporating PSA
[0125] cDNA encoding human PSA was cloned from the cell line T47-D
using a protocol similar to that described above for the cloning of
c-erbB2. Briefly, the T47-D cells were first stimulated with
Apigenin at 10-5 M as described by Rosenberg et al. (1998) Biochem
Biophys Res Commun. 248: 935-939. mRNA was then extracted and cDNA
synthesis, ligation into PinPoint.TM. and transformation of E.
coli. performed as described in Example 4. Clones expressing PSA
were identified using an anti-PSA antibody. Biotinylated PSA
antigen was prepared from E. coli transformed with the PinPoint.TM.
vector expressing biotinylated PSA according to the protocol
described in Example (5), part (A). The assay for PSA
autoantibodies was then performed according to the protocol
described in Example (5), part (B).
[0126] An autoantibody assay using the methods described above was
carried out on patients with prostate cancer using c-myc, p53,
c-erbB2, PSA and MUC 1 individually and as a panel. The results are
shown in FIG. 15 and confirm the increased sensitivity of such a
panel for detection of prostate cancer.
Example 16
Other Tumour Marker Antigens
[0127] CAl25 can be affinity purified from the ovarian cancer cell
line OVRCAR-3 (available from the ATCC) using Mab VK-8, as
described by Lloyd, K. O. et al. (1997) Int. J. Cancer. 71:
842-850.
[0128] APC protein is expressed by the colorectal cancer cell line
SW480 (available from the ATCC) as described by Munemitsu, S. et
al. (1995) PNAS 92: 3046-3050.
Sequence CWU 1
1
2137DNAArtificial SequenceSynthetic 1gacaggatcc ggatggattt
atctgctctt cgcgttg 37232DNAArtificial SequenceSynthetic 2gcggccgccc
tcatgtaggt ctccttttac gc 32
* * * * *