US20130090251A1 - Tumour Markers - Google Patents
Tumour Markers Download PDFInfo
- Publication number
- US20130090251A1 US20130090251A1 US13/438,344 US201213438344A US2013090251A1 US 20130090251 A1 US20130090251 A1 US 20130090251A1 US 201213438344 A US201213438344 A US 201213438344A US 2013090251 A1 US2013090251 A1 US 2013090251A1
- Authority
- US
- United States
- Prior art keywords
- cancer
- autoantibodies
- tumour marker
- tumour
- patients
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/811—Test for named disease, body condition or organ function
- Y10S436/813—Cancer
Definitions
- 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.
- 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.
- 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.
- 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)
- CCA carcinoembryonic antigen
- 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.
- the present inventors have sought to identify markers which do not depend on tumour bulk per
- tumour marker protein 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.
- 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.
- 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.
- 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 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).
- tumour markers 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.
- 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:
- 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.
- 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.
- 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.
- 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.
- 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.
- HRP horseradish peroxidase
- 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.
- antigenic fragment as used herein means a fragment which is capable of eliciting an immune response.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 PinPointTM 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.
- 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.
- 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
- 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.
- 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.
- 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.
- 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.
- p53 or c-erbB2 are not necessarily essential.
- suitable panels could be selected from the following:
- colorectal cancer suitable panels could be selected from the following:
- suitable panels could be selected from the following:
- suitable panels could be selected from the following:
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- MUC1 isolated from normal individuals 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.
- ABC MUC1 MUC1 protein isolated from serum of patients with advanced breast cancer
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the presence of anti-MUC1 autoantibodies in the patient's serum after treatment may be indicative of recurrence of disease.
- kits suitable for performing the methods for the detection of autoantibodies described herein.
- 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.
- 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
- Panel C anti-MUC1 autoantibodies.
- the dotted line represents the cut-off value for normality.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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.
- 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.
- 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.
- 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).
- established tumour markers reflecting tumour bulk e.g. CA15-3 and CEA
- 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).
- 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.
- 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.
- 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.
- 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.
- 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.
- ABC MUC1 was purified from pooled sera taken from 20 patients with advanced breast cancer using immunoaffinity chromatography as follows:
- 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° C. with rolling. The beads were then packed by centrifugation and the supernatant removed.
- PBS phosphate buffered saline
- 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° 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° 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° 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).
- 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:
- cDNA for p53 E. coli clone pBH53, deposited in the American Type Culture Collection under accession number 79110
- the PinPointTM 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 PinPointTM vectors are described in detail in the Promega Protocols and Applications Guide obtainable from Promega Corporation, Madison Wis., USA.
- Fusion proteins expressed from the PinPointTM 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.
- 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.
- 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.
- 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-GoTM T primed first strand cDNA synthesis kit. cDNA/mRNA was then blunt end ligated into the EcoRV site of the PinPointTM 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 ⁇ g/ml DNase 1; 40 ⁇ g/ml lysozyme).
- 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.
- E. coli transformed with the appropriate PinPointTM 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° 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 ⁇ 50,000 g and the resultant particle-free supernatant assayed by avidin ELISA to confirm the presence of biotinylated protein.
- control assays were performed following the procedure described above but using a sample of protein induced from E. coli transformed with a control PinPointTM vector containing an out-offrame cDNA instead of the particle free supernatant containing biotinylated antigen.
- control assays were performed following the procedure described above but using a sample of protein induced from E. coli transformed with a control PinPointTM vector containing an out-offrame cDNA instead of the particle free supernatant containing biotinylated antigen.
- FIG. 1 shows the results of the assays for autoantibodies specific to MUC1, c-erbB2 and p53.
- the dotted line represents the cut-off value for normality.
- 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.
- 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 +/ ⁇ .
- 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.
- 71% of patients were scored as strongly positive for breast cancer i.e. positive in at least two assays
- 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.
- 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 PinPointTM 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.
- 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 PinPointTM 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.
- Biotinylated c-myc and ras antigens were prepared from E. coli transformed with the appropriate PinPointTM 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).
- 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. Following-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:
- tumour marker immunoglobulin
- Serum levels of the tumour marker protein CA15-3 were not found to be predictive of recurrent disease.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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%.
- assaying for autoantibodies to MUC1 appeared to add predictive power to any combination of assays.
- 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.
- 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.
- 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.
- 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.
- FIGS. 8-10 show 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).
- 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.
- 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.
- FIG. 11 shows the break down of detection rates between urologically benign disorders (‘benign’) and the three stages of bladder cancer.
- ‘benign’ urologically benign disorders
- 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.
- stage PT1/2 and PT3/4 disease had previously received systemic therapy.
- 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:
- Biotinylated truncated BRCA1 antigen is then prepared from E. coli transformed with the appropriate PinPointTMplasmid 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).
- 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.
- 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 PinPointTM 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 PinPointTM 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).
- 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.
- 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.
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.
Description
- 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. §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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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.
- 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:
-
- (a) contacting a sample of bodily fluids from said mammal with a panel of two or more distinct tumour marker antigens;
- (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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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™ 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.
- 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.
- 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.
- 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 includeMUC 1 and/or c-myc, for colorectal cancer ras and/or APC, for prostate cancer PSA and/orBRCA 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: -
- p53 and
MUC 1 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-erB2 and/or
MUC 1 and/or c-myc and/or BRCA2 and/or PSA; - p53 and BRCA2 with optional c-erbB2 and/or
MUC 1 and/or c-myc and/or BRCA1 and/or PSA; - c-erbB2 and
MUC 1 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
MUC 1 and/or c-myc and/or BRCA2 and/or PSA; - c-erbB2 and BRCA2 with optional p53 and/or
MUC 1 and/or c-myc and/or BRCA1 and/or PSA;
- p53 and
- In the case of colorectal cancer suitable panels could be selected 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
- In the case of prostate cancer suitable panels could be selected from the following:
-
- p53 and PSA with optional BRCA1 and/or c-erbB2; c-erbB2 and PSA with optional p53 and/or BRCA1.
- In the case of ovarian cancer suitable panels could be selected from the following:
-
- p53 and CAl25 with optional c-erbB2 and/or BRCA1; c-erbB2 and CAl25 with optional p53 and/or BRCA1.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- The contents of all documents, articles and references cited herein are incorporated herein by reference.
- The present invention will be further understood with reference to the following Examples and the accompanying Figures in which:
-
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. -
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. -
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. -
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). -
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 byStage 1—i.e. lymph node negative andStage 2—i.e. lymph node positive and patients with metastatic cancer at 100% confidence. -
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 byStage 1—i.e. lymph node negative andStage 2—i.e. lymph node positive and patients with metastatic cancer at 95% confidence. -
FIG. 7 : shows the sensitivity for primary breast cancer in an analysis of autoantibody levels in a series of healthy controls and patients withStage 1 orStage 2 primary breast cancer at 95% confidence. -
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. -
FIG. 9 : shows the autoantibody levels in further samples from the second patient inFIG. 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). -
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). -
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. - * 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.
- ** Other malignancies were:—lung cancer, skin cancer, adenocarcinoma of unknown primary. Evidence of other neoplasia consisted of:—pleural effusion, ovarian cysts, colon polyps.
-
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. -
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. -
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. -
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. - ABC MUC1 was purified from pooled sera taken from 20 patients with advanced breast cancer using immunoaffinity chromatography as follows:
- 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° 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° 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° 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° 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). - 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:
-
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 - Normal urinary MUC1 is available from Dr M. R. Price, Cancer Research Laboratories, The University of Nottingham, University Park, Nottingham. NG7 2RD, United Kingdom.
- 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 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 × 10−7 1.7 × 10−9 115D8 1 × 10−8 3.38 × 10−8 C595 2.4 × 10−8 2.5 × 10−8 - 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™ plasmid vector (Promega Corporation, Madison Wis., USA) using standard molecular biology techniques. The PinPoint™ 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™ vectors are described in detail in the Promega Protocols and Applications Guide obtainable from Promega Corporation, Madison Wis., USA.
- Fusion proteins expressed from the PinPoint™ 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.
- 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.
- 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.
- 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™ T primed first strand cDNA synthesis kit. cDNA/mRNA was then blunt end ligated into the EcoRV site of the PinPoint™ 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 μg/
ml DNase 1; 40 μg/ml lysozyme). - Screening for colonies expressing anti-c-erbB2 reactive protein was carried out as follows:
-
- 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. - 2. Incubate nitrocellulose filter for 2 hours at room temperature with mouse anti-c-erbB2 antibody (Ab-3 from Oncogene Research Products, Calbiochem).
- 3. Wash the filter three times in TNT then incubate overnight at 4° C. with anti-mouse HRP conjugate.
- 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 μl 30% H2O2). - 5. After development (approximately 20 minutes treatment with chloronaphthol as described in step 4) wash filter with water and allow to air dry.
- 1. Wash nitrocellulose filter three times in TNT (10 mM Tris/
- 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.
- E. coli transformed with the appropriate PinPoint™ 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° 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 ˜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
-
- 1. Standard 96 well microtitre assay plates were coated with avidin, using 50 μl of a 1 μg/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.
- 2. Particle free supernatant containing the appropriate biotinylated antigen (prepared as described in section (1) above) was plated out at 50 μl 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.
- 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 μl 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.
- 4. 50 μl 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.
- 5. 50 μl 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.
- 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™ 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.
-
-
- 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 μl 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.
- 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 μl per well, and incubated for 60 minutes at room temperature with shaking. The plate was then washed four times with PBS/Tween.
- 3. 50 μl 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.
- 4. 50 μl 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.
-
-
- 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.
- 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
-
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 -
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). - 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.
- 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™ 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.
- 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™ 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.
- Biotinylated c-myc and ras antigens were prepared from E. coli transformed with the appropriate PinPoint™ 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).
- 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 - 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 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 - 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. - 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 . -
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. - 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. - 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 thestage 1 group; and 50-60% instage 2. However, by combining the results from all four assays, 76% ofstage stage 2 cancers were positive in one or more assay. The overall detection rate for early breast cancer (i.e. bothstage 1 andstage 2 cancers) using this system was 82%. In bothstage 1 andstage 2 cancer, assaying for autoantibodies to MUC1 appeared to add predictive power to any combination of assays. - 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 ). -
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. - 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.
- 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 astage 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 withstage 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 withstage 1 tumours that have not yet recurred, only 30% were positive in two or more autoantibody assays. -
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 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 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 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 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 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% - This study was carried out in order to assess whether autoantibody assays could be useful in the earlier detection of recurrent disease.
- 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 inFIG. 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. - 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.
- 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.
-
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 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 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 - 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. - 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:
-
(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 - The PCR product obtained using these primers was then cloned into the PinPoint™ 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.
- Biotinylated truncated BRCA1 antigen is then prepared from E. coli transformed with the appropriate PinPoint™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).
-
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. - 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™ 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™ 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).
- 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 inFIG. 15 and confirm the increased sensitivity of such a panel for detection of prostate cancer. - 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.
- 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.
Claims (7)
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)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/438,344 US20130090251A1 (en) | 1998-05-11 | 2012-04-03 | Tumour Markers |
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9810040.7A GB9810040D0 (en) | 1998-05-11 | 1998-05-11 | Blood borne tumour markers |
GB9810040.7 | 1998-05-11 | ||
PCT/GB1999/001479 WO1999058978A2 (en) | 1998-05-11 | 1999-05-11 | Tumour markers |
US70009201A | 2001-05-16 | 2001-05-16 | |
US11/953,237 US8114604B2 (en) | 1998-05-11 | 2007-12-10 | Tumour markers |
US13/349,348 US9696319B2 (en) | 1998-05-11 | 2012-01-12 | Tumour markers |
US13/438,344 US20130090251A1 (en) | 1998-05-11 | 2012-04-03 | Tumour Markers |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/349,348 Continuation US9696319B2 (en) | 1998-05-11 | 2012-01-12 | Tumour markers |
Publications (1)
Publication Number | Publication Date |
---|---|
US20130090251A1 true US20130090251A1 (en) | 2013-04-11 |
Family
ID=10831824
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/700,092 Expired - Fee Related US7402403B1 (en) | 1998-05-11 | 1999-05-11 | Tumour markers |
US11/953,237 Expired - Fee Related US8114604B2 (en) | 1998-05-11 | 2007-12-10 | Tumour markers |
US13/349,348 Expired - Fee Related US9696319B2 (en) | 1998-05-11 | 2012-01-12 | Tumour markers |
US13/438,344 Abandoned US20130090251A1 (en) | 1998-05-11 | 2012-04-03 | Tumour Markers |
Family Applications Before (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/700,092 Expired - Fee Related US7402403B1 (en) | 1998-05-11 | 1999-05-11 | Tumour markers |
US11/953,237 Expired - Fee Related US8114604B2 (en) | 1998-05-11 | 2007-12-10 | Tumour markers |
US13/349,348 Expired - Fee Related US9696319B2 (en) | 1998-05-11 | 2012-01-12 | Tumour markers |
Country Status (12)
Country | Link |
---|---|
US (4) | US7402403B1 (en) |
EP (3) | EP1710253A3 (en) |
AT (1) | ATE314651T1 (en) |
AU (1) | AU3838899A (en) |
CY (1) | CY1115366T1 (en) |
DE (1) | DE69929185T2 (en) |
DK (2) | DK1078264T3 (en) |
ES (2) | ES2443096T3 (en) |
GB (1) | GB9810040D0 (en) |
PT (2) | PT1078264E (en) |
SI (1) | SI1078264T1 (en) |
WO (1) | WO1999058978A2 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060094069A1 (en) * | 2002-11-14 | 2006-05-04 | Robertson John Forsyth R | Tumour marker proteins and uses thereof |
US20080108084A1 (en) * | 1998-12-10 | 2008-05-08 | University Of Nottingham | Cancer Detection Methods and Reagents |
US20080213921A1 (en) * | 2006-09-13 | 2008-09-04 | Robertson John F R | Immunoassay Methods |
US20080305476A1 (en) * | 2005-05-27 | 2008-12-11 | Onc-Immune Ltd. | Immunoassay Methods |
US20090176319A1 (en) * | 2007-12-24 | 2009-07-09 | Onclmmune Limited | Calibrator For Immunoassays |
US20110045508A1 (en) * | 2008-01-31 | 2011-02-24 | The Brigham And Women's Hospital, Inc. | Urinary CA125 Peptides as Biomarkers of Ovarian Cancer |
US20110086061A1 (en) * | 2000-06-14 | 2011-04-14 | Onclmmune Limited | Cancer Detection Methods and Reagents |
US9696319B2 (en) | 1998-05-11 | 2017-07-04 | Oncimmune Ltd. | Tumour markers |
US9714938B2 (en) | 2005-05-27 | 2017-07-25 | Oncimmune Ltd. | Immunoassay methods |
Families Citing this family (33)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040072264A1 (en) * | 2000-12-22 | 2004-04-15 | Morris David W. | Novel compositions and methods for cancer |
BR0207290A (en) * | 2001-02-16 | 2005-06-07 | Immunivest Corp | Method for analyzing a patient for the presence of a malignancy, malignant cell isolated from cultured cells, tumor vaccine, molecule associated with altered tumor diathesis, methods for determining changes in molecules associated with tumor diathesis as a means to predict the efficacy of therapy, as a means for analyzing appropriate dosage for therapy, as a means for monitoring therapy effectiveness, as a means for analyzing cancer progression, for performing a whole body biopsy on a patient, and for identifying changes in a tumor cell circulating for cells present in an in situ tumor mass, and test kit for analyzing a patient sample for the presence of a non-hematopoietic malignant cell |
US7214498B2 (en) | 2001-03-23 | 2007-05-08 | Benaroya Research Institute At Virginia Mason | Tumor associated antigens and methods of using the same |
US7270960B2 (en) * | 2001-08-29 | 2007-09-18 | Pacific Northwest Research Institute | Diagnosis of ovarian carcinomas |
US20050221305A1 (en) * | 2001-11-09 | 2005-10-06 | Benaroya Research Institute At Virginia Mason | Antigen panels and methods of using the same |
AU2003266236A1 (en) * | 2002-07-22 | 2004-02-09 | Nemod Immuntherapie Ag | Method for the production of an immunostimulating mucin (muc1) |
JP4628418B2 (en) * | 2004-03-08 | 2011-02-09 | アメリカ合衆国 | Autoantibody detection for cancer diagnosis |
US7858323B2 (en) | 2004-06-09 | 2010-12-28 | The Regents Of The University Of Michigan | Phage microarray profiling of the humoral response to disease |
SE0402536D0 (en) * | 2004-10-20 | 2004-10-20 | Therim Diagnostica Ab | Immunoregulation in cancer, chronic inflammatory and autoimmune diseases |
WO2006132587A1 (en) * | 2005-06-10 | 2006-12-14 | Forskarpatent I Syd Ab | Tumour marker in brca2 associated breast cancer |
CA2632190A1 (en) * | 2005-11-10 | 2007-07-12 | University Of Kentucky | Lung cancer diagnostic assay |
JP2009541259A (en) * | 2006-06-20 | 2009-11-26 | ブラッコ・イメージング・ソシエタ・ペル・アチオニ | Method for producing specific antibody against saccharide antigen |
KR20090034961A (en) * | 2006-07-08 | 2009-04-08 | 유니버시티 오브 켄터키 리서치 파운데이션 | Lung cancer diagnostic assay |
JP5495159B2 (en) * | 2006-09-13 | 2014-05-21 | オンクイミューン リミテッド | Improved immunoassay method |
GB2441824A (en) * | 2006-09-13 | 2008-03-19 | Oncimmune Ltd | Immunoassay method |
US8741581B2 (en) | 2009-04-27 | 2014-06-03 | Technion Research And Development Foundation Ltd. | Markers for cancer detection |
EP2446272A2 (en) | 2009-06-23 | 2012-05-02 | Eventus Diagnostics Israel Ltd. | A method and system for the detection of cancer |
US20130189707A1 (en) * | 2010-01-26 | 2013-07-25 | Svetlana Alexandrovna Sergeeva | Method of determination of autoantibody level by means of enzyme immunoassay |
US20110237461A1 (en) * | 2010-03-17 | 2011-09-29 | The Regents Of The University Of Michigan | Using phage epitopes to profile the immune response |
WO2011120015A2 (en) * | 2010-03-26 | 2011-09-29 | Armune Biosciences, Inc. | Method and system of particle-coupled phage epitope |
US9141756B1 (en) | 2010-07-20 | 2015-09-22 | University Of Southern California | Multi-scale complex systems transdisciplinary analysis of response to therapy |
CA2806381C (en) | 2010-08-13 | 2019-11-05 | Joshua Labaer | Biomarkers for the early detection of breast cancer |
US20130040839A1 (en) * | 2011-06-27 | 2013-02-14 | Ambergen, Inc. | Method for Diagnosing or Determining the Prognosis of Colorectal Cancer (CRC) Using Novel Autoantigens: Gene Expression Guided Autoantigen Discovery |
WO2013023162A2 (en) * | 2011-08-10 | 2013-02-14 | Genus Oncology, Llc | Anti-muc1 antibodies for cancer diagnostics |
US11066508B2 (en) | 2014-08-18 | 2021-07-20 | Basf Se | Polyester-modified polybutadienols for producing polyurethane elastomers and thermoplastic polyurethanes |
JP2018517892A (en) | 2015-04-10 | 2018-07-05 | アプライド プロテオミクス,インク. | Protein biomarker panel to detect colorectal cancer and advanced adenoma |
WO2018191645A1 (en) * | 2017-04-14 | 2018-10-18 | The Regents Of The University Of California | Methods and devices for performing autoantibody assays |
CN109448790B (en) * | 2018-11-19 | 2021-11-09 | 上海科技大学 | Storage medium, equipment and device for determining tumor immunotherapy marker |
GB2600701A (en) | 2020-11-04 | 2022-05-11 | Oncimmune Ltd | Antibody assay |
WO2022013321A2 (en) | 2020-07-14 | 2022-01-20 | Oncimmune Limited | Antibody assay |
CN112345755B (en) * | 2020-09-23 | 2023-06-16 | 杭州凯保罗生物科技有限公司 | Biomarker for breast cancer and application thereof |
WO2023091714A1 (en) * | 2021-11-18 | 2023-05-25 | Greenwich Lifesciences, Inc. | Methods for assessing an immune response and predicting cancer recurrence and assessment of current and prior cancer status |
GB2622246A (en) | 2022-09-08 | 2024-03-13 | Oncimmune Germany Gmbh | Antibody assay |
Family Cites Families (59)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4241044A (en) | 1978-12-28 | 1980-12-23 | Abbott Laboratories | Method of diagnosing cancer by detecting elevated anti-T antibody activity |
NZ211453A (en) * | 1984-03-22 | 1989-01-06 | Biotechnology Research Enterpr | Aryl azides, their preparation and use in the detection, localisation and isolation of polynucleotides |
EP0236606B1 (en) | 1986-03-10 | 1992-06-24 | Imre Corporation | Immune complex assay |
WO1989001153A1 (en) | 1987-07-24 | 1989-02-09 | Xoma Corporation | Methods for breast cancer detection |
US4937185A (en) | 1987-07-31 | 1990-06-26 | The Ohio State University Research Foundation | Detection of circulating antibodies to a cancer marker protein |
US7282345B1 (en) | 1989-08-04 | 2007-10-16 | Schering Ag | C-erbB-2 external domain: GP75 |
US5110911A (en) | 1989-11-02 | 1992-05-05 | Biomira, Inc. | Human tumor-associated thomsen-friedenreich antigen |
US5157020A (en) | 1990-05-24 | 1992-10-20 | Research Corporation Tech., Inc. | Synthetic senescent cell antigen |
DK9091D0 (en) | 1991-01-18 | 1991-01-18 | Novo Nordisk As | PROCEDURE FOR MANUFACTURING ANTIBODIES |
DE4120412C1 (en) | 1991-06-20 | 1993-01-07 | Henning Berlin Gmbh Chemie- Und Pharmawerk, 1000 Berlin, De | |
US6280962B1 (en) | 1991-11-25 | 2001-08-28 | Yoreh Biotechnologies Ltd. | Whole blood/mitogen assay for the early detection of a subject with cancer and kit |
US6322989B1 (en) * | 1991-11-25 | 2001-11-27 | Yoreh Biotechnologies, Ltd. | Whole blood/mitogen assay for the early detection of a subject with ovarian or breast cancer and kit |
US5885793A (en) | 1991-12-02 | 1999-03-23 | Medical Research Council | Production of anti-self antibodies from antibody segment repertoires and displayed on phage |
CA2118015A1 (en) | 1992-04-14 | 1993-10-28 | Jeffrey R. Marks | Method of detecting tumors containing complexes of p53 and hsp70 |
US5801005A (en) * | 1993-03-17 | 1998-09-01 | University Of Washington | Immune reactivity to HER-2/neu protein for diagnosis of malignancies in which the HER-2/neu oncogene is associated |
US5763164A (en) * | 1993-04-16 | 1998-06-09 | Northwestern University | Immunogenic cancer proteins and peptides and methods of use |
US5747268A (en) * | 1993-04-22 | 1998-05-05 | Dade International Inc. | Tumor marker control |
US5744144A (en) | 1993-07-30 | 1998-04-28 | University Of Pittsburgh University Patent Committee Policy And Procedures | Synthetic multiple tandem repeat mucin and mucin-like peptides, and uses thereof |
US5721105A (en) | 1994-02-20 | 1998-02-24 | B.R.A.H.M.S. Diagnostica Gmbh | Method for the immunological determination of proteins and kit for carrying out the method |
JPH07294530A (en) | 1994-04-20 | 1995-11-10 | Nippon Igaku Rinshiyou Kensa Kenkyusho:Kk | Detection of antibody in body fluids against pathogn |
CA2148971A1 (en) | 1994-05-26 | 1995-11-27 | Izak Bahar | Calibrator matrix |
JPH10505819A (en) | 1994-06-24 | 1998-06-09 | ヴラディミール ピー. トーチリン | Use of autoantibodies for treatment and prevention of tumors |
IL110464A0 (en) | 1994-07-26 | 1994-10-21 | Univ Ramot | Novel proteins for the diagnosis, imaging, and therapy of human cancer |
US5561049A (en) | 1994-09-21 | 1996-10-01 | Behringwerke Ag | Method for detecting antibodies |
US5876728A (en) * | 1995-02-15 | 1999-03-02 | Howard David Kass | Natural composition extracted from plants used in the treatment of cancer |
AUPN568095A0 (en) | 1995-09-27 | 1995-10-26 | Austin Research Institute, The | Anti-Galalpha(1,3)Gal antibody binding peptides |
GB9521544D0 (en) * | 1995-10-20 | 1995-12-20 | Univ Dundee | Activation of P53 protein and therapeutic applications thereof |
JPH09189702A (en) * | 1996-01-09 | 1997-07-22 | Hitachi Chem Co Ltd | Method for measuring antimucin antibody, cancer measuring method, and cancer diagnostic medicine |
JPH09229933A (en) | 1996-02-27 | 1997-09-05 | Kokichi Sugano | Autoantibody detecting method for p53 protein |
CA2263503C (en) * | 1996-08-16 | 2012-04-10 | The Johns Hopkins University School Of Medicine | Melanoma cell lines expressing shared immunodominant melanoma antigens and methods of using same |
EP1019727A1 (en) | 1997-06-03 | 2000-07-19 | Amdl, Inc | Immunoassay for the detection of cancer |
DE19805815C1 (en) | 1998-02-13 | 1999-11-11 | Ansgar W Lohse | Diagnostic for the detection of autoimmune hepatitis |
JP4083855B2 (en) | 1998-02-16 | 2008-04-30 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | Lung cancer prognosis test method |
GB9810040D0 (en) | 1998-05-11 | 1998-07-08 | Univ Nottingham | Blood borne tumour markers |
AUPP349098A0 (en) | 1998-05-13 | 1998-06-04 | South Eastern Sydney Area Health Service | A method of monitoring pancreatic tissue viability |
FR2780161B1 (en) | 1998-06-17 | 2000-09-29 | Centre Nat Rech Scient | METHOD FOR EARLY DIAGNOSIS OF CANCER BASED ON THE SEARCH FOR AUTO-ANTIBODIES DIRECTED AGAINST THE CSK PROTEIN AND KIT FOR ITS IMPLEMENTATION |
DE69941627D1 (en) | 1998-11-05 | 2009-12-17 | Univ Michigan | FOR CANCER |
US6387639B1 (en) * | 1998-11-10 | 2002-05-14 | Sloan-Kettering Institute For Cancer Research | Ma family polypeptides and anti-Ma antibodies |
GB9827228D0 (en) | 1998-12-10 | 1999-02-03 | Univ Nottingham | Cancer detection method and reagents |
US7807810B2 (en) | 1999-04-08 | 2010-10-05 | The Ohio State University Research Foundation | Nucleic acids encoding the major outer membrane protein of the causative agent of human granulocytic ehrlichiosis and peptides encoded thereby |
US6645465B2 (en) | 1999-08-06 | 2003-11-11 | Michigan, University Of The Regents | Annexin proteins and autoantibodies as serum markers for cancer |
US20020064801A1 (en) | 1999-12-01 | 2002-05-30 | Ryan Jeffrey R. | Novel and practical serological assay for the clinical diagnosis of leishmaniasis |
US6667160B2 (en) | 2000-03-15 | 2003-12-23 | Kenneth D. Fine | Method for diagnosing immunologic food sensitivity |
US20030138860A1 (en) | 2000-06-14 | 2003-07-24 | Robertson John Forsyth Russell | Cancer detection methods and reagents |
US20030232399A1 (en) | 2000-06-14 | 2003-12-18 | Robertson John Forsyth Russell | Cancer detection methods and reagents |
US20020137097A1 (en) | 2000-12-13 | 2002-09-26 | Bio-Rad Laboratories, Inc. | Standard diluent for multiplex assays |
US7745141B2 (en) | 2001-06-21 | 2010-06-29 | New York University | Mycobacterial proteins as early antigens for serodiagnosis and vaccines |
US20030049692A1 (en) | 2002-09-16 | 2003-03-13 | Norman Latov | Detection of anti-glycolipid antibodies by latex agglutination assay |
GB2395270B (en) | 2002-11-14 | 2006-08-16 | Univ Nottingham | Tumour marker proteins and uses thereof |
US7283245B2 (en) * | 2004-01-20 | 2007-10-16 | General Electric Company | Handheld device with a disposable element for chemical analysis of multiple analytes |
JP2005352771A (en) * | 2004-06-10 | 2005-12-22 | Hitachi Software Eng Co Ltd | Pattern recognition system by expression profile |
US20060141547A1 (en) | 2004-11-16 | 2006-06-29 | Das Hasi R | Novel diagnostic marker, a diagnostic kit and a method for diagnosis of rheumatoid arthritis |
GB2426581A (en) | 2005-05-27 | 2006-11-29 | Univ Nottingham | Immunoassay methods |
WO2007032634A1 (en) * | 2005-09-12 | 2007-03-22 | Industry Foundation Of Chonnam National University | A method for production of mature natural killer cell |
TWI304443B (en) | 2005-12-30 | 2008-12-21 | Nat Health Research Institutes | Alpha-enolase specific antibody and method of use |
CN101632020B (en) | 2006-09-13 | 2013-11-27 | 昂西免疫有限公司 | Improved immunoassay methods |
JP5495159B2 (en) | 2006-09-13 | 2014-05-21 | オンクイミューン リミテッド | Improved immunoassay method |
WO2008103188A1 (en) * | 2007-02-23 | 2008-08-28 | Alfalight, Inc | High efficiency partial distributed feedback (p-dfb) laser |
GB0725239D0 (en) | 2007-12-24 | 2008-02-06 | Oncimmune Ltd | Calibrator for autoantibody assay |
-
1998
- 1998-05-11 GB GBGB9810040.7A patent/GB9810040D0/en not_active Ceased
-
1999
- 1999-05-11 DK DK99921014T patent/DK1078264T3/en active
- 1999-05-11 ES ES05028132.8T patent/ES2443096T3/en not_active Expired - Lifetime
- 1999-05-11 AU AU38388/99A patent/AU3838899A/en not_active Abandoned
- 1999-05-11 AT AT99921014T patent/ATE314651T1/en active
- 1999-05-11 WO PCT/GB1999/001479 patent/WO1999058978A2/en active IP Right Grant
- 1999-05-11 EP EP05028131A patent/EP1710253A3/en not_active Withdrawn
- 1999-05-11 PT PT99921014T patent/PT1078264E/en unknown
- 1999-05-11 SI SI9930885T patent/SI1078264T1/en unknown
- 1999-05-11 ES ES99921014T patent/ES2257048T3/en not_active Expired - Lifetime
- 1999-05-11 EP EP99921014A patent/EP1078264B1/en not_active Expired - Lifetime
- 1999-05-11 PT PT50281328T patent/PT1731619E/en unknown
- 1999-05-11 DE DE69929185T patent/DE69929185T2/en not_active Expired - Lifetime
- 1999-05-11 DK DK05028132.8T patent/DK1731619T3/en active
- 1999-05-11 EP EP05028132.8A patent/EP1731619B1/en not_active Revoked
- 1999-05-11 US US09/700,092 patent/US7402403B1/en not_active Expired - Fee Related
-
2007
- 2007-12-10 US US11/953,237 patent/US8114604B2/en not_active Expired - Fee Related
-
2012
- 2012-01-12 US US13/349,348 patent/US9696319B2/en not_active Expired - Fee Related
- 2012-04-03 US US13/438,344 patent/US20130090251A1/en not_active Abandoned
-
2014
- 2014-03-07 CY CY20141100182T patent/CY1115366T1/en unknown
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9696319B2 (en) | 1998-05-11 | 2017-07-04 | Oncimmune Ltd. | Tumour markers |
US20080108084A1 (en) * | 1998-12-10 | 2008-05-08 | University Of Nottingham | Cancer Detection Methods and Reagents |
US20110086061A1 (en) * | 2000-06-14 | 2011-04-14 | Onclmmune Limited | Cancer Detection Methods and Reagents |
US20060094069A1 (en) * | 2002-11-14 | 2006-05-04 | Robertson John Forsyth R | Tumour marker proteins and uses thereof |
US8592169B2 (en) | 2002-11-14 | 2013-11-26 | Oncimmune Limited | Tumour marker proteins and uses thereof |
US20080305476A1 (en) * | 2005-05-27 | 2008-12-11 | Onc-Immune Ltd. | Immunoassay Methods |
US8722339B2 (en) | 2005-05-27 | 2014-05-13 | Oncimmune Ltd. | Immunoassay methods |
US9714938B2 (en) | 2005-05-27 | 2017-07-25 | Oncimmune Ltd. | Immunoassay methods |
US9719984B2 (en) | 2005-05-27 | 2017-08-01 | Oncimmune Ltd. | Immunoassay methods |
US8574848B2 (en) | 2006-09-13 | 2013-11-05 | Oncimmune Ltd. | Immunoassay methods |
US8927223B2 (en) | 2006-09-13 | 2015-01-06 | Oncimmune Ltd. | Immunoassay methods |
US20080213921A1 (en) * | 2006-09-13 | 2008-09-04 | Robertson John F R | Immunoassay Methods |
US20090176319A1 (en) * | 2007-12-24 | 2009-07-09 | Onclmmune Limited | Calibrator For Immunoassays |
US20110045508A1 (en) * | 2008-01-31 | 2011-02-24 | The Brigham And Women's Hospital, Inc. | Urinary CA125 Peptides as Biomarkers of Ovarian Cancer |
US8642347B2 (en) | 2008-01-31 | 2014-02-04 | The Brigham And Women's Hospital, Inc. | Urinary CA125 peptides as biomarkers of ovarian cancer |
Also Published As
Publication number | Publication date |
---|---|
EP1731619A3 (en) | 2007-09-26 |
SI1078264T1 (en) | 2006-06-30 |
DK1078264T3 (en) | 2006-05-15 |
DE69929185T2 (en) | 2006-08-31 |
DK1731619T3 (en) | 2014-03-24 |
ES2443096T3 (en) | 2014-02-17 |
ATE314651T1 (en) | 2006-01-15 |
US20120115749A1 (en) | 2012-05-10 |
WO1999058978A3 (en) | 2000-01-20 |
US20080153113A1 (en) | 2008-06-26 |
EP1710253A3 (en) | 2007-10-31 |
GB9810040D0 (en) | 1998-07-08 |
EP1078264A2 (en) | 2001-02-28 |
EP1710253A2 (en) | 2006-10-11 |
EP1078264B1 (en) | 2005-12-28 |
ES2257048T3 (en) | 2006-07-16 |
WO1999058978A2 (en) | 1999-11-18 |
CY1115366T1 (en) | 2017-01-04 |
US9696319B2 (en) | 2017-07-04 |
AU3838899A (en) | 1999-11-29 |
EP1731619B1 (en) | 2013-12-18 |
EP1731619A2 (en) | 2006-12-13 |
PT1078264E (en) | 2006-05-31 |
US8114604B2 (en) | 2012-02-14 |
US7402403B1 (en) | 2008-07-22 |
DE69929185D1 (en) | 2006-02-02 |
PT1731619E (en) | 2014-03-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9696319B2 (en) | Tumour markers | |
US20170052186A1 (en) | Cancer detection methods and reagents | |
EP1137943B1 (en) | Cancer detection method and reagents | |
US5652115A (en) | Method of detecting tumors containing complexes of p53 and HSP70 | |
JP5711196B2 (en) | Use of HE4 and other biochemical markers to determine ovarian cancer | |
CA2597271A1 (en) | Methods of detecting ovarian cancer | |
US20030138860A1 (en) | Cancer detection methods and reagents | |
US5443956A (en) | Detection, quantitation and classification of RAS proteins in body fluids and tissues | |
CA2427523A1 (en) | Predicting agent for metastasis | |
US8206897B2 (en) | Assay for soluble CD200 | |
IL129236A (en) | Monoclonal antibodies against a human act and serine protease complex | |
US5346813A (en) | Method of screening for small cell lung carcinoma | |
WO2021246153A1 (en) | Method and reagent for detecting pancreatic cancers | |
EP0662612A1 (en) | A method of using novel antibodies for the detection of benign and malignant tumors | |
US20030022259A1 (en) | Method for immunologically measuring RCAS1 and kit for measuring the same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ONCIMMUNE LTD., UNITED KINGDOM Free format text: NUNC PRO TUNC ASSIGNMENT;ASSIGNORS:ROBERTSON, JOHN F.R.;GRAVES, CATHERINE R.L.;PRICE, FRANCES R.;SIGNING DATES FROM 20080123 TO 20080124;REEL/FRAME:027980/0293 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |