Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-12-16
2004-01-13
Mosher, Mary E. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200, C436S064000
Reexamination Certificate
active
06677128
ABSTRACT:
INTRODUCTION
The present invention relates to a method for identification of cellular protein antigens to which patients with cancer, or patients at risk for cancer, may develop autoantibodies. The method of the invention involves the use of patient derived sera for the identification of the cellular protein antigens using two-dimensional gel electrophoresis followed by Western Blot analysis. The identification of such protein antigens provides novel markers that can be utilized for screening, for diagnostics and prognosis of disease. The invention also provides for the use of the identified protein antigens in immunoassays designed to detect the presence of serum antibodies to the specific protein antigens in sera from individuals that may harbor such antibodies. The invention further relates to the use of the identified antigens as immunogens for stimulation of an immune response in patients expressing such protein antigens. The invention is demonstrated by way of example in which elevated levels of circulating autoantibodies reactive against a tumor specific antigen were identified in sera derived from a lung cancer patient. In addition, elevated levels of circulating autoantibodies reactive against several specific &bgr;-tubulin isoforms were detected in the sera of neuroblastoma patients.
BACKGROUND OF THE INVENTION
Autoantibodies to normal or abnormal cellular proteins are known to be produced by patients in certain diseases such as autoimmune diseases and cardiovascular-related disorders, in some cases even before the disease has produced overt symptoms. However, such autoantibodies have rarely, if ever, been observed in individuals with cancer. Such antibodies to tissue proteins, e.g. p53, may serve as early markers for different types of cancer or for other illnesses. Their detection or the detection of their corresponding antigens in serum or other tissues and body fluids may have utility as indicators of risk for particular types of cancer or for other diseases, as diagnostic markers or as prognostic indicators.
The detection of autoantibodies to cellular antigens and the identification of proteins that have elicited autoantibodies has been accomplished using a variety of approaches. For example, Proliferating Cell Nuclear Antigen (PCNA) was first described as a nuclear antigen which bound antibodies from some patients with lupus erythematosus (Miyachi, K., Fritzler, M. J., and Tan, E. M., 1978, J. Immunol 121:2228-2234). It was subsequently observed that resting lymphocytes did not react with the antibody, in contrast to mitogen stimulated lymphocytes which displayed nuclear staining. This ultimately led to the identification of the protein, designated PCNA which is recognized by this autoantibody in lupus (Tan. E. M., Ogata, K., and Takasaki, Y. 1987, J. Rheumatol., 13:89-96). In some other cases, candidate proteins are singled out and investigated with respect to their ability to induce antibodies in patients, as was investigated for p53 (Crawford, L. V., Firm, D. C. Bulbrook, R. D., 1984, Int J Cancer 30:403-408). In addition, a technique called SEREX relies on serological analysis of recombinant cDNA expression libraries to identify tumor antigens (Old, L., et al. 1998, J. Exp. Med. 187:1163-1167). Thus, many approaches have been followed to search for proteins against which autoantibodies may be produced.
The combination of two different electrophoresis methods (so called “two dimensional” or “2D”-electrophoresis) has been widely utilized to separate proteins in complex mixtures such as tissues or body fluids. The first electrophoresis step generally separates proteins based on their charge. The second electrophoresis step generally separates proteins based on their molecular weight. The use of high resolution two-dimensional electrophoresis allows the simultaneous separation of up to several thousand individual proteins, providing an overall protein map of the protein mixture analyzed. The separated proteins can be visualized in the gel by means of staining with a variety of staining compounds including Coomassie blue or silver. Alternatively, mixtures containing isotopically labeled proteins such as with
25
S methionine, can be visualized by means of autoradiography.
Methods have been developed for the identification of protein(s) that react with a specific antibody among a large number of proteins separated by two-dimensional electrophoresis. The technique of Western blotting can readily reveal the protein with which the antibody reacts if the protein is sufficiently abundant and the antibody is sufficiently specific and with a sufficiently high titer, i.e., high affinity and avidity. The use of whole sera that may contain unknown antibodies against unknown protein antigens present in tumors or precancerous lesions for Western blotting of two dimensional gels has not been reported. Such technology may theoretically be complicated by a large measure of nonspecific reactivity, making it difficult to interpret results. Thus, the methods of the present invention, using Western blotting of two-dimensional gel electrophoresis of complex protein mixtures for the identification of novel antigens for which autoantibodies are present in sera of patients with tumors or with precancerous lesions, is novel.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a procedure for the identification of cellular protein antigens and for the detection of antibodies to specific cellular protein antigens in the serum of patients with cancer or with precancerous lesions. The identification of such protein antigens provides novel markers that can be used for screening, diagnostics and prognosis of disease.
The invention comprises separating antigen-containing protein mixtures by two-dimensional gel electrophoresis followed by transfer of the separated proteins onto a membrane. Specific antigens in the protein mixture are detected by treatment of the membrane with a patient's sera followed by detection of specifically bound antibody by use of a second labeled antibody which specifically binds the first antibody. Separated protein antigens are considered disease specific antigens if they show prominence in the presence of sera suspected of harboring autoantibodies compared to control sera. The source of proteins for two-dimensional analysis includes unfractionated tumors, isolated cancer cells or tumor infiltrating cells or cultured cell lines or subcellular protein fractions such as secreted proteins, membrane proteins, cytosolic or nuclear proteins.
The present invention also relates to the use of the identified protein antigens in immunoassays designed to detect the presence of serum antibodies to the specific protein antigens. Such immunoassays can be utilized for screening, for diagnostics and prognosis of disease. In accordance with the invention, measurement of antibody levels in a patient's sample can be used for the early diagnosis of diseases such as cancer. Moreover, the monitoring of serum antibody levels can be used prognostically to stage progression of the disease.
Additionally, the present invention further relates to the use of the identified protein antigens as immunogens for stimulation of an host immune response against the tumor cells. It is expected that such an approach can be used as a method for inhibiting tumor cell growth or facilitating tumor cell killing in individuals with specific cancers.
In a specific embodiment of the invention described herein, circulating autoantibodies reactive against specific &bgr;-tubulin isoforms, and their cleavage products, were detected in the sera of patients with neuroblastoma. The finding that &bgr;-tubulin isoforms are immunogenic in neuroblastoma patients provides a basis for development of diagnostic methods for neuroblastoma and other cancers in which these &bgr;-tubulin isoforms are expressed, as well as a means for monitoring prognosis of various therapeutic treatments for the disease. In addition the discovery that specific &bgr;-tubulin isoforms are expressed in tumor cells pro
Hanash Samir M.
Hinderer Robert
Misek David
Prasannan Latha
Baker & Botts L.L.P.
Mosher Mary E.
Regents of the University of Michigan
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