WO2000063231A1 - Lymphocyte platelet binding factor as a serum marker for cancer - Google Patents

Lymphocyte platelet binding factor as a serum marker for cancer Download PDF

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Publication number
WO2000063231A1
WO2000063231A1 PCT/US2000/009738 US0009738W WO0063231A1 WO 2000063231 A1 WO2000063231 A1 WO 2000063231A1 US 0009738 W US0009738 W US 0009738W WO 0063231 A1 WO0063231 A1 WO 0063231A1
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Prior art keywords
lpbf
cancer
sample
subject
lymphocyte
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PCT/US2000/009738
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French (fr)
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Eytan Barnea
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Bioincept, Inc.
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Priority to AU42336/00A priority Critical patent/AU4233600A/en
Publication of WO2000063231A1 publication Critical patent/WO2000063231A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966Animal cells
    • G01N33/56972White blood cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/52Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to screening methods for diagnosis and prognosis of cancer in a subject by means of detecting increased levels of lymphocyte platelet binding factor (LPBF) in sera and other biological fluids of the subject.
  • the method of the invention involves the use of subject derived serum samples, or other biological fluid samples, to determine the occurrence and level of LPBF in the sample.
  • the level of LPBF in the sample can be assayed using, for example, a lymphocyte/platelet binding assay.
  • the invention is based on the discovery that serum derived from subjects having cancer contains elevated levels of a factor that increases the level of binding of platelets to lymphocytes.
  • the present invention further provides for kits for carrying out the above described screening methods. Such kits may be used to screen subjects for increased levels of LPBF, as a diagnostic, predictive or prognostic indicator of cancer.
  • the invention is demonstrated by way of examples in which increased levels of LPBF were identified in serum samples derived from subjects with cancer.
  • a number of cellular proteins have been demonstrated to occur at increased levels in body fluids of subjects with different types of cancer.
  • the increased levels of such proteins in cancer subjects provides diagnostic and prognostic assays for the presence of cancer.
  • elevated serum levels of prostate specific antigen (PSA) is frequently used as an indicator of the presence of prostate cancer in subjects.
  • PSA prostate specific antigen
  • the occurrence of antibodies to tumor derived proteins as in the case of mutant p53 has also been noted to occur in some cancers.
  • Lymphocyte/platelet binding assays have been successfully used to detect a number of different factors in the serum of subjects.
  • lymphocyte/platelet binding assays have been used to detect preimplantation factor (PIF) in the serum of pregnant females as described in U.S. Patent No.5,646,003.
  • PIF preimplantation factor
  • the presence of PIF in serum is measured by its ability to function as an enhancer of lymphocyte binding to platelets.
  • detection of PIF activity is dependent on the addition of anti-CD2 antibodies to the reaction mixture.
  • the presence of PIF is an early positive indicator of pregnancy and remains positive throughout the first trimester of pregnancy.
  • assays for PIF have been found to be useful for predicting the spontaneous loss of pregnancies.
  • the invention provides methods for detecting increased levels of LPBF as a diagnostic or prognostic indicator of cancer.
  • the invention comprises assays developed to detect the level of LPBF in a subject's serum sample using assays such as lymphocyte/platelet binding assays.
  • Isolation and identification of proteins constituting LPBF made possible by the discovery that increased levels of LPBF may be detected using lymphocyte/platelet binding assays, permits their application to development of diagnostic and therapeutic tools.
  • diagnostic and therapeutic tools include antibodies that bind to LPBF and which may be used in immunoassays for detection of LPBF levels in samples of subjects.
  • the invention further provides for pre-packaged diagnostic kits which will be conveniently used in clinical settings, to diagnose patients having cancer or a predisposition to developing cancer.
  • the kits will also be utilized to monitor the efficiency of compounds used for treatment of cancer.
  • increased levels of LPBF were detected in serum samples derived from subjects with a variety of different cancers.
  • the finding that levels of LPBF are increased in the serum of cancer subjects provides a basis for development of diagnostic and prognostic methods as well as a means for monitoring the efficacy of various therapeutic treatments for cancer.
  • the present invention achieves a highly desirable objective, namely providing methods for the diagnostic and prognostic evaluation of subjects with cancer and the identification of subjects exhibiting a predisposition to developing cancer.
  • the assays of the invention comprise methods designed to detect increased levels of LPBF in serum or other biological fluids of a subject.
  • the invention encompasses a method for diagnosis and prognosis of cancer in a subject comprising: (a) detecting lymphocyte/platelet binding factor (LPBF) in a biological fluid sample derived from a subject; and (b) comparing the level of LPBF detected in the subject's sample to the level of LPBF detected in a control sample, wherein an increase in the level of LPBF detected in the subject's sample as compared to control samples is an indicator of a subject with cancer or at increased risk for cancer.
  • LPBF lymphocyte/platelet binding factor
  • LPBF LPBF
  • samples should originally be in, or rendered into, fluid form.
  • Such samples may include cell homogenates such as tumor cell homogenates, or tumor aspirates.
  • biological fluids such as urine, cerebrospinal fluid, synovial fluid, peritoneal fluid, pleural effusion, pericardial effusion, saliva, tears or sera, in which secreted proteins are localized can be used to screen for increased levels of LPBF expression.
  • the present invention also provides for kits for carrying out the above- described methods.
  • the methods can be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one reagent for detecting LPBF, such as an anti-LPBF antibody.
  • the diagnostic kits will comprise components for performing lymphocyte/platelet binding assays.
  • the present invention is based on the discovery that levels of LPBF in serum are increased in subjects with a wide variety of different cancers .
  • measurement of levels of LPBF proteins can be used for the early diagnosis of diseases such as cancer. Moreover, the monitoring of LPBF levels can be used prognostically to stage the progression of the disease and to evaluate the efficacy of compounds used to treat a cancer subject.
  • the detection of LPBF in a liquid sample from a subject can be accomplished by any of a number of methods. Preferred diagnostic methods for the detection of LPBF in a liquid sample from a subject can involve, for example, a lymphocyte/platelet binding assay.
  • Such an assay for LPBF in the liquid sample of a subject comprises the following steps:
  • the detection of LPBF expression can also be used to monitor the efficacy of potential anti-cancer compounds during treatment.
  • the level of LPBF expression can be determined before and during treatment.
  • the efficacy of the compound can be followed by comparing LPBF expression throughout the treatment.
  • Compounds exhibiting efficacy are those which decrease the level of LPBF expression as treatment with the compound progresses.
  • the present invention is demonstrated by way of example wherein elevated levels of LPBF were detected in serum samples derived from cancer subjects.
  • increased levels of LPBF were detected in serum samples derived from colon, lung, breast, head, neck, lymphoma, liver, pancreas, bone and cervical cancer patients.
  • the detection and/or quantitative measurement of LPBF in patient can be used in screening of subjects who are at risk for developing certain types of cancers or other proliferative disorders in which LPBF is over expressed.
  • LPBF ANTIBODIES Native LPBF protein can be purified from natural sources, by known protein purification techniques including chromatography (e.g., ion exchange, affinity and sizing column chromatography) centrifugation, differential solubility or by any other standard technique for the purification of proteins. LPBF activity may be detected during the purification techniques using lymphocyte/platelet binding assays.
  • LPBF protein may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen.
  • antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • antibodies to the LPBF protein are produced.
  • antibodies to a domain of the LPBF protein are produced.
  • LPBF protein it may not be necessary to purify LPBF protein for production of antibodies to the LPBF protein.
  • a sample containing a mixture of proteins, including LPBF may be used as an immunogen to generate antibodies that immunospecifically bind to LPBF.
  • the antibodies may be screened based on their ability to recognize a specific domain of the LPBF protein and inhibit LPBF activity as determined using a lymphocyte/platelet binding assay.
  • Various procedures known in the art may be used for the production of polyclonal antibodies to a LPBF protein or derivative or analog. In a particular embodiment, rabbit polyclonal antibodies to an epitope of a LPBF protein are obtained.
  • various host animals are immunized by injection with the native LPBF protein, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, etc.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette- Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette- Guerin
  • Corynebacterium parvum bacille Calmette- Guerin
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV hybridoma technique to produce human monoclonal antibodies may be used to produce monoclonal antibodies.
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies may be screened for their ability to bind directly to an epitope of the LPBF protein.
  • antibodies which recognize a specific domain of a LPBF protein and inhibit the activity of the LPBF protein may be screened for using a lymphocyte/platelet binding assay.
  • one assay uses such an assay, one assays hybridomas for a product which specifically binds to LPBF and inhibits the LPBF mediated binding of lymphocytes to platelets.
  • IMMUNOASSAYS FOR DETECTION OF LPBF Immunoassays wherein LPBF is detected by its interaction with a LPBF specific antibody may be used as preferred diagnostic methods.
  • Antibodies useful in the present invention can be used to quantitatively detect the presence of LPBF.
  • reagents other than antibodies, such as, for example, polypeptides that bind specifically to LPBF can be used in assays to detect the level of LPBF expression.
  • Immunoassays to be used in the practice of the invention include but are not limited to assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • a biological sample which may contain LPBF, such as serum or other biological fluids in which secreted proteins localize, is obtained from a subject suspected of having a particular cancer or risk for cancer.
  • Immunoassays for detection of LPBF typically comprise contacting the biological sample, such as a serum sample derived from a subject, with an anti-LPBF antibody under conditions such that specific antigen-antibody binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody.
  • a biological sample such as a serum sample derived from a subject
  • an anti-LPBF antibody under conditions such that specific antigen-antibody binding can occur
  • detecting or measuring the amount of any immunospecific binding by the antibody can be used to detect the presence and increased expression of LPBF wherein the detection of increased expression of LPBF is an indication of a diseased condition.
  • the levels of LPBF in a serum sample are compared to norms established for normal individuals and for subjects with a variety of non-cancerous or pre-cancerous disease states.
  • the biological sample such as a serum sample is brought in contact with a solid phase support or carrier, such as nitrocellulose, for the purpose of immobilizing any proteins present in the sample.
  • a solid phase support or carrier such as nitrocellulose
  • the support is then washed with suitable buffers followed by treatment with detectably labeled LPBF specific antibody.
  • the solid phase support is then washed with the buffer a second time to remove unbound antibody.
  • the amount of bound antibody on the solid support is then determined according to well known methods. Those skilled in the art will be able to determine optional assay conditions for each determination by employing routine experimentation.
  • the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or by visual means.
  • an appropriate substrate preferably a chromogenic substrate
  • Enzymes that can be used to detectable label the antibody include, but are not limited to, horseradish peroxidase and alkaline phosphates.
  • the detection can also be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme.
  • Detection of LPBF specific antibodies may also be accomplished using a variety of other methods. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect LPBF expression through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986).
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • the antibody may also be labeled with a fluorescent compound.
  • fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine.
  • a bioluminescent compound may be used to label the LPBF antibody. The presence of a bioluminescence protein is determined by detecting the presence of luminescence.
  • Important bioluminescence compounds for purposes of labeling are luciferin and luciferase, for example.
  • a kit according to the invention may comprise components which detect and/or measure LPBF antigen in the biological sample of a subject.
  • LPBF is detected and/or measured by enzyme linked immunoabsorbent assay (ELISA)
  • ELISA enzyme linked immunoabsorbent assay
  • such components may comprise an antibody directed to an epitope of LPBF which can be used to detect and/or quantitate the level of LPBK expression in the biological sample.
  • the antibody itself may be detectably labeled with a radioactive, flourescent, colorimetric or enzymatic label.
  • the kit may contain a labeled secondary antibody.
  • 8 ml of blood from a healthy O+ donor was diluted 1 : 1 with PBS and was layered over 12 ml of Histopaque column in a 50 ml test tube and centrifuged at 2400 rpm for 20 minutes to isolate lymphocytes plus platelets by density gradient. Following centrifugation the buffy coat was removed, diluted 1 :1 with PBS and washed twice with PBS at 1100 rpm for 10 minutes, each time removing the supernatant. During each washing, 2 ml of double distilled water was added to lyse the residual red blood cells. The recovered lymphocytes and platelets were diluted in PBS to a density of 10 12 cells per ml.
  • the serum samples were either used fresh or frozen at -20 °C.
  • the lymphocyte/platelet binding assays were carried out in microcentrifuge tubes in a total volume of 50 ⁇ l. 30 ⁇ l of the cell suspension (lymphocytes plus platelets) was added to 20 ⁇ l of cancer patient serum. The sample was incubated at room temperature for 10 minutes, agitating every 2 minutes. 10 ⁇ l of the sample was withdrawn and placed on a slide with a cover slip.
  • lymphocytes were counted at 400x magnification using a Nikon inverted microscope. The number of lymphocytes with, or without, three or more bound platelets were counted. A total of 200 cells in three different fields were counted. Data are expressed as percent platelet-bound lymphocytes per total bound and non-bound lymphocytes.
  • Statistical analysis was carried out by using chi-square analysis with Fischers Exact Test and ANOVA one way analysis of variance. A difference of P ⁇ 0.05 was considered significant. Less than 5% was considered a negative result and greater than 15% of the lymphocytes displaying a rosetted formation with 3 or more bound platelets indicated the presence of cancer.
  • lymphocyte/platelet binding assay In three instances where the patients' own lymphocytes and platelets were used in the lymphocyte/platelet binding assay, no quantitative differences were observed and LPBF results were similar to that obtained with the donor blood. In one patient, the presence of infectious mononucleosis also failed to affect LPBF in the assay.
  • the culture media of MCF-7 human breast cancer cells was tested using the lymphocyte/platelet binding assay.
  • the culture media was negative for

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Abstract

The present invention relates to screening methods for diagnosis and prognosis of cancer in a subject by means of detecting increased levels of lymphocyte platelet binding (LPBF) factor in sera and other biological fluids of the subject. The method of the invention involves the use of subject derived serum samples, or other biological fluid samples, to determine the occurrence and level of LPBF in the sample. The level of LPBF in the sample can be assayed using, for example, a lymphocyte/platelet binding assay. The invention is based on the discovery that serum derived from subjects having cancer contains elevated levels of a factor that increases the level of binding of platelets to lymphocytes. The present invention further provides for kits for carrying out the above described screening methods. Such kits will be used to screen subjects for increased levels of LPBF, as a diagnostic, predictive or prognostic indicator of cancer. The invention is demonstrated by way of examples in which increased levels of LPBF were identified in serum samples derived from subjects with cancer.

Description

LYMPHOCYTE PLATELET BINDING FACTOR AS A SERUM MARKER FOR CANCER
SPECIFICATION
1. INTRODUCTION The present invention relates to screening methods for diagnosis and prognosis of cancer in a subject by means of detecting increased levels of lymphocyte platelet binding factor (LPBF) in sera and other biological fluids of the subject. The method of the invention involves the use of subject derived serum samples, or other biological fluid samples, to determine the occurrence and level of LPBF in the sample. The level of LPBF in the sample can be assayed using, for example, a lymphocyte/platelet binding assay. The invention is based on the discovery that serum derived from subjects having cancer contains elevated levels of a factor that increases the level of binding of platelets to lymphocytes. The present invention further provides for kits for carrying out the above described screening methods. Such kits may be used to screen subjects for increased levels of LPBF, as a diagnostic, predictive or prognostic indicator of cancer. The invention is demonstrated by way of examples in which increased levels of LPBF were identified in serum samples derived from subjects with cancer.
2. BACKGROUND OF THE INVENTION A number of cellular proteins have been demonstrated to occur at increased levels in body fluids of subjects with different types of cancer. The increased levels of such proteins in cancer subjects provides diagnostic and prognostic assays for the presence of cancer. For example, elevated serum levels of prostate specific antigen (PSA) is frequently used as an indicator of the presence of prostate cancer in subjects. The occurrence of antibodies to tumor derived proteins as in the case of mutant p53 has also been noted to occur in some cancers. However, there is little knowledge that allows prediction of which proteins in tumors may be antigenic or may occur at increased levels in body fluids.
Lymphocyte/platelet binding assays have been successfully used to detect a number of different factors in the serum of subjects. For example, lymphocyte/platelet binding assays have been used to detect preimplantation factor (PIF) in the serum of pregnant females as described in U.S. Patent No.5,646,003. The presence of PIF in serum is measured by its ability to function as an enhancer of lymphocyte binding to platelets. In contrast to the lymphocyte/platelet binding assays described herein, detection of PIF activity is dependent on the addition of anti-CD2 antibodies to the reaction mixture. The presence of PIF is an early positive indicator of pregnancy and remains positive throughout the first trimester of pregnancy. In addition, assays for PIF have been found to be useful for predicting the spontaneous loss of pregnancies.
3. SUMMARY OF THE INVENTION It is an object of the present invention to provide methods for the diagnostic and prognostic evaluation of cancer, for the identification of subjects possessing a predisposition to cancer, and for monitoring patients undergoing treatment of cancer, based on the detection of increased levels of LPBF in biological fluid samples of subjects. The invention provides methods for detecting increased levels of LPBF as a diagnostic or prognostic indicator of cancer. The invention comprises assays developed to detect the level of LPBF in a subject's serum sample using assays such as lymphocyte/platelet binding assays.
Isolation and identification of proteins constituting LPBF, made possible by the discovery that increased levels of LPBF may be detected using lymphocyte/platelet binding assays, permits their application to development of diagnostic and therapeutic tools. Such tools include antibodies that bind to LPBF and which may be used in immunoassays for detection of LPBF levels in samples of subjects.
The invention further provides for pre-packaged diagnostic kits which will be conveniently used in clinical settings, to diagnose patients having cancer or a predisposition to developing cancer. The kits will also be utilized to monitor the efficiency of compounds used for treatment of cancer.
In a specific embodiment of the invention, increased levels of LPBF were detected in serum samples derived from subjects with a variety of different cancers. The finding that levels of LPBF are increased in the serum of cancer subjects provides a basis for development of diagnostic and prognostic methods as well as a means for monitoring the efficacy of various therapeutic treatments for cancer.
4. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Lymphocyte Platelet Binding Assays of Cancer Patients' Sera. Each sample contained 20 μl of sera from each patient with cancer. Samples
# 2, 22, 28, 39 and 49 represent negative controls and sample #12 represents a healthy patient. Figure 2. Lymphocyte Platelet Binding Assays of Cancer Patients' Sera (Blind
Test). All lettered samples represent negative controls. Figure 3. Lymphocyte Platelet Binding Assays of Cancer Patients' Sera (Blind
Test). Each sample contained 20μl of serum and 3 μl of CD2. Samples were labeled 1-30 [1-12 = cancer samples; 13-30 = negative controls]. 20 samples were chosen and randomly relabeled 1-20. Figure 4. Dose Dependence of Lymphocyte Platelet Binding Cancer Sample #2. Figure 5. CD2 Independence of Cancer Samples. Samples contained 20 μl of serum from cancer patient #22 and increasing amounts of antiCD2 antibody. Control levels were 20% of lymphocytes with 3 bound platelets in the positive case and 5% in the negative case. Figure 6. Heat Inactivation of LPBF. Samples contained serum from cancer patient #23. Control levels for this experiment were 19% of lymphocytes with 3 bound platelets in the positive case and 4% in the negative case. Figure 7. Effect of cancer patient sera on PIF activity. Addition of sera from small lung cancer patient had no significant effect on PIF activity produced by pregnancy sera. Control levels were
20 and 5 for positive and negative controls, respectively. Figure 8. Effect of cancer patient sera in PIF activity. Addition of sera from a bone cancer patient had no significant effect on
PIF activity produced by pregnancy sera. Control levels were 18 and 5 for positive and negative controls, respectively. Figure 9. HPLC Separation of Head and Neck Cancer Patient Sera. Negative control values for these fractions ranged from 4-6% of lymphocytes with 3 bound platelets. The region of activity is between fractions 26 and 32.
5. DETAILED DESCRIPTION OF THE INVENTION The present invention achieves a highly desirable objective, namely providing methods for the diagnostic and prognostic evaluation of subjects with cancer and the identification of subjects exhibiting a predisposition to developing cancer. The assays of the invention comprise methods designed to detect increased levels of LPBF in serum or other biological fluids of a subject.
Specifically, the invention encompasses a method for diagnosis and prognosis of cancer in a subject comprising: (a) detecting lymphocyte/platelet binding factor (LPBF) in a biological fluid sample derived from a subject; and (b) comparing the level of LPBF detected in the subject's sample to the level of LPBF detected in a control sample, wherein an increase in the level of LPBF detected in the subject's sample as compared to control samples is an indicator of a subject with cancer or at increased risk for cancer.
A wide variety of subject samples that may contain LPBF(s) can be prepared or assayed for the levels of LPBF. Such samples should originally be in, or rendered into, fluid form. Such samples may include cell homogenates such as tumor cell homogenates, or tumor aspirates. In a preferred embodiment biological fluids such as urine, cerebrospinal fluid, synovial fluid, peritoneal fluid, pleural effusion, pericardial effusion, saliva, tears or sera, in which secreted proteins are localized can be used to screen for increased levels of LPBF expression.
The present invention also provides for kits for carrying out the above- described methods. The methods can be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one reagent for detecting LPBF, such as an anti-LPBF antibody. Alternatively, the diagnostic kits will comprise components for performing lymphocyte/platelet binding assays. The present invention is based on the discovery that levels of LPBF in serum are increased in subjects with a wide variety of different cancers .
5.1. ASSAYS FOR DETECTION OF LPBF EXPRESSION In accordance with the invention, measurement of levels of LPBF proteins can be used for the early diagnosis of diseases such as cancer. Moreover, the monitoring of LPBF levels can be used prognostically to stage the progression of the disease and to evaluate the efficacy of compounds used to treat a cancer subject. The detection of LPBF in a liquid sample from a subject can be accomplished by any of a number of methods. Preferred diagnostic methods for the detection of LPBF in a liquid sample from a subject can involve, for example, a lymphocyte/platelet binding assay. Such an assay for LPBF in the liquid sample of a subject comprises the following steps:
(a) removing cells and/or cellular debris from the liquid sample ;
(b) providing blood lymphocytes containing platelets;
(c) admixing said liquid sample, lymphocytes and platelets; and
(d) determining in the admixture the percentage of lymphocytes bound to platelets; whereby a percentage significantly higher than the percentage in a normal control sample indicates the presence of LPBF in the serum. A test is considered positive when the percentage of lymphocytes bound to platelets is greater than 15%. Either autologous or heterologous lymphocytes may be used in the practice of the invention. Unlike, the lymphocyte/platelet binding assay used to detect PIF in the serum of pregnant females, the present assay for LPBF does not require the presence of anti- CD2 antibodies (see, U.S. Patent No. 5,646,003).
The detection of LPBF expression can also be used to monitor the efficacy of potential anti-cancer compounds during treatment. For example, the level of LPBF expression can be determined before and during treatment. The efficacy of the compound can be followed by comparing LPBF expression throughout the treatment. Compounds exhibiting efficacy are those which decrease the level of LPBF expression as treatment with the compound progresses.
The present invention is demonstrated by way of example wherein elevated levels of LPBF were detected in serum samples derived from cancer subjects. In particular, increased levels of LPBF were detected in serum samples derived from colon, lung, breast, head, neck, lymphoma, liver, pancreas, bone and cervical cancer patients. The detection and/or quantitative measurement of LPBF in patient can be used in screening of subjects who are at risk for developing certain types of cancers or other proliferative disorders in which LPBF is over expressed.
5.2. PREPARATION OF LPBF ANTIBODIES Native LPBF protein can be purified from natural sources, by known protein purification techniques including chromatography (e.g., ion exchange, affinity and sizing column chromatography) centrifugation, differential solubility or by any other standard technique for the purification of proteins. LPBF activity may be detected during the purification techniques using lymphocyte/platelet binding assays.
According to the invention, LPBF protein, its fragments or other derivatives, or analogs thereof, may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. In a specific embodiment, antibodies to the LPBF protein are produced. In another embodiment, antibodies to a domain of the LPBF protein are produced.
Alternatively, it may not be necessary to purify LPBF protein for production of antibodies to the LPBF protein. For example, a sample containing a mixture of proteins, including LPBF, may be used as an immunogen to generate antibodies that immunospecifically bind to LPBF. The antibodies may be screened based on their ability to recognize a specific domain of the LPBF protein and inhibit LPBF activity as determined using a lymphocyte/platelet binding assay. Various procedures known in the art may be used for the production of polyclonal antibodies to a LPBF protein or derivative or analog. In a particular embodiment, rabbit polyclonal antibodies to an epitope of a LPBF protein are obtained. For the production of antibody, various host animals are immunized by injection with the native LPBF protein, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette- Guerin) and Corynebacterium parvum.
For preparation of monoclonal antibodies directed toward a LPBF protein sequence or analog thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV hybridoma technique to produce human monoclonal antibodies (Cole et al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) may be used to produce monoclonal antibodies.
In the production of antibodies, screening for the desired antibody is accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay). Antibodies may be screened for their ability to bind directly to an epitope of the LPBF protein. Alternatively, antibodies which recognize a specific domain of a LPBF protein and inhibit the activity of the LPBF protein may be screened for using a lymphocyte/platelet binding assay. Using such an assay, one assays hybridomas for a product which specifically binds to LPBF and inhibits the LPBF mediated binding of lymphocytes to platelets. For selection of an antibody that specifically binds a first LPBF homolog but which does not specifically bind a different LPBF homolog, one selects on the basis of positive binding to the first LPBF homolog and a lack of binding to the second LPBF homolog.
5.3. IMMUNOASSAYS FOR DETECTION OF LPBF Immunoassays wherein LPBF is detected by its interaction with a LPBF specific antibody may be used as preferred diagnostic methods. Antibodies useful in the present invention can be used to quantitatively detect the presence of LPBF. In addition, reagents other than antibodies, such as, for example, polypeptides that bind specifically to LPBF can be used in assays to detect the level of LPBF expression.
Immunoassays to be used in the practice of the invention include but are not limited to assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. A biological sample which may contain LPBF, such as serum or other biological fluids in which secreted proteins localize, is obtained from a subject suspected of having a particular cancer or risk for cancer. Immunoassays for detection of LPBF typically comprise contacting the biological sample, such as a serum sample derived from a subject, with an anti-LPBF antibody under conditions such that specific antigen-antibody binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody. In a specific aspect, such binding of antibody, for example, can be used to detect the presence and increased expression of LPBF wherein the detection of increased expression of LPBF is an indication of a diseased condition. The levels of LPBF in a serum sample are compared to norms established for normal individuals and for subjects with a variety of non-cancerous or pre-cancerous disease states.
In an embodiment of the invention, the biological sample, such as a serum sample is brought in contact with a solid phase support or carrier, such as nitrocellulose, for the purpose of immobilizing any proteins present in the sample. The support is then washed with suitable buffers followed by treatment with detectably labeled LPBF specific antibody. The solid phase support is then washed with the buffer a second time to remove unbound antibody. The amount of bound antibody on the solid support is then determined according to well known methods. Those skilled in the art will be able to determine optional assay conditions for each determination by employing routine experimentation.
One of the ways in which LPBF specific antibodies can be detectably labeled is by linking the antibody to an enzyme, such as for use in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD; Noller, A., et al., 1978, J. Clin. Pathol. 31 :507-520; Butler, J.E., 1981, Meth. Enzymol. 73:482-523). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or by visual means. Enzymes that can be used to detectable label the antibody include, but are not limited to, horseradish peroxidase and alkaline phosphates. The detection can also be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme.
Detection of LPBF specific antibodies may also be accomplished using a variety of other methods. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect LPBF expression through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. The antibody may also be labeled with a fluorescent compound. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine. Likewise, a bioluminescent compound may be used to label the LPBF antibody. The presence of a bioluminescence protein is determined by detecting the presence of luminescence. Important bioluminescence compounds for purposes of labeling are luciferin and luciferase, for example.
5.4. KITS The present invention further provides for kits for carrying out the above-described assays. The assays described herein can be performed, for example, by utilizing pre-packaged diagnostic kits, comprising at least a LPBF antibody reagent (for detection of LPBF protein), which can be conveniently used, e.g., in clinical settings to diagnose disorders such as cancer.
In a first series of nonlimiting embodiments, a kit according to the invention may comprise components which detect and/or measure LPBF antigen in the biological sample of a subject. For example, where LPBF is detected and/or measured by enzyme linked immunoabsorbent assay (ELISA), such components may comprise an antibody directed to an epitope of LPBF which can be used to detect and/or quantitate the level of LPBK expression in the biological sample. The antibody itself may be detectably labeled with a radioactive, flourescent, colorimetric or enzymatic label. Alternatively, the kit may contain a labeled secondary antibody.
In a second series of nonlimiting embodiments, a kit according to the invention may comprise components which detect and/or measure LPBF mediated lymphocyte/platelet binding activity. Such components may comprise blood lymphocytes containing platelets and a control serum sample. 6. EXAMPLE: INCREASED LEVELS OF LPBF DETECTED IN THE SERUM OF CANCER PATIENTS The following subsection describes experimental data relating to the detection of increased levels of LPBF in the serum of cancer patients.
6J. MATERIALS AND METHODS
6.1.1. PREPARATION OF LYMPHOCYTES Histopaque and Dulbecco's Phosphate Buffered Saline (PBS) were obtained from Sigma (St. Louis, MO).
8 ml of blood from a healthy O+ donor was diluted 1 : 1 with PBS and was layered over 12 ml of Histopaque column in a 50 ml test tube and centrifuged at 2400 rpm for 20 minutes to isolate lymphocytes plus platelets by density gradient. Following centrifugation the buffy coat was removed, diluted 1 :1 with PBS and washed twice with PBS at 1100 rpm for 10 minutes, each time removing the supernatant. During each washing, 2 ml of double distilled water was added to lyse the residual red blood cells. The recovered lymphocytes and platelets were diluted in PBS to a density of 1012 cells per ml.
6.1.2. LYMPHOCYTE/PLATELET BINDING ASSAY The serum samples were either used fresh or frozen at -20 °C. The lymphocyte/platelet binding assays were carried out in microcentrifuge tubes in a total volume of 50 μl. 30 μl of the cell suspension (lymphocytes plus platelets) was added to 20 μl of cancer patient serum. The sample was incubated at room temperature for 10 minutes, agitating every 2 minutes. 10 μl of the sample was withdrawn and placed on a slide with a cover slip.
All lymphocytes were counted at 400x magnification using a Nikon inverted microscope. The number of lymphocytes with, or without, three or more bound platelets were counted. A total of 200 cells in three different fields were counted. Data are expressed as percent platelet-bound lymphocytes per total bound and non-bound lymphocytes. Statistical analysis was carried out by using chi-square analysis with Fischers Exact Test and ANOVA one way analysis of variance. A difference of P <0.05 was considered significant. Less than 5% was considered a negative result and greater than 15% of the lymphocytes displaying a rosetted formation with 3 or more bound platelets indicated the presence of cancer.
6.1.3. PREPARATION OF CULTURE MEDIA FROM MCF-7
HUMAN BREAST CANCER CELLS 1.25 x 103 MCF-7 cells were plated in 1 ml of medium in a 24 well cell culture plate and incubated for 96 hours at 37°C, 5% CO2 in RPMI medium 1640 (GibcoBRL;Rockville, MD) and 5% fetal bovine serum (GibcoBRL;Rockville, MD). The media was collected after 4 days, separated from the cells and tested for LPBF activity.
6.1.4. ANALYTICAL GEL FILTRATION Gel permeation chromatography (GPC) was performed as follows. 0.5 ml sera sample was passed through a Shodex KW-803 column using PBS buffered system and a flow rate of 0.5 ml/min. 1 ml fractions a minute were collected. Collected fractions were lyophilized and assayed for LPBF activity.
6.2. RESULTS 49 cancer patients' serum was analyzed using the lymphocyte/platelet binding assay, 46 of which displayed a clear indication of increased binding of lymphocytes to platelets when compared to negative controls (Figure 1). These patients represented a broad spectrum of diagnoses including breast, colon, lung, head and neck, lymphoma, liver, pancreas, bone, and cervical cancer (Table 1). TABLE 1
Figure imgf000015_0001
The results described above were confirmed in two blind tests (Figure
2, 3). In addition, the cancer patient sera reacted in the lymphocyte/platelet binding assay in a dose dependent manner (Figure 4). These cancer samples were originally assayed using the lymphocyte/platelet bind assay utilized to detect Preimplantation Factor (PIF) which includes an incubation with an anti-CD2 monoclonal antibody. However, further investigation revealed that the cancer samples caused an autorosette formation of lymphocytes and platelets independent of the presence of anti-CD2 antibody (Figure 5). The factor(s) present in the cancer patients' sera that causes this reaction is heat labile, as demonstrated in Figure 6, indicating that the factor(s) is a protein, or that the binding of platelets to lymphocytes is enzyme mediated. In three instances where the patients' own lymphocytes and platelets were used in the lymphocyte/platelet binding assay, no quantitative differences were observed and LPBF results were similar to that obtained with the donor blood. In one patient, the presence of infectious mononucleosis also failed to affect LPBF in the assay.
To determine the origin of the factor(s) responsible for the observed rosette formation, the culture media of MCF-7 human breast cancer cells was tested using the lymphocyte/platelet binding assay. The culture media was negative for
LPBF activity indicating that the active factor(s) was not secreted by the cancer cells.
Using positive and negative controls, small volumes of the cancer patient sera had no effect on the LPBF on 20 μl of pregnancy positive sera (Figures 7, 8). HPLC techniques were used to determine the region of biological activity and to isolate the LPBF factor(s) responsible for the observed phenomenon. Using an analytical gel filtration column, a small quantity of head and neck cancer patient serum was separated in a PBS buffer. Collected samples were frozen and then defrosted. A total of 30 samples were assayed, 20 μ of each fraction, and activity was found in fractions 26-32 (Figure 9). The present invention is not to be limited in scope by the embodiments disclosed in the examples which are intended as an illustration of one aspect of the invention and any method which is functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Various publications are cited herein, the contents of which are hereby incorporated by reference in their entirety.

Claims

1. A method for diagnosis and prognosis of cancer in a subject comprising:
(a) detecting LPBF in a biological fluid sample derived from a subject; and
(b) comparing the level of LPBF detected in the subject's sample to the level of LPBF detected in a control sample, wherein an increase in the level of LPBF detected in the subject's sample as compared to a control sample is an indicator of the presence of cancer.
2. The method of claim 1 wherein the LPBF protein is detected using a lymphocyte binding assay.
3. The method of claim 1 wherein the LPBF protein is detected using polyacrylamide gel electrophoresis.
4. The lymphocyte binding assay of claim 2 comprising:
(a) obtaining a biological fluid sample from a subject;
(b) removing cells and cellular debris from the fluid sample;
(c) providing blood lymphocytes and platelets;
(d) admixing said fluid sample, lymphocytes and platelets; and (g) determining in the admixture the percentage of lymphocytes bound to platelets; whereby a percentage significantly higher than the percentage in a normal control sample indicates the presence of LPBF in the serum.
5. A method for diagnosis and prognosis of cancer in a subject comprising: (a) contacting a biological sample obtained from the subject with an anti-LPBF antibody under conditions such that a specific antigen-antibody binding can occur; and
(b) detecting or measuring the amount of immunospecific binding by the antibody, wherein an increase in the level of immunospecific binding by the antibody detected in the biological sample obtained from the subject as compared to a control sample is an indicator of the presence of cancer.
6. The method of claim 5, wherein the amount of immunospecific binding by the antibody is measured by an immunoprecipitation assay.
7. The method of claim 1 or 5, wherein the sample is a serum sample.
8. A kit for diagnosis and prognosis of cancer in a subject comprising a component for detecting the presence LPBF in a biological sample.
9. The kit of claim 8 wherein the component for LPBF is an anti-LPBF antibody.
10. A kit for diagnosis and prognosis of cancer in a subject comprising the components necessary for conducting a lymphocyte platelet binding assay.
11. The kit of claim 10 comprising donor blood lymphocytes and platelets, activated complement and a control serum sample.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
US5646003A (en) * 1994-03-23 1997-07-08 Barnea; Eytan R. Preimplantation factor

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5646003A (en) * 1994-03-23 1997-07-08 Barnea; Eytan R. Preimplantation factor

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