Title:  Annexin proteins and autoantibodies as serum markers for cancer

United States Patent:  6,645,465

Issued:  November 11, 2003

Inventors:  Hanash; Samir M. (Ann Arbor, MI); Misek; David (Ann Arbor, MI); Hinderer; Robert (Flint, MI); Beer; David (Chelsea, MI); Brichory; Franck (Ann Arbor, MI)

Assignee:  Michigan, University of the Regents (Ann Arbor, MI)

Appl. No.:  370337

Filed:  August 6, 1999

Abstract

The present invention relates to screening methods for diagnosis, prognosis, or susceptibility to cancer in a subject by means of detecting the presence of serum autoantibodies to specific annexin protein antigens in sera from subjects. The present invention also provides screening methods for diagnosis and prognosis of cancer in a subject by means of detecting increased expression levels of annexin proteins in biological samples of the subject. The method of the invention can also be used to identify subjects at risk for developing cancer. The method of the invention involves the use of subject derived biological samples to determine the occurrence and level of expression of annexin proteins or expression of annexin derived peptides or antigens, and/or the occurrence and level of circulating autoantibodies to specific annexin protein antigens. The present invention further provides for kits for carrying out the above described screening methods. Such kits can be used to screen subjects for increased levels of annexin proteins, or for the detection of autoantibodies to annexin proteins, as a diagnostic, predictive or prognostic indicator of cancer.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide screening 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 elevated levels of annexin autoantibodies in biological samples of subjects. The invention also provides methods for detecting overproduction of annexin proteins and/or overproduction of groups of annexin proteins as a diagnostic or prognostic indicator of cancer.

The present invention relates to diagnostic evaluation and prognosis of cancer by detecting autoantibodies to annexin protein antigens in the serum of subjects with cancer or with precancerous lesions. The detection of increased serum levels of autoantibodies to annexin proteins constitutes a novel strategy for screening, diagnosis and prognosis of cancer.

The present invention provides for the use of the annexin protein antigens in immunoassays designed to detect the presence of serum autoantibodies to the annexin protein antigens. Such immunoassays can be utilized for diagnosis and prognosis of cancer. In accordance with the invention, measurement of annexin autoantibody levels in a subject's serum can be used for the early diagnosis of cancer. Moreover, the monitoring of serum autoantibody levels can be used prognostically to stage progression of the disease.

The invention further relates to assays developed to detect the level of annexin proteins in a subject's sample. Such assays include immunoassays wherein the annexin proteins are detected by their interaction with anti-annexin specific antibodies. For example, annexin antibodies or fragments of antibodies may be used to quantitatively detect the presence and amount of annexin proteins in a subject's sample.

The invention also relates to the use of annexin proteins as antigens to immunize patients suffering from diseases characterized by increased expression levels of the annexin protein antigens. Stimulation of an immunological response to such antigens, is intended to elicit a more effective attack on tumor cells; such as inter alia inhibiting tumor cell growth or facilitating the killing of tumor cells. The identification of autoantibodies to annexin protein antigens associated with particular cancers provides a basis for immunotherapy of the disease.

The invention further provides for pre-packaged diagnostic kits which can be conveniently used in clinical settings to diagnose patients having cancer or a predisposition to developing cancer. The kits can also be utilized to monitor the efficiency of agents used for treatment of cancer. In one embodiment of the invention, the kit comprises components for detecting and/or measuring the levels of autoantibodies directed toward annexin antigens in a sample. In a second embodiment, the kit of the invention comprises components which detect and/or measure annexin antigens in the biological sample.

The present invention is based on the discovery that expression levels of Annexin I and II are increased in tumor tissue samples derived from subjects with pulmonary adenocarcinoma or squamous cell lung cancer. Additionally, increased levels of autoantibodies against Annexin I and II were detected in the serum of the subjects with lung adenocarcinoma and squamous cell carcinoma. The finding that levels of annexin proteins and annexin autoantibodies are increased in samples derived from 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.

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 annexin protein production, or the presence of annexin autoantibodies, in serum or other biological samples from a subject. For purposes of the present invention, annexin proteins are characterized by a canonical motif in which a stretch of approximately 70 amino acids is repeated at least four times (Wallner, B. P. et al., 1986, Nature 320:77-80; Weber, K. and Johnson, N., 1986, FEBS Lett. 203: 95-98; Saris, C. J. M. et al., 1986, Cell 46:201-212; Huang K. S. et al., 1986, Cell 46:191-199).

Specifically, the invention encompasses a method for diagnosis and prognosis of cancer in a subject comprising:

(a) quantitatively detecting annexin protein in a biological sample derived from a subject; and

(b) comparing the level of protein detected in the subject's sample to the level of protein detected in a control sample,

wherein an increase in the level of annexin protein 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. In addition to detecting annexin protein, annexin derived peptides, antigens or differentially modified annexin proteins may be detected for diagnosis and/or prognosis of cancer.

A wide variety of protein mixtures that may contain annexin proteins can be prepared or assayed for the level of protein expression. In a preferred embodiment tumor tissue samples, including but not limited to lung tissue samples, derived from subjects suspected of, or predisposed to cancer can be used to screen for increased levels of annexin protein production.

The present invention encompasses a method for diagnosis and prognosis of a subject with cancer, comprising:

(a) contacting an antibody-containing biological sample derived from a subject, with a sample containing annexin protein antigens under conditions such that an immunospecific antigen-antibody binding reaction can occur; and

(b) detecting the presence of immunospecific binding of autoantibodies present in the subject's biological sample to the annexin protein,

wherein the presence of immunospecific binding of autoantibodies indicates the presence of cancer in the subject.

In a specific embodiment of invention, annexin proteins, including but not limited to Annexin I and II and peptides derived therefrom, are purified and utilized to screen a subject's serum for the presence of circulating autoantibodies to such protein antigens, by means of sensitive and rapid immunoadsorbent assays or by other procedures.

The present invention also provides for kits for carrying out the above-described methods. The methods can be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least a reagent for detecting annexin protein such as an anti-annexin antibody. Alternatively, the diagnostic kits comprise an annexin protein or antigenic protein fragment for detection of annexin autoantibodies in a subject derived sample.

The present invention is based on the discovery that levels of Annexin I and II proteins are increased in adenocarcinoma and squamous cell lung tumors derived from subjects with cancer. Additionally, increased levels of circulating autoantibodies reactive against annexin proteins have been detected in the serum of subjects having adenocarcinoma and squamous cell lung tumors.

5.1. Assays for Detection of Annexin Production

In accordance with the invention, measurement of levels of annexin proteins in samples derived from a subject can be used for the early diagnosis of diseases such as cancer. Moreover, the monitoring and quantitation of annexin protein levels can be used prognostically to stage the progression of the disease and to evaluate the efficacy of agents used to treat a cancer subject.

The detection of annexin proteins in a sample from a subject can be accomplished by any of a number of methods. Preferred diagnostic methods for the detection of annexin proteins in the biological sample of a subject can involve, for example, immunoassays wherein annexin proteins are detected by their interaction with an annexin specific antibody. Antibodies useful in the present invention can be used to quantitatively or qualitatively detect the presence of annexins or antigenic fragments thereof. In addition, reagents other than antibodies, such as, for example, polypeptides that bind specifically to annexin proteins can be used in assays to detect the level of annexin protein expression. Alternatively, detection of annexin proteins may be accomplished by detection and measurement of levels of biological properties associated with annexin proteins, such as for example, phopholipase A, and anticoagulant activity.

Immunoassays useful 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 annexin proteins, such as lung tissue or other biological tissue, is obtained from a subject suspected of having a particular cancer or risk for cancer. Aliquots of whole tissues, or cells, are solubilized using any one of a variety of solubilization cocktails known to those skilled in the art. For example, tissue can be solubilized by addition of lysis buffer comprising (per liter) 8 M urea, 20 ml of Nonidet P-40 surfactant, 20 ml of ampholytes (pH 3.5-10), 20 ml of 2-mecaptoethanol, and 0.2 mM of phenylmethylsulfonyl fluoride (PMSF) in distilled deionized water.

Immunoassays for detecting expression of annexin protein typically comprise contacting the biological sample, such as a tissue sample derived from a subject, with an anti-annexin antibody under conditions such that an immunospecific antigen-antibody binding reaction 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 production of annexin proteins wherein the detection of increased production of annexin proteins is an indication of a diseased condition. The levels of annexin protein in a biological sample are compared to norms established for age and gender-matched 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 tissue extract 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 annexin 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 annexin 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.; Voller, 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 phosphatase. Detection can also be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme.

Detection of annexin 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 annexin protein 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 annexin antibody. The presence of a bioluminescence protein is determined by detecting the presence of luminescence. Important bioluminescence compounds for purposes of labeling are luciferin, luciferase and aequorin.

In a specific embodiment of the invention, the levels of annexin proteins in biological samples can be analyzed by two-dimensional gel electrophoresis. Methods of two-dimensional electrophoresis are known to those skilled in the art. Biological samples, such as tissue samples, are loaded onto electrophoretic gels for isoelectric focusing separation in the first dimension which separates proteins based on charge. A number of first-dimension gel preparations may be utilized including tube gels for carrier ampholytes-based separations or gels strips for immobilized gradients based separations. After first-dimension separation, proteins are transferred onto the second dimension gel, following an equilibration procedure and separated using SDS PAGE which separates the proteins based on molecular weight. When comparing biological samples derived from different subjects, multiple gels are prepared from individual biological samples (including samples from normal controls).

Following separation, the proteins are transferred from the two-dimensional gels onto membranes commonly used for Western blotting. The techniques of Western blotting and subsequent visualization of proteins are also well known in the art (Sambrook et al, "Molecular Cloning, A Laboratory Manual", 2^nd Edition, Volume 3, 1989, Cold Spring Harbor). The standard procedures may be used, or the procedures may be modified as known in the art for identification of proteins of particular types, such as highly basic or acidic, or lipid soluble, etc. (See for example, Ausubel, et al., 1999, Current Protocols in Molecular Biology, Wiley & Sons, Inc., N.Y.). Antibodies that bind to the annexin proteins are utilized in an incubation step, as in the procedure of Western blot analysis. A second antibody specific for the first antibody is utilized in the procedure of Western blot analysis to visualize proteins that reacted with the first antibody.

The detection of annexin protein levels in biological samples can also be used to monitor the efficacy of potential anti-cancer agents during treatment. For example, the level of annexin protein production can be determined before and during treatment. The efficacy of the agent can be followed by comparing annexin expression throughout the treatment. Agents exhibiting efficacy are those which decrease the level of annexin protein production as treatment with the agent progresses.

The present invention is demonstrated by way of example wherein elevated levels of annexin proteins have been detected in tissue samples derived from patients with lung adenocarcinoma and squamous cell lung carcinoma. In particular, increased levels of Annexin I and II were detected in samples derived from lung cancer patients. The detection and/or quantitative measurement of annexin proteins in biological samples can be used in screening of subjects who are at risk for developing certain types of cancers or other proliferative disorders in which the annexin proteins are overexpressed. In addition, qualitative differences in the pattern of occurrence in serum or biological fluids of different members of the annexin family of proteins can be used as a screening, diagnostic or prognostic indicator of cancer or cancer risk.

5.2. Assays for Detection of Anti-Annexin Autoantibodies

The present invention provides diagnostic and prognostic methods for diseases such as cancer based on detection of circulating annexin autoantibodies in a subject. The method is validated by the use of a biological sample from a subject with cancer and from age and gender matched controls, without cancer. A biological sample which may contain autoantibodies, such as serum, is obtained from a subject suspected of having a particular cancer or suspected of being predisposed to developing cancer. A similar body fluid is obtained from a control subject that does not have cancer.

In accordance with the invention, measurement of autoantibodies reactive against the annexin protein antigens can be used for the early diagnosis of diseases such as cancer. Moreover, the monitoring of autoantibody levels can be used prognostically to stage the progression of the disease. The detection of autoantibodies in a serum sample from a patient can be accomplished by any of a number of methods. Such methods include immunoassays which 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.

Such an immunoassay is carried out by a method comprising contacting a serum sample derived from a subject with a sample containing the annexin protein antigens under conditions such that an immunospecific antigen-antibody binding reaction can occur, and detecting or measuring the amount of any immunospecific binding by the autoantibody. In a specific aspect, such binding of autoantibody by tissue sections, for example, can be used to detect the presence of autoantibody wherein the detection of autoantibody is an indication of a diseased condition. The levels of autoantibodies in a serum sample are compared to the levels present in an analogous serum sample from a subject not having the disorder.

The immunoassays can be conducted in a variety of ways. For example, one method to conduct such assays involves anchoring of annexin protein onto a solid support and detecting anti-annexin antibodies specifically bound thereto. The annexin proteins to be utilized in the assays of the invention can be prepared via recombinant DNA techniques well known in the art. For example, a DNA molecule encoding an annexin protein or an antigenic fragment thereof can be genetically engineered into an appropriate expression vector for large scale preparation of annexin protein. It may be advantageous to engineer fusion proteins that can facilitate labeling, immobilization or detection of the annexin protein. See, for example, the techniques described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Alternatively, the annexin protein may be purified from natural sources, e.g., purified from cells, using protein separation techniques well known in the art. Such purification techniques may include, but are not limited to molecular sieve chromatography and/or ion exchange chromatography. In practice, microtitre plates are conveniently utilized as the solid support for the annexin proteins. The surfaces may be prepared in advance and stored.

The present invention is demonstrated by way of example wherein elevated levels of circulating autoantibodies reactive against several annexin protein antigens have been detected in the sera of cancer patients. The detection and/or quantitative measurement of circulating anti-annexin autoantibodies in serum can be used in screening of subjects who are at risk for cancer or other proliferative disorders in which annexin protein levels are increased.

5.3. Immunotherapy

The invention also relates to the use of annexin proteins as antigens to immunize patients suffering from diseases characterized by the production of the annexin protein antigens. Stimulation of an immunological response to such antigens, is intended to elicit a more effective attack on tumor cells; such as inter alia inhibiting tumor cell growth or facilitating the killing of tumor cells. The identification of autoantibodies to annexin protein antigens associated with particular cancers provides a basis for immunotherapy of the disease.

The patient may be immunized with the annexin protein antigens to elicit an immune response which facilitates killing of tumor cells or inhibiting tumor cell growth. The annexin protein antigens can be prepared using the methods described above for purification of proteins.

In an embodiment of the invention an immunogen comprising a purified annexin protein antigen to which a patient with cancer has developed autoantibodies, is used to elicit an immune response. For administration, the annexin protein antigen should be formulated with a suitable adjuvant in order to enhance the immunological response to the protein antigen. Suitable adjuvants include, but are not limited to mineral gels, e.g. aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and potentially useful human adjuvants such as BCG (bacilli Calmett-Guerin) and (Corynebacterium parvum). Many methods may be used to introduce the formulations derived above; including but not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous.

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 an annexin peptide (for detection of Annexin autoantibodies) or an annexin antibody reagent (for detection of annexin 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 comprises components for detecting and/or measuring human IgG antibodies directed toward annexin antigen. As one example, where the antibodies are detected and/or measured by enzyme linked immunoabsorbent assay (ELISA), such components may comprise target antigen, in the form of at least one and preferably a plurality of different annexin antigens or epitopes thereof, linked to a solid phase, and a means for detecting a human antibody bound to target antigen. Such means for detection may be, for example, an antibody directed toward the constant region of human IgG (e.g., rabbit anti-human IgG antibody), which may itself be detectably labeled (e.g., with a radioactive, fluorescent, colorimetric or enzyme label), or which may be detected by a labeled secondary antibody (e.g., goat anti-rabbit antibody).

In a second series of nonlimiting embodiments, a kit according to the invention may comprise components which detect and/or measure annexin antigens in the biological sample of a subject. For example, where annexin proteins are detected and/or measured by enzyme linked immunoabsorbent assay (ELISA), such components may comprise an antibody directed to epitopes of the annexin proteins which can be used to detect and/or quantitate the level of annexin expression in the biological sample. The antibody itself may be detectably labeled with a radioactive, flourescent, colorimetric or enzyme label. Alternatively, the kit may contain a labeled secondary antibody.

Claim 1 of 6 Claims

We claim:

1. A method for diagnosing lung cancer in a subject comprising:

(i) contacting a serum sample derived from the subject with one or more protein antigens wherein said protein antigens are Annexin I and Annexin II protein antigens; and

(ii) detecting immunospecific binding of Annexin I or Annexin II autoantibodies in the serum sample,

wherein an increase in the level of immunospecific binding detected in step (ii) as compared to a control serum sample is an indicator of lung cancer.

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