TW200827718A - Alpha-enolase specific antibody and method of use - Google Patents

Abstract

This invention relates to a method of monitoring cancer development by determining the abundance of alpha-enolase protein wherein increased abundance is an indication of the severity of cancer. In another embodiment, the invention relates to a method of detecting cancer malignancy by determining the abundance of alpha-enolase antibodies in a sample wherein low levels of such antibodies indicates the malignancy of cancer. Also provided is a method of suppressing tumor growth by inducing the anti-ENO1 immune response.

Description

200827718 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a method for monitoring the development of cancer by measuring the content of α-enolase in cancer cells, and the present invention also relates to a method A method for detecting the presence or absence of cancer and the degree of deterioration thereof by measuring the richness of anti-enolase antibodies in serum. Further, a method for inhibiting tumor growth by inducing an anti-α-enolase immunoreaction is also disclosed in the specification. [Prior Art] Tumors arise from a single hyperplasia of a single cell and then into a cancer cell (Collins, F.S. and Trent, J.M. 2001. Cancer Genetics· Harrison's Principles of Internal Medicine 503·). Cancer is characterized by the spontaneous growth of tumor cells and transfer to other tissues (Fenton, RG and Longo, DL 2001. Cell Biology of Cancer. Harrison's Principles of Internal Medicine 509.) o Tumor cells produce immune systems Identification of specific antigens, these tumor cell-associated antigens contain mutated normal cellular proteins, abnormally expressed cellular proteins, abnormal cell surface proteins and oncogene viral proteins, but not only this (Abbas, AK, Lichtman, AH, And Pober 5 JS 2000. Cellular and Molecular Immunology 386-391). Therefore, in theory, the immune system treats these tumor-associated antigens as foreign invaders and destroys tumor cells without harming normal cells. Accordingly, if these tumor-associated antigens can be identified, 5 200827718 will contribute to the clinical application of cancer in clinical prognosis and treatment.

The degree of cancer deterioration can be determined by the condition of pleural effusion (Pleural Effusion), which is the excess water produced in the space between the lungs and the chest wall (Light, RW· 2000· Disorders of the Pleura, Mediastinum). , and Diaphragm· Harrison's Principles of Internal Medicine 1513-1514). Lung cancer, breast cancer, and lymphoma can cause about 75% of patients with malignant pleural effusions (Light, R.W. 2000. Disorders of the Pleura, Mediastinum, and Diaphragm. Harrison's Principles of Internal Medicine 1513-1514). Malignant effusion is usually caused by the infiltration of lymphocytes and tumor cells. Some immune complexes associated with lung tumors (Andrews, BS·, Arora, NS, Shadforth, MF, Goldberg, SK, and Davis, JSt 1981. The role of immune complexes in the pathogenesis of pleural effusions· Am Rev Respir Dis 124:115-120· ; Zeng, CQ·, Alpert, LC” and Alpert, E. 1992. Characterization of a lung cancer-associated auto-antigen. Int J Cancer 52:523-529.; Gorsky, Y·, Weiss , I·, and Sulitzeanu, D. 1977. Complexes of breast-cancer-associated antigen (s) and corresponding antibodies in pleural effusions from a patients with breast cancer. Isr J Med Sci 13:844-847.) or autoantibodies For example, anti-p53 antibody (Lai, C丄·, Tsai, CM·, Tsai, Τ·Τ·, Kuo, Β·Ι·, Chang, K_T·, Fu, HT, Perng, RP? and Chen, JY 1998 Presence of serum anti-p53 antibodies is associated with pleural effusion and 6 200827718

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y poor prognosis in lung cancer patients· Clin Cancer Res 4:3025-3030·), antinuclear antibody (Wang, DY·, Yang, PC·, Yu, WL·, Kuo, SH·, and Hsu ,Ν·Υ· 2000. Serial antinuclear antibodies titer in pleural and pericardial fluid. Eur Respir J 15:1106-1110.) and anti-L-Myc antibody (Yamamoto, A·, Shimizu, E_, Sumitomo, K·, Shinohara L.A., Namikawa, 0, Uehara, H., and Sone, S. 1997. L-Myc overexpression and detection of auto-antibodies against L-Myc in both the serum and pleural effusion from a patient with non-small cell Lung cancer. Intern Med 36: 724-727.), has been found in hydronephrosis and is associated with poor cancer prognosis. Some antigens associated with lung tumors are also found in malignant water, such as cytokeratine 19 fragments, neuron-specific enolase (EN02), squamous cell carcinoma antigen ( Miedouge, M., Rouzaud, P., Salama, G., Pujazon, MC., Vincent, C., Mauduyt, Μ·Α·, Reyre, J., Carles, P., and Serre, G. 1999. Evaluation Of seven tumour markers in pleural fluid for the diagnosis of malignant effusions. Br J Cancer 81:1059-1065·) and soluble HLA_I (Amirghofran, Z·, Sheikhi, AK, Kumar, PV, and Saberi Firouzi, M. 2002. Soluble HLA class I molecules in malignant pleural and peritoneal effusions and its possible role on NK and LAK cytotoxicity. J Cancer Res Clin Oncol 128: 443-448. Epub 2002 Aug 2009.) and so on. 7 200827718 SUMMARY OF THE INVENTION The present invention is directed to a method of monitoring the development of cancer comprising determining the extent to which cancer cells are enriched with alpha-enolase, wherein the increase in abundance is associated with the severity of the cancer ( Severe) related. In another embodiment, the degree of enrichment is determined by measuring the binding of the alpha-leanolase specific disassembly to the alpha-enolase. The present invention also provides a method for producing an α-enolase-specific antibody, which comprises obtaining primers containing sequence number: 1 and SEQ ID NO: 2 or degenerate variants thereof. Cloning a complementary deoxyribonucleic acid (cDNA), and obtaining a recombinant protein by expressing the complementary deoxyribonucleic acid, and then using the recombinant protein to prepare a plurality of antibodies or single antibodies Strain antibody. The invention further provides a method for detecting the degree of cancer and malignacy thereof, comprising determining the richness of an anti-α-enolase-specific antibody in a serum sample, wherein the low-level anti-α-enolase specific Sexual antibodies represent the presence and deterioration of cancer. Further, the present invention provides a method of inhibiting tumor growth comprising the step of inducing an anti-α-enolase immunoreaction. Additional objects and advantages of the invention will be set forth in part in the description which follows. The objects and advantages of the invention will be apparent from the <RTIgt; It is to be understood that the above description of the invention is not intended to limit the scope of the invention. The accompanying drawings, which are incorporated in the specification of the claims [Embodiment] In the present invention, a purified lung cancer effusion autoantibody is used as a probe to detect a tumor-associated antigen, i.e., p48 antigen, in malignant pleural effusion of a lung cancer patient. In the examples, detailed analysis by biochemical enrichment procedures and mass spectrometry confirmed that the p48 antigen is a human alpha enolase (alpha-enolase or EN01). - enolase in addition to the case of up-regulation in cancer patients, and further provides a method of monitoring the development of cancer, comprising determining the degree of enrichment of alpha-enolase in cancer cells, wherein the degree of enrichment Increases are associated with cancer progression. Specifically, in monitoring the development of cancer, the degree of enrichment of α-enolase in cancer cells of the more advanced stages of cancer is higher. Furthermore, the higher the enrichment of α-enolase in cells, the more likely it is to relapse. In another embodiment, the degree of enrichment is determined by measuring the binding of an alpha enolase specific antibody to an alpha enolase. The α-enolase specific anti-system utilizes a vector containing an α-enolase cDNA to transfect Escherichia coli and purify the recombinant α-deptanol produced therefrom, 200827718 for injection An animal is produced by inducing an immune response in the animal. The α-enolase cDNA is obtained from a primer having a SEQ ID NO: 2 and SEQ ID NO: 2 or a degenerate variant thereof. Further, the enolase-specific antibody may be a monoclonal antibody or a plurality of antibodies. In another embodiment, when monitoring cancer development, the richness can be determined by Western blot, surface staining, flow cytometry analysis, immunohistochemistry. Staining, quantitative reverse transcriptase-PCR, microarray analysis, or other suitable methods known to those of ordinary skill in the art. The present invention also provides a method for detecting cancer by measuring the richness of an anti-α-enolase-specific antibody in the present invention, wherein a low-level anti-α-enolase-specific antibody represents the presence of cancer And worsened. The sample can be a serum sample or a sample of pleural effusion. The so-called low-α-enolase-specific anti-system means that the content of the anti-α-enolase-specific antibody is statistically significantly lower than that of a healthy person (Ρ值&lt;〇·〇1). The richness of the anti-α-enolase-specific antibody can be known by the sandwich ELISA (enzyme linked immimosorbent assay), the Western blot method, or other common knowledge in the art. The appropriate method to measure. Furthermore, the cancer used to monitor the development of cancer may be non-small cell lung cancer. In one embodiment, non-small cell lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and 200827718 large cell carcinoma may also be used. The present invention describes a method for inhibiting tumor growth by inducing an immune response to an α-enolase antibody. It is induced by directly administering to the patient an anti-α-enolase-specific antibody or an α-enolase antigen to induce an anti-α-i enolase immunoreactivity. Comparing patients who developed high-valency α-enolase antiserum with those who did not produce antiserum, the former was found to be relatively small in tumor size. (a), noun definition: ® The term "isolated" or "purified" as used in this specification means the removal of a substance from its original environment (for example, if it exists naturally, Refers to the natural environment). For example, a polynucleotide or polypeptide that is found to be naturally present in a living animal is not isolated, but the same polynucleotide or polypeptide is separated from its native environment, It is single. The ligated polynucleotide may be part of a vector, and/or the singly isolated polynucleotide or singly isolated polypeptide may also be part of a composition as long as the B vector or the composition is not The isolated polynucleotide and the isolated environment in which the isolated polypeptide is originally present may be used. The term "antibody" as used in this specification is intended to be generally defined by its y, as well as immunoglobulins, autoantibodies, monoclonal antibodies and polyclonal antibodies, and fragments of antibodies. The antibody of the present invention w may be chimeric, humanized or human. The term "autoantibody" as used in the specification, 11 200827718 It means an antibody against its own antigen. For example, the immune system of an organism is induced by its own normal and antigenic endogenous body composition, producing antibodies against the composition of the organism itself. As used herein, the term "monoclonal antibody" means a chemically and immunologically homologous antibody produced by a hybridoma, see A Laboratory Manual, Harlowe And Lane, eds·, Cold Spring Harbor, Ν·Υ· (1998). The term "polyclonal antibody" as used in the present specification means an antibody produced by synthesizing plasma cells (B lymphocytes) of one or more of the antibodies formed by an immune reaction against the same antigen. These antibodies are usually made by the body of an animal immunized with an antigen. As used herein, the term "a-enolase specific antibody" means a highly specific antibody to human ENOl, but not to human EN02 and EN03. The term "anti-a-enolase antibody" as used in the present specification means an antibody that binds to an α-enolase.

The term "recombinant DNA" as used in this specification means that the arrangement of nucleic acid sequences does not occur in nature. More specifically, in the natural world, contiguous nucleic acid sequences identical to those of the DNA are not found, at least in order of arrangement, orientation or spacing. In addition, the present specification states that the standard DNA recombination and molecular cloning techniques mentioned are in the art of the prior art. For detailed reference, please refer to Sambrook et al. Molecular Cloning: A 12 200827718 laboratory Manual and Cold Spring Harbor Laboratory Press : Cold Spring Harbor, 1989· A “recombinant protein”. As for the term "recombinant protein", it refers to the protein produced by the use of recombinant DNA. As used herein, the term "degenerative variants" means any or all of the possible nucleic acid sequences which can be directly translated into amino acid sequences according to standard genetic codes and which are translated into amino acid sequences. The amino acid sequence is translated from the reference nucleic acid sequence or any genetic code that retains the function of the reference nucleic acid. The term "primer" as used in this specification means a polynucleotide chain which is capable of adding a deoxyribonucleotide to the chain end by the action of a DNA polymerase. As used herein, the term "effusions or effusion" means the exudation of fluid into a body cavity or tissue. For example, pleural effusion and tuberculous pleural effusion. The term "low level" as used in this specification refers to an anti-α-enolase antibody in a patient. The content was statistically significantly lower than that of healthy people, with a Ρ value of &lt; 0.01. As used herein, the term "patient" means a mammal, including but not limited to humans, primates, domesticated mammals, and mammals in laboratories, and the like. As used herein, the term "monitoring" means to detect and/or observe the progression of cancer by measuring the extent of alpha-enolase in cancer cells. 13 200827718 The term "abundance" as used in this specification means the degree of expression of alpha-enolase in single or multiple cells. In various embodiments, an increase in richness is associated with cancer progression. Cancer cells with advanced cancer are highly rich. In addition, the more abundant cells are more likely to have cancer recurrence. Richness can be measured by measuring the binding of α-enolase to α-enolase-specific antibodies, Western blot, surface staining, flow cytometry analysis , immunohistochemical staining (IHC), quantitative reverse transcriptase-PCR (RT-PCR) and/or microarray analysis, etc., but not limited to the above The method. (2) Autoantigen/autoantibody of lung cancer-α-enolase: In the present invention, a purified lung cancer effusion autoantibody is used as a probe to detect in malignant pleural effusion of lung cancer patients. Tumor-associated antigen, ρ48 antigen. In the examples, it was described that the p48 antigen was confirmed to be a human α-enolase (ENO1) by analysis of biochemical enrichment procedures and mass spectrometry. In the earliest studies, enolase was thought to be an enzyme involved in glycolytic metabolism, but recent evidence has shown that enolase is a multifunctional protein (Kim, JLW·, and Dang). , CV 2005. Multifaceted roles of glycolytic enzymes. Trends Biochem Sci 30: 142-150·). The study found that in the milk animal cells, there are three isomeric forms of enolase, namely α-enolase (ΕΝ01), β-enolase (ΕΝ03) and γ-enolase (ΕΝ02). The performance of the three enol 200827718 enzyme isomers is regulated by a tissue-specific approach. Among them, α-enolase is widely present in various tissues, and γ-enolase and β-enolase are mainly found in neurons (or neuroendocrine tissues) and muscle tissues (Pancholi, V. 2001· Multifunctional alpha-enolase: its role in diseases. Cell Mol Life Sci 58: 902-920.). These enolase isomers form heterodimers or homodimers, which catalyze the conversion of phosphoglycerides to acid enolpyruvate during glycolysis. In addition to the function of glycolytic action, the presence of α-enolase has recently been found on the cell surface, and its function as a receptor for plasminogen (Redlitz, A·, Fowler, BJ·, Plow) , EF·, and Miles, LA·1995· The role of an enolase-related molecule in plasminogen binding to cells· Eur J Biochem 227:407-415·), which means that the α-enolase on the cell surface may be It plays a role in tissue invasion. In the hypoxia state, α-sterolase is one of many up-regulated stress proteins, and its function is presumed to be protected by increasing anaerobic metabolism. Cell survival (Jiang, Β·Η·, Agani, F·, Passaniti, A” and Semenza, GL 1997. V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth Factor and enolase 1: involvement of HIF-1 in tumor progression. Cancer Res 57:5328-5335.) Using a selectable translation initiation codon (translation 15 200827718 start codon), a-enolase transcript ( Transcript) can be translated into a myk promoter-binding protein-1 (ΜΒΡ·1) with a molecular weight of 37 kDa, which is located in the nucleus and is thought to interact with the c-myc Ρ2 promoter. Binding, the c-myc gene cannot be translated and the cell growth is blocked (Ray, RB, and Steele, R. 1997. Separate domains of MBP-1 involved in c-myc promoter binding an d growth suppressive activity. Gene 186:175-180.) ° Studies have reported that high-valency autoantibodies against enolase are associated with a variety of different systemic or organ-specific autoimmune diseases (Gitlits) , VM·, Toh, BH·, and Sentry, JW·2001· Disease association, origin, and clinical relevance of autoantibodies to the glycolytic enzyme enolase. J Investig Med 49:13 8-145.). In these autoimmune diseases Among them, a-enolase-specific autoantibodies have been found in patients suffering from diseases such as kidney disease, viral hepatitis, and systemic lupus erythematosus. Although γ-enolase has been shown to be a product of several tumors (Gomm, SA·, Keevil, BG·, Thatcher, Ν·, Hasleton, PS, and Swindell, RS 1988. The value of tumour markers in lung cancer· Br J Cancer 58: 797-804.), but as far as the inventors are aware, there have been no autoantibody-related reports of alpha-enolase in any cancer patient. A recent study suggested that α-enolase may be an antigenic target for human oral squamous cancer cells (Miyazaki, A·, Sato, Ν., Takahashi, S·, Sasaki, A·, Kohama, G ·, Yamaguchi, A·, 16 200827718

Yagihashi, A·, and Kikuchi, K. 1997. Cytotoxicity of histocompatibility leukocyte antigen-DR8-restricted CD4 killer T cells against human autologous squamous cell carcinoma. Jpn J Cancer Res 88:191-197· ; Sato, N·, Nabeta, Y·, Kondo, H·, Sahara, H·, Hirohashi, Y·, Kashiwagi, K·, Kanaseki, T·, Sato, Y·, Rong, S·, Hirai, I·, et al· 2000. Human CD8 And CD4 T cell epitopes of epithelial cancer antigens. Cancer Chemother Pharmacol 46: S86-90_) and can be recognized by autologous CD4+ T cells. Therefore, enolase-specific humoral immunity may be associated with disease progression. To date, cancer-related retinopathy has been the only clinical case associated with α-enolase autoantibodies, but the mechanism for improving tumor immunity and how to cause visual symptoms is unclear. In general, large amounts of tumor cells or abnormal gene expression (Chen, vaccine·Τ······················ An important source of autoantigens for immune diseases. In the study of the present invention, the presence of an autoantibody of α-enolase was confirmed in cancer patients. A method of preparing an α-enolase specific antibody is further proposed in the study of the present invention. The method comprises using an extract comprising SEQ ID NO: 1 and SEQ ID NO: 2 or a degenerate variant thereof to obtain an α-enolase complementary deoxyribonucleic acid for transduction, and by expressing the complementary deoxyribonucleic acid A recombinant protein is obtained, and then the recombinant protein is used to prepare a plurality of antibodies. In addition, it is more isolated from the water of cancer patients to fight against humans. 17 200827718 Autoantibody of α-enolase. (C), using oc-enolase (ΕΝΟ 1) to monitor cancer development:

Although changes in alpha-enolase gene regulation have been observed in some types of cancer, the extent of their performance is quite controversial. In some highly tumorigenic or metastatic cell lines, the regulation of α-enolase at the gene level can be found in alveolar type II pneumocyte ( Peebles, Κ·Α., Duncan, MW·, Ruch, RJ·, and Malkinson, AM 2003. Proteomic analysis of a neoplastic mouse lung epithelial cell line whose tumorigenicity has been abrogated by transfection with the gap junction structural gene for connexin 43 , Gjal. Carcinogenesis 24:651-657·), small cell lung cancer (Zhang, L., Cilley, RE·, and Chinoy, MR 2000. Suppression subtractive hybridization to identify gene expressions in variant and classic small cell lung cancer cell lines. J Surg Res 93:108-119·), as well as head and neck cancer cells (Wu, W·, Tang, X·, Hu, W., Lotan, R., Hong, WK·, and Mao, L. 2002. Identification and Validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry Clin Exp Metastasis. 19:319-326.) o Similarly, previous studies of enzyme activity measurements in breast cancer studies have concluded that alpha-enolase may be involved in cancer conditions. Process (Hennipman, A., Smits, J., van Oirschot, Β·, van Houwelingen, JC·, Rijksen, G., Neyt, JP, Van Unnik, JA·, 18 200827718 and Staal, GE Glycolytic enzymes in breast Cancer, benign breast disease and normal breast tissue.251-263.) Recently, in a bioinformatics study using gene chips and ESTs databases, 18 of the 24 types of cancerous tumors overexpressed EN01. (Altenberg, B.? and Greulich, KO 2004. Genes of glycolysis are ubiquitously overexpressed in 24 cancer classes. Genomics 84:1014-1020·) o

The gene that activates the glycolytic enzyme is critical for cancer cells to adapt to the hypoxic microenvironment. In the absence of oxygen, many genes are activated by the hypoxia-inducible transcription factor (Semenza, GL·, Jiang, Β·Η·, Leung, SW·, Passantino, R·, Concordet, JP., Maire, P., and Giallongo, A. 1996. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J Biol Chem 271:32529- 32537.) is regulated, and α-enolase is one of them. Recently, some data show that carcinogenic AKT and Myc can directly activate glycolytic, and EN01 is one of the genes regulated by Myc (Kim, JW, Zeller, Κ·Ι·, Wang, Y·, Jegga, AG). ·, Aronow, BJ·, O'Donnell, Κ·Α·, and Dang, CV 2004. Evaluation of myc E-box phylogenetic footprints in glycolytic genes by chromatin immunoprecipitation assays. Mol Cell Biol 24:5923-5936·) o The loss of gene regulation of a-enolase can be found in some cancers and is often highly correlated with cancer progression (Peebles, KA, Duncan, 19 200827718).

MW·,Ruch,RJ·,and Malkinson,Α·Μ·2003. Proteomic analysis of a neoplastic mouse lung epithelial cell line whose tumorigenicity has been abrogated by transfection with the gap junction structural gene for connexin 43, Gjal. Carcinogenesis 24:651 -657. ; Zhang, L·, Cilley, RE·, and Chinoy, MR 2000. Suppression subtractive hybridization to identify gene expressions in variant and classic small cell lung cancer cell lines. J Surg Res 93:108-119. ; Altenberg, B., and Greulich, KO 2004. Genes of glycolysis are ubiquitously overexpressed in 24 cancer classes. Genomics 84:1014-1020·). The above research results are in response to Holland (Holland, JP., Labieniec, L., Swimmer, C., and Holland, MJ 1983. Homologous nucleotide sequences at the 5! termini of messenger RNAs synthesized from the yeast enolase and glyceraldehyde-3- J. Biol Chem 258:5291-5299.) and Giallongo (Giallongo, A·, Feo, S., Moore, R., Croce, CM·,and Showe,LC 1986· Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha-enolase. Proc Natl Acad Sci USA 83:6741-6745.) Early observation, showing a-enolase Increased performance on cells in the exponential growth phase, but only very low performance during the stationary phase of cell growth. 20 200827718 However, in a recent study on the relationship between the performance of α-enzymes and clinical outcomes, quite different conclusions were presented. The study used 9C12 monoclonal antibodies and found in patients with non-small cell lung cancer (Chang, YS·, Wu, W., Walsh, G., Hong, WK·, and Mao, L. 2003. Enolase- Alpha is frequently down-regulated in non-small cell lung cancer and predicts aggressive biological behavior. Clin Cancer Res 9:3641-3644.), whose performance of 〇1_enolase is down-regulated. The 9C12 monoclonal antibody was first developed by Redlitz's laboratory (Redlitz, A., Fowler, BJ, Plow, EF, and Miles, LA·1995. The role of an enolase-related molecule in plasminogen binding to cells). Eur J Biochem 227:407-415·), which can identify the 54kKDa and 48KDa proteins in U937 cell lysates, as well as the purified α-enolase protein in the human brain, meaning that in the above experiments, except for α- In addition to the enolase protein, an additional α-enolase-reiated molecule (ERM) is present. In addition, although the 9C12 monoclonal antibody did not cross-react with rabbit β-denselase in the aforementioned report (er〇ss reacti〇n), whether it has cross-reacted to γ-enolase has not been confirmed. Research published. In fact, neuron-specific enolase-gamma-enolase has also been confirmed in lung tissue. For example, in the above studies, enolase was also present in the tumor cell line of CA926. Thus, the inventors believe that the use of specific antibodies to individual enolase isomers to resolve the role of various enolase isomers in the pathogenesis of disease is an important key. The present invention demonstrates that the expression of the central enolase in cancer cells of lung cancer patients is regulated by 21 200827718, and further provides a method for monitoring the development of cancer, including measuring the enrichment of α-enolase in cancer cells, Increased richness is associated with cancer progression. Specifically, in monitoring the development of cancer, the degree of enrichment of α-enolase in cancer cells of more advanced cancers is higher. Furthermore, if the abundance of α-enolase in the cells is higher, the cancer is more likely to recur. In another embodiment, the degree of enrichment is determined by measuring the binding of an alpha enolase specific antibody to an alpha enolase. The α-enolase-specific anti-system transfects Escherichia coli with a vector containing an α-enolase cDNA, and purifies the recombinant α-enolase produced therefrom for injecting an animal. Manufactured by inducing an immune response in the animal. The α-enolase cDNA is obtained from an extractor having SEQ ID NO: 1 and SEQ ID NO: 2 or a degenerate variant thereof. Further, the α-enolase-specific antibody may be a monoclonal antibody or a plurality of antibodies. In another embodiment, when monitoring the development of cancer, the richness can be determined by Western blotting, surface staining, flow cytometry analysis, immunohistochemical staining, quantitative reverse transcriptase- PCR (quantitative reverse transcriptase-PCR), microarray analysis or other methods known to those skilled in the art are known. The present invention also provides a method for detecting cancer by measuring the richness of an anti-α-enolase-specific antibody in the present, wherein a low-level anti-enolase-specific antibody represents the presence and deterioration of cancer. The sample can be a serum sample or a sample of pleural effusion. The so-called low anti-α-enolase specific anti-system means that the content of the α-enolase-specific antibody is statistically significantly lower 22 200827718 The antibody content in healthy persons (P value &lt; 0.01). The enrichment of the α-enolase-specific antibodies can be measured by sandwich-type sandwich enzymes in combination with immunosorbent assays, western blotting or other methods known to those of ordinary skill in the art.

In addition, the cancer used to monitor the development of cancer may be non-small cell lung cancer, or non-small cells selected from adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Lung cancer D (IV), α-enolase function: The general view is that excessive expression of glycolytic enzymes, such as α-enolase, may promote anaerobic glycolytic action of cancer cells, and disease progression Highly relevant (Gatenby, RA·, and Gillies, RJ· 2004· Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4··891-899·). In the present invention, α-enolase is confirmed in hydrostatic tumor cells by obtaining an antiserum of a rabbit which is highly specific to human a-enolase but does not cross-react with other enolase isomers. There is a significant overexpression in the tumor portion of the lung cancer tissue sample. The distribution of α-enolase was also found on the cell surface of the tumor. Furthermore, the present inventors have also found that the degree of expression of α-enolase is closely related to clinical results in immunohistochemical staining studies. The inventors believe that alpha-enolase plays a specific role in immune regulation. The humoral immune response in cancer patients with a lower anti-α-enolase may be attributed to the excessive expression of cancer cells after cancer formation a- 23 200827718

Enolase, which produces an immunosuppressive effect. The production of immunosuppressive effects contributes to the metastasis of cancer cells. In general, the internal cells of the primary tumor lack sufficient nutrients and oxygen supply during the growth process, resulting in tumor necrosis. Necrotic tumor cells overexpress and release alpha-enolase prior to necrosis, and these alpha-enolases may be presented to the axillary lymphocytes or sputum lymphocytes by antigen-presenting cells. This type of autologous CD4+ T lymphocyte, which recognizes α-enolase and is considered an immune antigen, has recently been found in patients with oral squamous cell carcinoma (Sato, Na, Nabeta, Υ ·, Kondo, Η., Sahara, Η·, Hirohashi, Y., Kashiwagi, K·, Kanaseki, T·, Sato, Y·, Rong, S·, Hirai, I·, et al· 2000· Human CD8 and CD4 T cell epitopes of epithelial cancer antigens. Cancer Chemother Pharmacol 46:S86_90·) In addition to a-enolase, some tumor-associated antigens have also been found to be overexpressed in many types of cancer, and these antigens are later It has been shown to be functional in the regulation of tumor immunity. These antigens include indoleamine-2,3_dioxygenase (IDO) (Muller, AJ·, Duhadaway, JB·, Donover, PS·, Sutanto-Ward, E·, and Prendergast, GC 2005. Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Binl, potentiates cancer chemotherapy. Nat Med 11:312-319. Epub 2005 Feb 2013·), B7-H1 (Dong, H·, Strome , SE·, Salomao, DR” Tamura, H., Hirano, F., Flies, DB·, Roche, P_C·, Lu, J., Zhu, G., Tamada, K., et al. 2002. 24 200827718

Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 8:793-800. Epub 2002 Jun 2024·) and RCAS1 (Nakashima, M., Sonoda, K·, and Watanabe, T 1999. Inhibition of cell growth and induction of apoptotic cell death by the human tumor-associated antigen RCAS1· Nat Med 5: 938-942·). Taking B7-H1 as an example, in addition to the situation in which 80-90% of patients with over-exposure are found in patients with lung cancer or other different cancers, B7-H1 also promotes tumor-specific poisoning of T cells ( Tumor-specific cytotoxic T cell) The function of apoptosis, which in turn leads to the deterioration and metastasis of tumor cells. However, autoantibodies against B7-H1 have only been found in patients with autoimmune diseases, but no reports have been found in any cancer patients (Dong, H., Strome, SE·, Matteson, EL., Moder, KG·, Flies, DB·, Zhu, G·, Tamura, H., Driscoll, CL·, and Chen, L. 2003· Costimulating aberrant T cell responses by B7-H1 autoantibodies in rheumatoid arthritis. J Clin Invest 111 :363-370·) 〇 is similar to B7-H1. According to our preliminary study (not shown in some data), α-enolase is not only in lung cancer (about 95%), but also includes breast cancer (greater than 90%). There is an over-expression of bowel cancer (about 40%) and ovarian cancer (about 30%). However, the incidence of α-enolase autoantibodies in pleural effusions in lung cancer patients (7.4%: 3 out of 54 patients) was associated with patients with non-cancer-related diseases. The incidence (54.8 %: 17 out of 31 patients) was found to be extremely low in the incidence of alpha-enolase autoantibodies in pleural effusions of lung cancer 25 200827718. Therefore, α-dense alcoholase may play an important role in the regulation of tumor immunity.

In addition to the complete determination of the function of α-enolase in glycolysis, studies have indicated that it is involved in some other cellular physiological responses, such as transcriptional regulation or cell invasion. Some studies have confirmed the p37KDa's Myc promoter-binding protein-1 (MBP-1), which is a translated variant of α-enolase. ΜΒΡ-1 is a 'suppressor of gene transcription by Myc mediated by binding to the P2 promoter of the c-myc gene (Ray, R·, and Miller, DM· 1991. Cloning and Mol Cell Biol 11:2154-2161·), and is thought to manipulate its function through interaction with the nuclear histone deacetylase (Ghosh) , Α·Κ·, Steele, R.5 and Ray, RB 1999. MBP-1 physical associates with histone deacetylase for transcriptional repression. Biochem Biophys Res Commun 260:405-409.). MBP-1 induces cell death and growth inhibition in cell and tumor models (Ray, RB·, Steele, R., Seftor, E., and Hendrix, M. 1995) Human breast carcinoma cells transfected with The gene encoding a c-myc promoter-binding protein (MBP-1) inhibits tumors in nude mice· Cancer Res 55:3747-3751·). However, despite the 95% similarity between the coding sequences of MBP-1 and ENOl, our α-enolase antiserum was used in the analysis of Western blotting in the hydrolary tumor and control group cells. Any possible p37 protein was detected. In the analysis of immunohistochemical staining, some tumor cells (less than 5%) were found to have a positive nuclear reaction. Therefore, further study on whether EN01 plays a functional role in the nucleus will clarify α-enolase. Another important issue with the difference in cell function between ΜΒΡ-1. Another putative cellular function of alpha-enolase is as a receptor for hemoplasminogen and is thought to play a role in tissue invasion (Redlitz, Α·, Fowler, BJ, Plow, EF? and Miles) , LA 1995. The role of an enolase-related molecule in plasminogen binding to cells. Eur J Biochem 227:407-415·) o (V), inhibition of tumor growth: The present invention describes an induction of anti-α-ene The immunological reaction of an alcoholase to achieve a method of inhibiting tumor growth. It is based on the administration of an alpha-sterolase antigen to induce an anti-alpha-enolase immune response. Comparing patients who produced high-yield α-enolase antiserum with those who did not produce antiserum, the former was found to be relatively small in tumor size. Other embodiments of the invention will be apparent to those skilled in the art in the <RTIgt; The description and its embodiments are intended to be illustrative only, and the scope of the invention and the scope of the invention are defined by the scope of the appended claims. The present invention relates to technical or scientific terms, and the meaning of other words is defined by the general knowledge of those skilled in the art to which the present invention pertains, except as defined by the present invention. With respect to the range of values, in addition to what has been expressly specified by the present invention, the package 3; 1 is at any intermediate value between the south limit value and the lower limit value (mtervemng value), and the unit of the intermediate value may be marked to at least _ (8) The limit value is very early. Furthermore, the invention encompasses any intermediate value specified by him. Moreover, the present invention also encompasses each of the three ranges having a more south limit value, a lower limit value, or both, except where explicitly excluded from the specified value. • In addition, all values in the specification and additional patent claims for the range of reaction conditions, percentages, multi-peptide lengths, and multi-core (four) lengths, except as expressly specified in the present invention, may be used. The eight-valued ambiguity is modified. Therefore, the numerical parameters set forth in the specification and the appended claims are approximations and may vary somewhat depending upon the desired characteristics of the invention. The solution of each numerical parameter 7 ′ should at least explain the effective number of the disclosed value when using four people. The reduction is not limited to the interpretation of the equival of the scope of patent application. Although the numerical parameters presented in the examples of the specification are as accurate as possible ': None: How the experiment itself still has its inherent error in the measurement. It is to be noted that the words "a", "an" and "the", which are used in the scope of the application of the invention, may be used to refer to the plural. Thus, for example, reference to the scope of the patent application can be used to represent a plurality of such nucleotides, which can be used to refer to one or more reagents, or to the technical field to which the present invention pertains. Usually equal to what the knowledger knows. The invention will be further described below by way of examples. These embodiments 28 200827718 are merely illustrative of the invention and disclose various features of certain embodiments of the invention. Therefore, the following examples should not be construed as limiting the invention. 2. Examples: The techniques utilized in the practice of the present invention include common cell biology techniques, cell culture techniques, antibody techniques, and genetic engineering techniques. These techniques are all commonly used in the art and are described in detail in the literature. The following examples illustrate the application and development of α-enolase-specific antibodies in monitoring the development of different cancers, as well as the inhibition of tumor growth by inducing an anti-α-enolase immunoreaction. purpose. (1), Example 1 ·· General Method 1. Treatment of malignant pleural effusion and samples: Collect malignant from 54 patients with different lung cancer subtypes under the permission of the National Institutes of Health Human Research Committee (IRB) Pleural effusion. Of the 54 patients, 4 had small cell carcinoma, 45 had adenocarcinoma, and 5 had squamous cell carcinoma. In addition, pleural effusions were also collected from 23 patients with non-cancer-related diseases. Of the 23 patients, 12 had pneumonia, 5 had tuberculosis, and 5 had heart disease. After collecting pleural effusion using thoracocentesis, the pleural effusion was quickly placed in a centrifuge within two hours and centrifuged at 300 xg for 10 minutes to precipitate cells in pleural effusion. Reuse previous research 29 200827718 research report (Chen, YM·, Tsai, CM·, Whang-Peng, J., and Perng, RP 2002. Double signal stimulation was required for full recovery of the autologous tumor-killing effect of effusion- Associated lymphocytes. Chest 122: 1421-1427.) The serial gradient centrifugation described with Ficoll-Plaque and Percoll separates the tumor cells from the water-related lymphocytes in the sediment to obtain a tumor-rich tumor. Cell part (tumor_cell enriched fractions). Finally, the percentage of tumor cells was estimated using cytological examinations. In the above samples, the purity of the tumor cells measured by the cytometry method was between 70% and 90%. 2. Purification of antibodies: The immunoglobulin fraction in pleural effusions can be purified by ammonium sulfate precipitation. First, a volume of 〇·66 volume of ice-saturated ammonium sulfate solution was dropped into a volume of water, and mixing was continued for 1 hour at 4 ° C, then placed in a centrifuge and centrifuged at 10,000 x g for 10 minutes at 4 ° C. . The precipitate obtained by centrifugation was dissolved in 0.1 volume of distilled water, and then dialysis was continued for one night in phosphate buffer (containing 10% glycerol). On the next day, replace the buffer with a new one and then dialysis the sample overnight. 3, immunotransfer method: In order to detect ρ48, α-enolase, β-enolase, γ-enolase, first 30 200827718 50 pg of cell lysate separated by 10% SDS-PAGE . Then, select the applicable antibody as described below and detect it by Western blotting method. The determination of glyceraldehyde phosphate dehydrogenase (GAPDH) or β-actin (β-actin) It is used as a control group for the amount of protein added. The immunocomplex formed by Bailey was visualized by SuperSignal enhanced chemiluminescence (Pierce, Rockford, IL). The β-enolase and γ-enolase monoclonal antibodies were purchased from Abnova Co., Taiwan. Myc-labeled protein antibody (from 9E10 cell line), GAPDH antibody and β-actin antibody were purchased from Upstate, New York, USA.

Biotechnology, Biogenesis, UK and Sigma Co., USA. (B), Example 2: Using CA926's own water-collecting antibody to identify p48 antigen In order to detect the humoral immunity existing in pleural effusion, the purified CA926 self-hydrating antibody was used to carry out western ink of tumor cells in 54 lung cancer patients. Point method analysis. Tumor cells were isolated from pleural effusions with lung adenocarcinoma patients using the aforementioned continuous gradient centrifugation method. Thereafter, the cell lysate of the normal lung tissue was used as a control group, and Western blot analysis was performed. The results showed that in 54 lung cancer patients, CA926 hydrocolloid antibody could identify i or 2 antigens with large or abnormal performance in the tumor cells of 4 patients. However, in this test, 21 non-tumor related diseases did not detect specific antigens (test data were not included in the instructions). In addition, patient CA926 is one of the four patients with abnormal antigens detected. Its autoantibodies specifically identify the major protein of 48-kDa (p48) in tumor 31 200827718 cells. In the NHRI-L89 (L89) cancer cell line, the same p48 antigen also appeared. The presence of the p48 antigen was confirmed by a competition experiment using CA926 hydrocolloid antibody adsorbed in advance by L89 cell lysate. Therefore, in this study, L89 can be used as a source of p48 antigen. The experimental procedure is detailed below. First, 30 gg of p48-rich cell lysate obtained from CA926 and L89 tumor cells was first subjected to immunoblot test with CA926 antibody after electrophoresis. In addition, CA926 cell lysate was additionally immunodetected using CA926 antibody adsorbed by L89 cell lysate. The above immunoreaction was carried out in an incubation manifold (purchased from Hoefer, San Francisco, CA). The determination of glyceraldehyde 3-phosphate dehygrogenase (GADPH) was used as a control group for the amount of protein added. Molecular weight markers are indicated on the left in units of Kd. The results of the experiment are shown in Fig. 1. It is known that both CA926 and L89 tumor cells contain an antigen recognized by an antibody purified by CA926 pleural effusion. Pre-adsorption experiments confirmed that the antigen recognized by CA926 antibody in CA926 tumor cells also appeared in L89 tumor cells. From the molecular weight of the identified antigen, the CA926 antibody recognizes the p48 antigen. (C), Example 3: Purification and confirmation of p48 antigen 1. Confirmation of p48 antigen: Next, P48 anti-32 200827718 was confirmed by biochemical purification and mass spectrometry. First, the p48 antigen was purified by (1) DEAE anion column purification, (?) ammonium sulfate precipitation separation, and (3) SDS-PAGE separation. Prior to purification, the detergent-soluble L89 cell lysate was dialyzed and pre-cleared with centrifugation. In Figure 2A, the results of the purification of the deae anion column in the first step of the above experiment are shown. The cell lysate is first added to the DEae anion column. Thereafter, a NaCl solution having a gradually increasing concentration is used to elute the protein bound to the column. Right using 20 mM Tris-HCl solution (ΡΗ7·0) as a buffer for DEAE column separation, optimally binds 94.65% of the protein to the column, whereas ρ48 antigen cannot bind to the column. If the CA926 hydrophobic antibody is used as a ridge needle, the western blotting point of unb〇und (UB), washout (W) cell lysate, and fractionated elutants (Fl-F7) In the test, p48 antigen was detected in unbound and eluate. In Fig. 2A, the results of the purification of ammonium sulfate by the second step of the above experiment are shown. The unbound liquid containing the p48 antigen and the eluate can be further enriched with p48 by ammonium sulphate (不同 to 〇/〇) with different degrees of saturation. Western blotting with the CA926 antibody confirmed that the antigen was mainly concentrated in the 60-80% saturation of ammonium sulphate. In Fig. 2A, the results of the separation of the third step of the above experiment by SDS-PAGE are also shown. The cell lysate enriched by ammonium sulfate separation was further separated by two 10% SDS-PAGE. One of them was tested with the Western blot method using the CA926 antibody as a probe, and the other was analyzed by silver staining. The putative p48 protein band on the colloid treated with silver staining was cut out and subjected to mass spectrometry. In Figure 2B, the results of mass spectrometry are shown. The trypsin-hydrolyzed p48 protein was subjected to mass spectrometry (Matrix Science, Co) to obtain a MALDI-MS spectrum, and six randomly selected peptides were sequenced, and the P48 antigen was determined to be a human α-enolase. The results of the sequence alignment of the above peptides and the α-enolase in the protein database are shown below the second map. 2. Confirmation of ρ48 antigen: Further studies were performed to confirm that α-enolase is the binding target of the CA926 antibody. This experiment was confirmed by the method described in Example 4 using the α-enolase recombinant protein. In order to produce the α-enolase recombinant protein, the α-enolase gene was selected from NHRI-L89 cells by RT-PCR, and the gene-specific primers used are described below. : The preamble is 5,-GGTGGAATTCTATCTCTCTCAAGATCCAT-GCC-3* (sequence number: 1), and the inverted primer is SLACTCCATGGTTACTTGGCCAAGGGGTTTCTd1 (sequence number: 2). In addition, the α-enolase gene fragment produced by the above RT-PCR was hydrolyzed with EcoRI and Ncol, and then cloned into the pGEX-KG vector and expressed in Escherichia coli to produce a recombinant which was ligated to the GST tag sequence. Protein (GST-ENOl). Protein purification was then carried out by glutathione-immobilized affinity 34 200827718 chromatogi*aphy (Sigma, St-Louis). The GST-tagged protein is then screened by enzymatic hydrolysis of thrombin. Then, each of the GST-tagged proteins and the GST-tagged ... enolase recombinant protein (GST-EN01) were treated with (or not) thrombin (TB), respectively, with GST antibody and CA926 antibody ( Marked in the bottom of Figure 2C), the Western blot method is used. The result is shown in Figure 2C. As can be seen, the CA926 antibody specifically binds to α-enolase and its conjugating protein (fusi〇n pr〇tein) but does not bind to the GST tag sequence, indicating that α-enolase is the antigenic target of CA926 tumor cells. . (IV) Example 4: Specific identification of CA926 antibody isomers In mammalian cells, there are three enolase isoforms, namely α-enolase (ΕΝ01), β-enolase ( ΕΝ03) and γ-enolase (ΕΝ〇2). These three enolase isomers have approximately 84% identity in the protein sequence. Thus, the present invention is directed to the determination of enolase isomers in CA926 and L89 tumor cells.

1 &gt; RT-PCR

In order to determine the specificity of the identification of the isomers by CA926 antibody, human β-enolase was first isolated from human hearts (Strategene, La J〇lla, CA) and CΑ926 cDNA pools by RT-PCR. Γ-enolase gene. The isomer-specific primers used in the above gene selection are described below: the forward primer of γ-enolase is 5,-ATTGAATTCTTCCATAGAGAAGATCTGGGCCCGG-GA 35 200827718 GAT-3' (sequence number·· 3); and the reverse primer of γ-enolase is 5,-ATTGAATTCTCACAGCACACTGGGATTACGGAAG-3' (SEQ ID NO: 4); the pre-collector of β-enolase is 5,-AGGG-AATTCTGCCATGCAGAAAATCTTTGC -3' (SEQ ID NO: 5), and the inverted pull-up of β-enolase is 5, -ATTGAATTCTCACTT-GGCCTTCGGGTT-3' (Sequence J: 6). The resulting fragment was then hydrolyzed with EcoRI and cloned into the pBlueScript-myc vector and as described in previous studies (Shih, NY, Li, J., Karpitskii, V., Nguyen, A., Dustin, MLL., Kanagawa, 0·, Miner, JH, and Shaw, AS 1999. Congenital nephrotic syndrome in mice lacking CD2-associated protein. Science 286:312-315.), the vector is delivered to HeLa cells by infection with T7 vaccinia virus, and A lot of performance. In order to produce the Myc-tag recombinant α-enolase protein, the α-enolase gene was selected from NHRI-L89 cells by RT-PCR, and the gene-specific primers used were described. As follows: The pre-collision is 5, -GGTGGAATTCTATCTATTCTCAAGATCCAT-GCCJ (sequence number: 1), and the inverted pull-in is 5'-ACTCCATGGTTACTTGGCCAAGGGGTTTCT-3* (sequence number: 2). Next, the obtained PCR fragment was hydrolyzed by EcoRI, and then cloned into the pBlueScript-myc vector, and the vector was introduced into HeLa cells by the infection of T7 vaccinia virus as described above to give a large amount of expression. HeLa cells transfected with the above three constructs can express myc-a-36 200827718 enolase, myc-γ-dense alcoholase and myc-β-sterolase, respectively. The test results are shown in Figure 3A. Shown. Figure 3A shows the results of the analysis by RT-PCR. This test confirmed that CA926 cells can express α-enolase and γ-enolase, but cannot express β-enolase. L89 cells can express α-enolase in a large amount. In the above-mentioned RT-PCR analysis test, the β-actin gene was used as an internal control. 2. Immunoprecipitation and immunoprecipitation HeLa cells were expressed with myc-calibrated α-sterolase (myc-ENOl) and myc-calibrated γ-enolase (myc-EN02). Transfection was carried out with a vector such as myc-calibrated β-enolase (myc-EN03) and myc-calibrated empty vector (myc-Tag). Next, 3 〇pg of the above-mentioned transfected cell lysate was separately taken and separated by 10% SDS-PAGE electrophoresis, followed by ex-enolase specific antiserum and γ-enolase specific single plant. The antibody or the β-enolase-specific monoclonal antibody was used as a probe for the Western blot test. The α-enolase-specific rabbit antiserum was prepared by Kelowna Inc. of Taiwan using the above recombinant GST-α-enolase protein. In order to avoid cross-reactivity, the antiserum was treated with immobilized GST-resin and E. coli lysate expressing GST-tagged protein to remove GST-specific antibodies and other interfering antibodies. The specificity of rabbit antiserum for a single enolase isomer was tested by Western blotting and immunoprecipitation. The γ-enolase-specific monoclonal antibody and β-enolase-specific monoclonal antibody were purchased from 37 200827718 from Taiwan Abnova Co. The 70-α-enolase, γ-enolase, or β-enolase was first transfected into HeLa cell lysate at 10 °/. Separation by SDS-PAGE electrophoresis and immunoassay with antibodies. In the immunoprecipitation (IP) experiment, the above-mentioned transfected HeLa cell lysate was bound with α-enolase antiserum and then precipitated with Protein-G beads (Pierce, Rockford, 111). The probe is detected using an antibody specific for the myc-calibrated protein. • The results of the above experiments are shown in Figure 3B, where the antibody on the left side of the figure indicates the antibody used for immunoassay and the anti-clearing, while the right side of the figure indicates the detected α- The enolase is an endogenous alpha-enolase or myc-labeled alpha-sterolase of HeLa. As can be seen from the figure, in the cells transfected with vectors such as myc-ENOl, myc-EN02, myc-EN03, the endogenous α-enolase produced by the cells is recognized by the CA926 antibody. In addition, the CA926 antibody also recognizes myLa-tag α-diacoholase in the cell lysate of HeLa cells transfected with the myc-ENO1 vector. In the bottom two columns of the figure, the control group in the immunoassay was tested with anti-Myc antibody or anti-α-tubulin. The results of immunoassay using α-enolase, γ-enolase, and β-enolase specific antibodies were verified by transfection with vectors such as myc-ENO 1, myc-EN02, and myc-EN03. Cells, which do show specific enolase isoforms. As for the immunoprecipitation analysis, it was confirmed that the 'enolase-specific antibody has its specificity for recognizing the α-enolase. In summary, it was confirmed that CA926 antibody and rabbit antiserum can only recognize α-enolase and do not recognize γ-enolase or β-enolase. 38 200827718 (V), Example 5: Low-frequency performance of α-enolamine antibodies in cancer patients The following tests use Western blotting methods to analyze pleural effusions in cancer patients and non-cancer diseases. _ Enolase specific antibody performance. Among the cancer diseases ~ 'only 3 patients detected α-enolase specific antibodies' and 31 non-cancer patients, there are 侦 侦The α-enolase specific antibody was detected. The test results are shown in the figure (the test for cancer patients) and the fourth chart (for non-cancer patients, the test). The test results show that, with non-cancer Fortunately, the frequency of α-diasterase-specific antibodies measured in cancer patients is significantly lower. The asterisk (*) indicated in Figure 4 indicates weak immunity in the experiment (4). Activity, it is speculated that the immunomodulation mediated by enolase may occur in most cancer patients. In Figures 4 and 4, only 8 of 54 lung cancer patients were presented. Experimental results, with the results of 7 patients in 31 non-cancer patients. Using sandwich sandwich enzyme immunoassay (sandwich EUSA) to measure the level of enolase-specific antibodies in normal humans, patients with non-cancer-related diseases, and lung cancer patients. First, all 96 wells are used. An ELISA plate of immobilized GST monoclonal antibody (purchased from the company) was used to immobilize the purified GST label of 〇·2盹~611 with enolase or GST protein. Then, from normal people, respectively Patients with non-cancer-related diseases and lung cancer patients obtained serum or pleural effusion, and diluted 1:10 for ELISA to assess α-enolase specificity in patients with serum or 39 200827718 pleural effusion The level of the antibody. The anti-human IgG (called Jackson Lab) combined with HRP (Horseradish Peroxidase) was used to test the α-enolase antibody in the serum sample, and the receptor of HRP (ABST) (purchased from KPL) The company carried out the reaction to measure the absorbance at OD4G5. By comparing the number of 读取D4G5 read, the content of EN01 antibody is known. The analysis result of ELISA is shown in Figure 4C, the asterisk in the figure (*) represents Statistical significance in the case of Student's t-test (P value &lt; 〇·〇1). If the average α-enolase antibody content in normal human serum is compared with non-cancer disease There is no significant difference in the average α-enolase antibody content in pleural effusions. However, if the α-enzyme activity in normal human serum and pleural effusion in non-cancer patients The mean content was significantly different from the average α-enolase antibody content in pleural effusions of lung cancer patients (both Ρ values were &lt; 0.01). The above results confirmed that cancer patients have a lower α-enolase autoantibody content than non-cancer patients and normal people. (6), Example 6 · · Lung cancer tumor cells and tissues in the α-enolase overexpressed in the following experiments, the use of α-enolase antiserum immunotransfer method, α-enolase surface staining Surface staining and immunohistochemistry (IHC) are used to determine whether α-glycosidase in tumor cells has abnormal performance. The above experiments used two human lung cancer primary cells, normal human bronchial 200827718 epithelial cells (NHBE) and small airway epithelial (SAEC) as control groups. From the results of Western blotting and immunohistochemical analysis, it is strongly pointed out that the increase in α-enolase in lung cancer patients is a common phenomenon. 1. α-enolase antiserum immunotransfer analysis In order to determine the degree of expression of α-enolase, 17 patients were selected from 54 patients with lung cancer, and 31 patients were suffering from non-cancer related diseases. Six patients with a sufficient amount of stagnant cells were selected for examination. Cell lines such as L89, CA926, CA1207 and CA2730 were obtained from lung cancer patients, while NC13 and NC16 cells were obtained from non-cancer patients. Lung embryonic epithelial cells (WI38), normal human bronchial epithelial cells (NHBE) and small airway epithelial cells (small airway epithelial) purchased from Cambrex (Walkersville, MD) Cells, SAEC) are control groups. The cells used as the control group were cultured using an appropriate culture solution supplied by the supplier, and the number of culture passages was 2. Then, 30 pg of cell lysate was separated by 10% SDS-PAGE electrophoresis, and then subjected to immunotransfer analysis with α-enolase antiserum, which was immunized with β-actin specific antibody. The analysis department is used as an internal control. The above experimental results are shown in Fig. 5A. As can be seen from the figure, α·enolase antiserum can be identified in L89, CA926, CA1207 and €82730 cells higher than NHBE, SAEC, NC13 and NC16 cells. Double the alpha enolase content. Accordingly, compared with non-cancer patients, the α-enolase in the cells obtained from the lung cancer patients in 200827718 was overexpressed. 2, α-enolase surface staining and flow cytometry: (1), cells: Sedimentary primary tumor cell line (CA926) from a 51-year-old female patient with lung adenocarcinoma. Subculture was carried out in a laboratory with a cell culture medium, and cells having a subculture of 2 were used as experiments of the present invention. The CA926 cell culture was prepared by adding 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 1 mM sodium pyruvate (Introgen, Grand Island, NY), 0.1 mM to DMEM. Non-essential amino acids (Sigma-Aldrich, St. Louis), 2 mM branic acid, 50 μg/ml streptomycin and 500 U/rnl penicillin. In addition, the 11111-1^89 cell line was derived from a 36-year-old hydronephrian tumor cell of a female patient with stage IV lung adenocarcinoma (cytokeritin (+)/calretinin (-)). The cells were cultured in RPMI 1640 culture medium, and 5% fetal bovine serum, 2 mM branic acid and antibiotics were added to the culture medium, and at least 40 subcultures were used for the experiment. (2) Surface staining and flow cytometry: For flow cytometry, cells cultured from cancer or non-cancer-related hydrops are first cultured in ALC-4 (Masuda, N., Fukuoka, M). ·, Takada, M., Kudoh5 S·, and Kusimoki, Y. 1991. Establishment and characterization of 20 human non-small cell lung cancer cell lines in a serum-free defined medium (ACL-4). Chest 100:429- 438·) and RPMI-1640 medium (1:1) 42 200827718 One generation or two passages. The intact cells were then stained with α-enolase antibody (1:300 dilution) and antibody-free, respectively, and visualized with goat anti-rabbit serum bound to Cy2 (Jackson Lab). The analysis was then carried out on a FACScan flow cytometer (Becton Dickinson), and the expression of α-enolase was known by the measured fluorescence intensity. The test results of the above experiments are shown in Figure 5, in which the control group of sputum and SAEC cells, whether treated with α-enolase antibody or antibody-free staining, showed no significant difference in the measured spectra. The amount of alpha-enolase present on the surface of these cells did not reach a detectable level. However, in the test of L89 and CA926 cells, if the spectrum measured by the α-enolase antibody was compared, the peak was shifted to the right compared with the antibody-free staining, indicating that L89 and CA926 cells were observed. It will express α-enolase on the cell surface. This test result confirms the argument that the aforementioned α-enolase plays a certain role in the transfer. 3. Immunohistochemical staining analysis: (1) Preparation of tissue samples and clinical characteristics of patients: The acquisition of tissue treated with paraffin was approved by the hospital's ethics committee. The formalin-immobilized and paraffin-embedded tissues were obtained from 80 patients with non-small cell lung cancer (NSCLC), 40 of whom had squamous cell carcinoma (SCC) and 31 patients. He has adenocarcinoma (AD), 4 patients with adenosquamous cell carcinoma, and 5 patients with large cell carcinoma. The qualitative nature of tumor cell histology is based on WHO classification, and the number of stages of disease is judged by tumor size and lymph node metastasis (node 43 200827718 metastasis). None of the patients received adjuvant or neoadjuvant chemotherapy. Surgery was performed at the Taichung Veterans General Hospital from January 2000 to December 2001 and was fully tracked. (2) Immunohistochemical staining (ihc) analysis:

To determine the performance of α-enolase in lung tumors, immunohistochemical staining analysis was performed on samples of 80 patients using the usual analytical methods. Briefly, tissue sections (about 4 um thick) were placed on a poly-L-lysine-coated fragment and air dried and dewaxed. It was then placed in 50% sterol containing 35% hydrogen peroxide for about 30 minutes to stop the activity of endogenous peroxidase. The sections were dehydrated again, washed with phosphate buffer, and then incubated with α-enolase antiserum (1:2000 dilution). In addition, 'α-enolase adsorbed by pre-immunized serum, anti-GST-tagged protein, or GST-EN01 antigen (1:2000 dilution) pre-immobilized with 1 mg of membrane Anti-jinqing, the stained sections obtained by processing the same sample can also be used to detect the specificity of α-enolase antiserum. After the above-mentioned section was treated with a biotinylated secondary antibody, the sections were treated with 3,3-diaminobenzidine solution (Dak〇, Carpinteda CA), and then ABC complex (ABC complex) was used. To image "and counter-stained with hematoxylin" 〇 immunohistochemistry results are shown in Figure 6, in this experiment, 76 of the 80 samples showed α-ene Alcoholase. A and B in Fig. 6 are samples from patients with adenocarcinoma (AdenoCA) and are laterally treated with ... enolase anti-44 200827718 serum or alpha-partamase antiserum previously immobilized with immobilized gst-enoi antigen. Cross-sectional image showing the specificity of the α-enolase antiserum. As for the c in Fig. 6, it is the alveolar epidermal cell image that looks normal at the proximal end of the tumor, and does not show enhanced positive immunostaining on the cytoplasm or nucleus or on the cell membrane (refer to the cultivar in Figure 6c). The box arrow points to). However, alveolar epidermal cells located at the distal end of the tumor present a degraded basal staining. In Fig. d, the adenocarcinoma and cells in another patient are also strongly expressed on the cytoplasm (X-paraffinase immunostaining. ^, tissue slice is magnified 100 times (Fig. 6) Ad) and 200 times (in the insertion box of c_d in Fig. 6), the scale bar is ι〇〇μιη long. (VII), Example 7: α-enolase expression degree and human lung cancer cells Correlation between cell invasiveness First screening of human lung cancer cell line (CL1-0) with low tissue invasion ability by using Transwell assay (chu, YW, Yang, PC·, Shyu, YC) Hendrix, MJ·, Wu, R., and Wu, cw 1997. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 17: 353-36). Next, as described above, reuse The transmembrane experiment was performed five times to obtain a highly invasive subunit of CL1-5 cells (Chu, YW·, Yang, PC·, Shyu, YC·, Hendrix, MJ·, Wu, R). ·, and Wu, CW· 1997· Selection of invasive and metastatic subpopulat Isolation from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 45 200827718 17:353-36) and further screening of the lung metastasis ability of CL1-5 cell line, 4 more times After screening in vivo, the subunits sub-sub-line were obtained, Chu, YW·, Yang, PC·, Shyu, YC, Hendrix, MJ, Wu, R·, and Wu, CW 1997· Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 17:353-36) 〇In order to know that a-enolase is in CL 1-0 cells, CL1-5 cells and CL1-5F4 The performance of the cells is therefore determined using the Western blot method. After lysing the cell lysates of the aforementioned three cells, they were placed in a centrifuge and centrifuged at a speed of 5000 rpm for 10 minutes. Then, 30 pg of cell lysate was separately separated by 10% SDS-PAGE electrophoresis, and transferred to nitrocellulose membrane, and α-enolase rabbit antiserum (diluted 1:5000). The reaction was carried out for 1 hour to detect the expression of α-enolase, and the rabbit antiserum source of α-enolase was as described above. At the same time, the β-actin gene was used as an internal control. The results of the above experiments are shown in Fig. 7A. From the experimental results, it is confirmed that the α-ene is confirmed in the process of screening for invasive ability as the concentration of the protein band detected by the anti-α-enolase antibody increases. The degree of alcoholase performance also increases. Next, each cell strain was tested by a transmembrane experiment to find out the extent of its ability to invade a microporous membrane (purchased from Becton Dickison, Franklin Lakes, NJ) covered with extracellular mesenchyme (matrigel). First, cells (2 x 104 cells) were seeded in the upper trough of the two test cell systems and cultured for 24 hours in a lower trough containing 46 200827718 with 10% FBS. The two test slots are separated by a microporous membrane (with a pore size of 8 μηι) covered with matrigel. At the end of the culture period, the membrane coated with matirigel was metered by a microscope. The above experimental results are shown in Fig. 7B. It can be seen from the figure that the cell invasive ability is significantly increased after a series of screening processes. As can be seen from the comparison of Figures 7A and 7B, the degree of expression of α-enolase in the cells is significantly increased by the screening process, and the cell invasive ability is also enhanced. Accordingly, cells highly expressing α-enolase also have a large invasive ability. From this, it can be seen that the increase in the degree of expression of α-enolase is closely related to the invasive ability of cells. (VIII), Example 8: Low-level expression of α-enolase is related to the low invasive ability of cells. In order to test whether the decrease in α-enolase performance leads to a decrease in cell invasive ability, RNA interference is used. The α-enolase is down-regulated or bound to it using an anti-α-enolase antibody. 1. RNA interference method: First, 2 pg of the empty vector and shRNA complementary to the _ enolase sequence (purchased from Open Biosystems) were transfected into CL1-5F4 cells, respectively, to obtain a stable clone. The ShRNA sequence complementary to the human α-enolase gene is as follows: 5,-AGCTGTTGAGCACATCAATAAA-3' (SEQ ID NO: 7) 〇 Next, after 24 hours of transfection, 2 pg is used. 111&gt;〇111}^11 antibiotics were screened for fine 47 200827718 cells, and one cell line (named vector control, VC) was selected from CL 1-5F4 cells transfected with empty vector, and transfected with α-浠Of the CL1-5F4 cells of the shRNA complementary to the alcoholase sequence, three cell lines (designated C4, C5 and C8) were selected. The decline in the expression of α-enolase in C4, C5 and C8 cell lines was confirmed by the Western blot method. The test results are shown in Fig. 8. First, 30 gg transfected empty vector (VC cells) and CL1-5F4 cell lysates transfected with α-enolase-encoding shRNA (C4, C5 and C8 cells) were separated by 10% SDS_PAGE. It is then transferred to a nitrocellulose membrane. The expression of α-enolase was detected by an anti-α-enolase antibody. At the same time, β-actin was used as a control group for the amount of protein added. Compared with the cell lines not transfected with the shRNA vector, the C-, C5 and C8 cell lines showed significantly lower α-enolase levels. As for the transfer ability of CL1-0, CL1-5 and the above three cell lines (C4, C5 and C8), the in vitro transmembrane test was used. In this test, the microporous membrane was not covered with matrige. The in vitro transmembrane experimental test method for cell invasive ability is as described above. In the transfer ability test, 5 x 104 cells were implanted in the upper tank of the two test tank systems, and cultured in a lower tank containing 10% FBS for 6 hours. The two test tanks are separated by a microporous membrane (pore size 8 μιη). Next, the cells transferred to the filter and the other side of the membrane were stained and counted by a microscope. In the test for cell invasiveness, 2 x 10 4 cells were first implanted in a matrigel-coated filter, followed by a test for 24 hours as described in Example 7. The two test tanks are separated by a matrigel microporous membrane (pore size 8 μιη) on 48 200827718. After the completion of the culture period, the filter was stained and counted by a microscope. The above test results are shown in Figures 9 and 9 and the CL1-0 cell line is used as a control group to represent cells with low metastatic ability and low invasive ability. From this result, it was found that about 146.5 ± 31.58 CL1-5F4 cells metastasized, and about 1.5 ± 1.59 CL1-5F4 cells were invaded. As for CL1-5F4 cells (VC) transfected with an empty vector, the test results were similar to those of CL1-5F4 cells. However, cells transfected with shRNA (C4 and C5) showed a significant decrease in cell transfer and invasion compared to cells transfected with empty vector (VC). This test result confirmed that if the α-enolase is down-regulated, the cell transfer ability and invasive ability of CL1-5F4 can be lowered. 2. In vivo testing of human lung cancer cells: In order to test the effect of α-enolase in the regulation of CL1-5 cells, on the growth rate and metastasis of tumors in vivo, therefore, the same as above, the empty vector or The shRNA complementary to the α-enolase sequence was transfected into CL1-5F4 cells, and the vector controll cell line was obtained by the former, and the C4 and C8 cell lines were obtained by the latter. To determine the effect of α-enolase on tumor growth, a stable cell line of IxlO6 was injected subcutaneously into 5 NOD/SCID mice, and the size of the tumor was measured. After that, the growth of the tumor is monitored every two to three days. The size of the tumor is determined by multiplying the square of the short diameter by the long diameter and multiplying by one-half (short idameters2xlong diameterxl/2). Five mice were included in each test group. 49 200827718 The above experimental results are shown in Fig. 10. As can be seen from the figure, the growth curves of CL 1-5F4 cells, VC cells and C4 cells are quite similar, but the growth rate of C8 cells is slow. From the results of this experiment, it can be seen that if the cells are injected into NOD/SCID mice by subcutaneous injection, the decrease in the performance of α-enolase will only have a slight effect on the growth rate of the tumor. In order to understand the effect of α-enolase on the metastatic situation, a stable cell line of 2χ106 was injected into 5 NOD/SCID mice via the tail vein, and after 28 days, the lung nodules were measured. The number of nodules). The above test results are shown in Fig. 11. As shown in the figure, in the vector control of CL1-5F4 cells (Vector control) transfected with empty vector, the number of lung metastases is about 85±35, as for CL1 transfected with shRNA. In the -5F4 cells (C4, C8, respectively), the number of lung metastases was 31 ± 15 and 26 ± 10. From this, it can be seen that when the expression amount of α-enolase is lowered, the situation of lung metastasis is greatly reduced. 3. In vivo testing of mouse cancer cells: B16F1 mouse melanoma cells (murine melanmoa cells) were injected intravenously into the tail of C57BL/6 mice to form metastatic pulmonary nodules and 5 times. B16F1-L5 cell line was obtained after colonization. The procedure for screening highly metastatic tumor cells was to inject B16F1 cells into the tail vein of C57BL/6 mice to form metastatic tumor nodules in the lungs of the rats. Next, after the tumor nodules were excised and ground, a single cell suspension solution was prepared, and the obtained tumor cells (designated 50 200827718 B16F1-L1) were cultured in vitro. Next, b16F1_Li tumor cells were collected and/mainly injected into another C57BL/6 mouse. The in vivo screening process of the aforementioned highly metastatic tumor cells was repeated four times to produce a Bi6F1_L5 cell line. As shown in the results of the experiment shown in Fig. 12A, it was found that the C57BL/6 mice that had been intravenously injected with B16F1-L5 produced more tumor nodules with the parental cell line 516?1 (P値&lt;0.05)'. As shown, the number of lung metastases in B16F1_L5 cells was 172, and the number of lung metastases in parental cells B16F1 was 52. In addition, the western blot test results by the mouse enolase (mEN()1) antibody are shown in Fig. 12B. As can be seen from the figure, the expression of α_enolase in B16F1 cells is only b16F1_L5 cells. 75%. The test with α_tubulin (...plus (4) (8) antibody at the bottom of the figure) is used as an internal control group for the amount of protein added to normalize the performance of mENOl. To produce α-enolase performance Decreased stable cell line, therefore, the arna complementary to the X-vector and α-luciferase sequence was transfected into B16F1-L5 cells to produce a control cell line (vect〇r contro cells) and α- Two cell strains regulated by enolase (1G9 and 2E11 cells), and in order to test whether the low performance of α-enolase is associated with a decrease in the degree of cell metastasis, so 2 cells of cells were injected via the tail vein into C57BL/6. In mice, the number of tumor nodules was measured in μ days after injection. Each test group contained only mice. The above test results are shown in Fig. 13A, and the aforementioned human lung cancer cell line. The results of test 5 are similar to 'when paired (the expression of X-leanolase can reduce the number of metastatic tumor nodules in the lungs (P値 is less than 〇〇〇5). As shown in Figure 51 200827718, Dyeing empty carrier B16F1-L In 5 cells (Vector control), the number of lung metastases was approximately 235 ± 109, as for B16F1-L5 cells transfected with shRNA (1G9 and 2Ell) (5, ATGTAGACACCGAAGTGAT-3) (SEQ ID NO: 8), The number of lung metastases was 36±24 and 24±27, respectively. In addition, as shown in Fig. 13B, in the Western blot test conducted by mENO1 antibody, 1G9 and 2E11 cells were α-enolase. The performance is only 90% and 85% of the control group cells (VC). Similarly, the test with α-tubulin antibody at the bottom of the figure is the internal control group as the amount of protein added. To normalize the performance of mENOl. 4. Antibody Blockade: In the following examples, in order to test the effect of α-enolase on cell invasive ability, The test was carried out by mixing the _ enolase antibody and the GST antibody as a control group with CL1-5F4 cells. First, 2xl04 CL1-5F4 cells were cultured at 37 ° C with an enolase antibody or GST antibody, respectively. After 30 minutes, the cultured cells were implanted according to the above steps. In the upper trough of the two test cell systems, the cell invasion test was performed. The test results are shown in Fig. 14. In the figure, the cell invasive ability of cells cultured with α-enolase antibody is dose-related. From this, it was found that when cultured with an α-enolase antibody, the invasive ability of CL1-5F4 cells was remarkably inhibited. 52 200827718 (9), Example 9: Up-regulation of α-enolase performance and clinical results 1. Quantitative and statistical analysis of α-enzyme activity: To understand the performance of α-enolase regulation and patients The relationship between clinical outcomes was analyzed using several different statistical methods. This study was performed on 80 patients with NSCLC who underwent postoperative follow-up for 60 months, and the median follow-up period for all patients was 29.5 months. The staining of α-enolase in water-storing cells was observed at a magnification of 200 times, using the QuickC score method (Barnes, DM·, Harris, WH·, Smith, P., Millis, RR·, And Rubens, RD 1996· Immunohistochemical determination of oestrogen receptor: comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients. BrJ Cancer 74: 1445-1451·), semi-quantitative analysis, and by two Pathologists performed independently in a blinded manner. Conflicting scores are determined by discussion microscope under microscope observation. Normal lung cell sections and tumor control group sections (purchased from BioGenex, San Ramon, CA) were included in this experiment. In summary, this rapid scoring method takes into account the intensity and distribution of alpha-enolase immunoreactivity in tumors. Among them, in terms of intensity score, negative, weak, moderate, and strong staining results are represented by 0, 1, 2, and 3, respectively; in terms of the distribution score, the tumor ratio showing a positive staining reaction is determined according to the following scores. :0 % = 0, 1-25 % = 1, 26-50 % = 2, 51-75 % = 3 and 76-100 % = 4. In combination with the intensity score and distribution score of the positive staining responders obtained above, a quick score ranging from 0 to 7 is finally given. 53 200827718 In addition, the number of patients and the corresponding intensity and distribution of the α-enolase staining reaction are shown in Table 1. Table 1 〇 (negative) 1 (weak) 2 (medium) 3 (strong) minutes 0 (0%) 0 0 0 0 1 (1-25%) 0 0 2 2 2 (26-50%) 0 0 8 16 3 (51-75%) 0 5 14 17 4(76-100%) 0 5 4 7 In addition, in terms of survival analysis, the statistical date of progression-free survival (PFS) is determined by the surgery day. Calculate the cancer recurrence day or death day. The patient who survived without cancer recurrence was counted to the last review. The statistical date of overall survival (OS) is calculated from the day of surgery to death. The correlation between the degree of expression of α-enolase (quick scoring &lt; 5 or ^ 5) and clinical parameters was calculated by Fisher's exact test univariate and Logistic regression multivariate methods. Survival curves for OS and PFS were plotted using the Kaplan-Meier method. In addition, the log-rank test is used to analyze the difference in pairwise survival rates. 2. Relationship between α-enolase performance and cancer development: The test results of the above experiments are shown in Table 2 and Figure 15. 54 200827718

Table 2 ENOl performance variables all (%) Score &lt; 5 (%) Score S 5 (%) P value (single variable) P値 (multivariate) Fast score 80 (100) 17 (21) 63 (79) 1.0 0.926 Intermediate age (years) &lt;65 29(36) 6(21) 23(79) 1.0 0.926 ^65 51(64) 11(22) 40(78) Gender male 69(86) 15(22) 54(78 ) 1.0 0.789 Female 11 (14) 2 (18) 9 (82) Smoking habits no 25 (31) 8 (32) 17 (68) 1.0 0.665 There are 55 (69) 15 (27) 40 (73) histological classification scale BCC 40(50) 8(20) 32(80) 1.0 0.785 Non-squamous cell carcinoma 40(50) 9(22) 31(78) Adenocarcinoma 31(39) 6(19) 25(81) 0.787 0.742 Non-adenocarcinoma 49(61) 11(22) 38(78) Number of cancer stages I + II 51(64) 15(29) 36(71) 0.022 0.018* III 29(36) 2(7) 27(93) Recurrence None 35(44) 12(34) 23(66) 0.015 0.012* There are 45(56) 5(11) 40(89) * indicates a statistically significant difference (P value &lt;〇·〇5) in the above 80 Of the patients, 30 died of lung cancer, and the median OS was 10 months (from 2 months to 42 months); 45 patients had a recurrence of 55 200827718 calendar disease. Using the Quick Score method (Rhodes, A., Jasani, B., Barnes, DM, Bobrow, LG, and Miller, KD 2000· Reliability of immunohistochemical demonstration of oestrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring Systems· J Clin Pathol 53:125-130·) Semi-quantitative analysis of a-enolase performance (&lt; or ^5), the results showing that age, gender, smoking habits or histological subcategories are not There is a statistical difference in the performance of a-enolase. In contrast, the number of tumors and recurrence are highly correlated with the extent of EN01 performance. Patients with stage III (93%) or relapse (89%) had significantly higher a-enolase performance than patients with stage I/II (71%) or no recurrence (66%). (P values are 〇·〇18 and 〇·〇ΐ2, respectively). According to the survival analysis by the Kaplan-Meier method, the performance of a-enolase was highly correlated with PFS or 0s of all patients (Fig. 15A and 15B). Patients with a high performance-enolase tumor (quick score -5) were highly associated with poor OS and PFS (P = 0.0027 舆 0.0035, respectively).

In addition, since the rapid scoring results of most cancer stage I11 tumors are more than 5, if the a-enolase is highly expressed, the criterion for poor prognosis is likely to be biased. Accordingly, in order to avoid the occurrence of this deviation, only the patients in the first stage of cancer were further analyzed. Based on the analysis results shown in Figures 15C and 15D, it was consistently demonstrated that the degree of expression of a-enolase was inversely related to PFS and OS (p values of 0.0015 and 0.064, respectively). Accordingly, these experimental data can be used to support the role of alpha-enolase in prognosis in patients with NSCLC 56 200827718, even in patients with stage UII cancer.

The Cox multivariate analysis method is used to analyze the patient's alpha-enolase table. Current, tumor stage and / or cancer recurrence, age, gender, smoking habits or histological subcategory for PFS and OS The impact. As a result of the analysis, it was found that only when the performance of α-enolase was quickly scored -5, it was associated with poor PFS (P値 of 0.031). Conversely, cancer staged/epumpnial patients showed a better PFS trend (P値 0.076). In addition, the low performance of α-enol • enzymes and the absence of cancer recurrence are important factors in obtaining better OS (Ρ値 0.008 and 0.003, respectively). These analyses further indicate that the performance of α-enolase is effective as a prognostic indicator for survival in NSCLC patients. (10), Example 10: Low-level anti-α-enolase-specific antibodies in serum of patients with non-small cell lung cancer, respectively, using sandwich sandwich enzyme immunoassay (sandwich # ELISA assay) The level of serum α-enolase-specific antibodies in patients with non-cancer-related diseases and those suffering from non-small cell lung cancer. First, GST-specific antibodies were plated in each well on the ELISA plate, and then 0.2 pg/well of pure GST-α-enolase recombinant protein was added to each well of the ELISA plate to make GST-α-enol The enzyme recombinant protein is fixed on it. A-enolase-specific antiserum (ENO1-specific IgG) was used as a standard to establish a standard curve of α-enolase-specific antibody content. The serum to be tested was obtained from 5 normal subjects, 21 patients with non-cancerous 57 200827718-related diseases, and 35 patients with non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma). ). These serum samples were then diluted 1:10 and subjected to an ELISA assay in which anti-human IgG from HRP-conjugated goats was used to detect ENO1 antibodies in serum samples and reacted with HRP receptors (ABST) at OD4G5. The absorbance value was measured to show the content of the dilute alcoholase-specific antibody. The above experimental results are shown in Figure 16. It can be seen from the figure that the content of α-enolase-specific antibodies in serum of patients with non-small cell lung cancer is statistically significantly lower than that of normal and non-cancer related diseases. (Ρ值&lt;〇.〇1). (11), Example 11: Inducing the production of anti-α-enolase Humoral immunity response can inhibit tumor growth in the following examples, and the use of α-enolase on the cell surface can be utilized. The vaccination/protection model and the treatment model of the ML1 mouse liver cancer cell line were used to evaluate whether α-enolase can be used as a vaccine or antibody-based immunotherapy target. In order to produce the mouse ENOl recombinant protein, the mouseENO1 gene was cloned from ML-1 liver cancer cells by RT-PCR, and the gene-specific primers used were described as follows: 5,-AATTCTAGACTCTATTCTCAGGATCCA-3, and the inverted pull-in is 5,-AATAAGCTTTTATTTGCCAGGGGGTT-3'. In addition, the mouse EN01 fragment produced by the above RT-PCR was hydrolyzed with Xba I and Hind III, and then cloned into the pGEX-KG vector and expressed in E. coli in a large amount of 2008 20081818 to generate a GST-tagged sequence. mENOl recombinant protein (GST-mENOl). The protein was then purified by glutathione-immobilized affinity chromatography (Sigma 5 St. Louis). Further, as described above, the GST protein was obtained by purifying Escherichia coli transfected with the pGEX-KG vector. In the vaccination/prevention model, 20 pg of mouse EN01 antigen was administered to BalB/C mice 30 days before, 1 and 5 days before injection of ML-1 liver cancer cells (the number of mice tested with mENOl was 6) or 20 pg of GST antigen (5 mice with GST immunoassay), or no antigen (the number of mice as control group is 6). Thereafter, IxlO6 ML-1 liver cancer cells were injected into all BalB/C mice. In addition to measuring the tumor size of the immunized mice per week, blood collection was performed to detect the presence of anti-α-enolase antibodies. The results of the inoculation/prevention model are shown in Figure 17, and the mice inoculated with the mENO1 recombinant antigen produced anti-enolase antibodies (data not shown), and the growth rate of the tumors in mice immunized with GST-mENO1. Significantly slow down. In the treatment model, first, 1X106 ML-1 liver cancer cells were injected subcutaneously into BalB/C mice. Two weeks later (from day 14 after inoculation of liver cancer cells), the mouse adjuvant CpG 1826 (50 pg) was mixed with mENOl antigen (20 pg) to induce an anti-α-diffinase immune response. Rats in the control group were given the antigen-free adjuvant CpG 1826 (50 pg). Thereafter, a boost injection of the immunological response was performed on the 28th day and the 35th day with the same mixture, and the tumor size was measured after 14, 12, 28, 35, 42 days, 59 200827718, respectively. In addition, a western blot test was conducted on day 42 to detect the presence of anti-α-enolase antibodies in the serum of all mice. The above experimental results are shown in Figs. 18 and 19, respectively. In Fig. 18, the test results of Western ink method after 42 days of injection of ML-1 liver cancer cells in mice, wherein the left test results were from mice administered with mENOl antigen, and the test results on the right were controlled. Group of mice. From the test results, 6 of the mice receiving mENOl antigen and adjuvant injection, 3 of them can produce high amount of mENOl specific antibody in serum (left box, rows 1-3) (1:3000 dilution, in 10 § of mENO1 antigen was detected in 1 second. As for the other 3 mice that also received mENO1 antigen and adjuvant injection, anti-mENO1 antibody was not detected in serum (left box 4-6 row). As for the mice in the control group, no anti-mENOl antibody was produced in the serum (rows 1-6 of the right box). At the same time, in order to verify that each row in the Western blot test was added with the same amount of mENOl antigen, the Western blot was separately separated and tested with mENOl antibody (box below). In Figure 19, mice that produced anti-mENO1 antibodies (anti-mENOl Ab(+), ♦), mice that did not produce anti-mENO1 antibodies (anti-mENOl Ab(-),), and controls were shown. Group of rats (▲), the size of the tumor after several days of injection of ML-1 liver cancer cells. As can be seen, the mice which produced the anti-mENO1 antibody had a significantly smaller tumor size after 42 days of ML-1 liver cancer cell injection than the mice which did not produce the anti-mENO1 antibody and the control group. Accordingly, an anti-ENO antibody immune response that produces a strong 60 200827718 in vivo can help reduce tumor size. (11) M Example 12 - Immunohistochemical analysis of breast cancer tissue samples This phenomenon was observed in breast cancer patients, except for the up-regulation of ~enolase observed in non-small cell lung cancer cells. First, a sample of breast cancer tumors was collected, and then α-enolase was stained by immunohistochemical staining, and then evaluated by two well-trained pathologists based on the reaction intensity after enolase staining. To analyze the performance of the heart enolase. In this experiment, after obtaining postoperative tissue samples from breast cancer patients, dehydration and paraffin embedding were performed according to standard procedures. The 5 μιη thick tissue was stained with α-enolase antiserum and imaged by DAB according to the manual provided by the manufacturer (Dak〇). The results of immunohistochemistry experiments are shown in Figure 2. In the figure, τ and N represent the staining results of normal tissues near the tumor and the tumor, respectively. From the test results, 36 of the 58 tumor samples showed tumor cells showing moderate or strong staining results. However, all tumor samples did not show any staining on their cell membranes. Conversely, 20 normal interstitial tissues (str〇inai c〇unterparts) located near the tumor showed weak staining results. In addition, if compared with the expression of enolase in normal tissues near the tumor, 96% of the tumor samples (56 of 58 samples) showed up-regulation of α-enolase in their cancer tissues. Thus, it is speculated that the excessive expression of α-enolase may be associated with the tumorigenesis of breast cancer. The present invention has been described above with reference to a preferred embodiment. However, it is not intended to limit the invention, and various modifications and changes may be made without departing from the spirit and scope of the invention. The scope of the invention is defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; In Example 2, the results of the Western blot test were carried out on the cell lysate containing P48 using CA926 antibody or CA926 hydrocolloid antibody adsorbed in advance with L89 cell lysate. The results showed that in CA926 and L89 tumor cells, p48 antigen was the main immunological activity target of CA926 effusion antibody. 2A is a diagram showing the process of purifying p89 autologous antigen in the process of purifying p48 autoantigen by DEAE anion column purification, ammonium sulfate purification, and SDS-PAGE separation in Example 3 of the present invention. Western ink point test results. As a result, the p48 autoantigen has been purified from L89 cells. Fig. 2B is the result of mass spectrometry analysis of the purified p48 antigen. The p48 protein obtained in Figure 2A was subjected to trypsin hydrolysis and sequencing. As described in Example 3, the results of the peptide sequence analysis were consistent with the human α-enolase 62 200827718 sequence in the National Institutes of Health protein database, and it was revealed that the p48 antigen system was an α-enolase. The '2C diagram shows the Western blot test results of the α-enolase obtained by the transfer method in L89 cells in Example 3 of the present invention, and the GST-labeled α-enolase is being carried out. Before the Western blot test, _ treatment was performed with thrombin. Immunoassays were performed using GST antibody or CA926 antibody as probes, respectively. As a result, the α-enolase was the immunoreactive target of the CA926 hydrophobial antibody. Figure 3 is a diagram showing the results of RT-PCR analysis in Example 4 of the present invention for testing α-enolase, γ-enolase and β-enolase in L89 cells and CA926 tumor cells. Performance situation. In this RT-PCR analysis test, specific pull-ups of β-actin were used as internal controls. The results showed that L89 cells could express α-enolase, but could not express β-enolase and γ-enolase, while CA926 cells could express (1_enolase and 丫-enolase, but could not The β-enolase is shown. Figure 3 is a graph showing the results of Western blotting of the lysates of HeLa cells after transfection in Example 4 of the present invention. The α-olefins of HeLa cells were respectively labeled with myc- Alcoholase (myc-ENOl), myc-calibrated γ-sterolase (myc-EN02), myc-calibrated β-enolase (myc-EN03), and myc-calibrated empty vector (myc-Tag) After the vector is transfected, the cell lysis step is carried out. The above-mentioned transfected HeLa cell lysate product is first bound with α-enolase antiserum, and then the antibody specific for myc-calibrated protein is used as a probe. The detection showed that the α-enolase antiserum can recognize the α-enolase 0. The fourth and 48th lines are shown in the fifth embodiment of the present invention, from non-small cells 63 200827718 lung cancer (NSCLC) patients (Fig. 4A) The pleural effusion antibody obtained from non-cancer-related diseases (Fig. 4B), the α-enolase Western blot method As a result, the results showed that the non-cancer-related disease patients had more α-enolase-specific antibodies than the cancer patients. Fig. 4C is a view showing the fifth embodiment of the present invention, from a normal person The results of ELISA experiments performed on serum, patients with non-cancer-related diseases, and water-collecting cells obtained from lung cancer patients. The results showed that non-cancers were compared with non-small cell carcinoma (NSCLC) patients. The anti-α-enolase antibody carried by the related patient and the normal person has a high power price. Fig. 5 is a view showing the results of the Western blot test of the hydrous tumor cells in Example 6 of the present invention. L89, CA926, CA1207 and CA2730 are lung hydrostatic tumor cells obtained from lung cancer patients. NC13 and NC16 hydrocephalus cells are obtained from non-cancer patients. Normal human lung primary epithelial cells (normal human lung primary epithelial cells) ), normal bronchus/tracheal primary epidermal cells (NHBE), small airway primary epidermal cells (SAEC), and fetal embryonic epithelial cells (WI38) as control group, with α-enolase anti-blood Immunotransfer analysis was performed. Subsequently, after removal by antibody stripping, immunoassay was performed again on the same transfer film using β-actin-specific antibody as a control group for the addition amount. (l〇a(jing contr〇l). The results showed that the α-enolase in the hydroneedoma cells obtained from lung cancer patients was overexpressed. Figure 5 shows the flow cytometry of the lung cells of intact L89 cells, CA926 cells and control group after complete cell surface staining with α-enolase anti-64 200827718 serum in Example 6 of the present invention. Test results. The results showed that there were more α-dense alcoholases on the surface of cancer cells than the control group cells. Fig. 6 is a view showing the results of immunohistochemical staining of lung tumor tissues by the α-enolase antiserum in Example 6 of the present invention. In the figure, the position indicated by the arrow is the image of the alveolar epidermal cells at the proximal end of the tumor. It can be seen that the cytoplasm, the nucleus or the cell membrane may show an enhancement of immunostaining. Thus, in the lung tumor tissue, its α- The enolase expression shows an increased situation. Fig. 7 is a graph showing the results of Western blotting test of human lung cancer cell lines with an anti-α-enolase antibody in Example 7 of the present invention, wherein an actin-specific anti-system was used as a control group. The results showed that oc-enolase showed significant expression in CL1-5 and CL1-5F4 cells compared with CLi_〇 cells. Fig. 7B is a view showing the invasion of a human lung cancer cell line by a transmembrane experiment in Example 7 of the present invention. The results showed that the cell invasive ability of CL1_5 and CL1-5F4 cells was significantly increased compared with CL1_〇 cells. Figure 8 is a diagram showing the results of western blotting of the human lung cancer cell line of the lung cancer cell line of the human lung cancer cell line, wherein the human lung cancer cell line is subjected to ShRNA complementary to the α-enolase sequence. Transfection to reduce performance' and actin-specific anti-system as a control group. _ If not, the expression level of α-enolase was significantly lower in the cell line transfected with shRNA complementary to the α-pariferase sequence. 65 200827718 Figures 9A and 9B are diagrams showing transmembrane test results of human lung cancer cell cell metastasis (A) and invasion (B) ability in Example 8 of the present invention, wherein the human lung cancer cell line utilizes α-ene The shRNA complementary to the alcoholase sequence was transfected. From the results, it was revealed that the decrease in the expression of α-enolase was associated with a decrease in cell transfer ability and invasive ability. Fig. 10 is a view showing the observation of tumor growth after injecting a human lung cancer cell line transfected with an α-enolase-encoding shRNA into a NOD/SCID mouse in Example 8 of the present invention. The results show that a decrease in the performance of α-starlase has a slight effect on tumor growth. Figure 11 is a graph showing the results of pulmonary metastasis after injection of a human lung cancer cell line transfected with a shRNA complementary to an α-enolase sequence into NOD/SCID mice in Example 8 of the present invention. . As a result, compared with the control group, if a lung cancer cell line transfected with a shRNA complementary to the enolase sequence was injected into a mouse, the number of pulmonary metastase nodules produced was small. Figures 12A and 12B show the number of lung metastases measured after injection of mouse melanoma cells into C57BL/6 mice in Example 8 of the present invention, and α-enolase in mouse melanoma cells. The result of the Western blot method is 4, and the tubulin (a-tubulin) antibody is used as the internal control group of the Western blot method. The results show that the enolase expression in tumor cells is related to the number of lung metastases. Figure 13A is a graph showing the number of lung tumors measured after injection of mouse melanoma cells transfected with shRNA complementary to a cardiac enolase sequence into C57BL/6 | mice in Example 8 of the present invention. . The results are shown in 2008 20081818, when the performance of α-enolase is down-regulated, the number of metastatic nodules in the lungs also decreases. Fig. 13 is a view showing the results of Western blotting of the cell lysate of the shRNA which is complementary to the mENO1 sequence to the mouse melanoma cells in Example 8 of the present invention. The α-tubulin antibody was used as an internal control group of the Western blot method. The results showed that transfection of cells with shRNA complementary to the mENO1 sequence reduced the amount of a-sterolase expression. Fig. 14 is a graph showing the results of a transmembrane experiment in which cell invasion was controlled by an a-enolase-specific antibody in Example 8 of the present invention. The results show that the a-enolase-specific antibody can reduce the invasive ability of tumor cells and is related to the dose of the antibody provided. Figure 15 is a graph showing the results of Kaplan-Meier analysis of overall survival (OS) and progression-free survivals (PFS) of cancer patients in Example 9 of the present invention. The higher the degree of expression of tumor α-enolase, the lower the survival rate of cancer disease 10. Fig. 16 is a view showing the results of sandwich-type sandwich enzyme-binding immunosorbent assay (ELISA) in pleural effusion obtained from a patient with non-small cell lung cancer (NSCLC) in Example 10 of the present invention. The results showed that the content of α-enolase-specific antibodies in the serum of patients with non-small cell lung cancer was statistically significantly lower than that of normal and non-cancer related diseases. Figure 17 is a graph showing the amount of tumor size in BalB/C mice after inoculation with BalST/C mice by GST-mENO1 recombinant antigen and injection of liver cancer cells into 67 200827718 mice in Example 11 of the present invention. Test results. The results showed that the growth rate of the tumor was significantly inhibited in the mice inoculated with the GST-mENO1 recombinant protein. Fig. 18 is a graph showing the results of the Western blotting method of the mouse ~ enolase specific antibody in Example n of the present invention. Hepatoma cells were first injected into BalB/C mice, then mEN〇1 antigen was administered, and finally the serum obtained from BalB/c mice was tested. The results showed that about half of the mice (3 out of 6) produced a certain amount of mENO1-specific antibodies after administration of the old rat hepatoma cells and the mENO1 antigen. Fig. 19 is a graph showing the results of measuring the tumor size of the mouse liver cancer cells injected into BalB/C mice, followed by administration of mENOl antigen and BalB/C old m* in Example u of the present invention. As a result, the mice which produced the anti-antibody in Fig. 18 had a significantly smaller tumor size than the mice which did not produce the anti-mENO1 antibody. Fig. 20 is a view showing the intensity and distribution of a breast cancer tissue sample by immunohistochemical staining in Example 12 of the present invention. The results showed that the up-regulation of α-enolase was also observed in breast cancer patients. Table 1 shows the number of patients analyzed by immunohistochemical staining (IHC) after staining &amp; enolase according to the intensity and distribution of the heart enzyme enzymatic staining reaction. Table 1 illustrates the correlation between the degree of expression of the β-enamine α-enolase in the rapid scoring method and the clinical parameters of the patient in the rapid scoring method. 68 200827718 [Key component symbol description]

Claims (1)

200827718 X. Application for Patent Park: l A method for monitoring the development of cancer in cancer patients, which comprises: • obtaining cancer cells from patients; and b. determining the α-enolase in cancer cells Abundance; wherein this increase in richness is related to the severity of the cancer. 2. The method of claim 1, wherein the severity of the cancer is related to the number of cancers. 3. The method of claim 1, wherein the higher the number of cancer phases, the higher the expression of the α-enolase. 4. The method of claim 1, wherein the severity of the cancer is related to whether the cancer is prone to recurrence or whether a cancer recurrence occurs. 5. The method of claim 1, wherein the severity of the cancer is related to the survival rate of the cancer patient. 6. The method of claim 1, wherein the alpha-enolase is rich in the alpha-enolase and the oxime 1 by the method of claim 1, wherein the alpha-enolase is measured by the alpha-enolase and the oxime 1 Determined by binding between _ enolase specific antibodies. The method of claim 6, wherein the α-enol transspecific anti-system is produced by an α-enolase cNDA-induced immune reaction and the α-enolase cnda is It is obtained by a puller containing a sequence number··1 and a sequence number of 2 or a degenerate variant thereof. 8. The method of claim 1, wherein the richness is measured by a plurality of methods selected from the group consisting of Western blotting, surface staining and flow cytometry, immunohistochemical staining, Quantitative reverse transcriptase_polymerase chain reaction and microarray analysis. 9. The method of claim 1, wherein the cancer is non-small cell lung cancer. 10. The method of claim 9, wherein the non-small cell carcinoma is selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. 11. The method of claim 9, wherein the non-small cell cell carcinoma is an adenocarcinoma. 12. The method of claim 1, wherein the cancer is rectal cancer, breast cancer, and liver cancer. 13. A method for preparing an α-enolase-specific antibody, comprising: obtaining an α-enolase cDNA; 71 200827718 b· presenting the α-enolase CDNA to produce a recombinant protein; and c·utilizing The recombinant protein produces a monoclonal antibody or a plurality of antibodies specific for a cardiac enolase. 14. The method of claim 13, wherein the antibody is selected from one or more of the group consisting of a plurality of antibodies, monoclonal antibodies, human antibodies, antibodies, or active fragments of the above antibodies. The method of claim 13, wherein the 稀^-thinolase eNDA is obtained by a primer comprising the sequence number: i and SEQ ID NO: 2 or a degenerate variant thereof. 16. A method of detecting cancer, the method comprising: a. obtaining a sample containing an antibody from a patient; and b. determining a richness of an α-enolase-specific antibody in the sample; wherein the low degree is Anti-α-enolase specific antibodies represent the presence of a progressive cancer. [17] The method of claim 16, wherein the sample is a serum sample or a hydroponic water sample. 18. The method of claim 1 is a non-small cell lung cancer. The method of claim 16, wherein the cancer 72 200827718 19. As described in the scope of patent application 帛18, the cell-cell carcinoma system 遝ό余甲 the non-cancerous line k from adenocarcinoma, squamous cell carcinoma and large cell fine two:: gland: _18 items The method according to the method of claim 16, wherein the method of claim 16 is the method of claim 16, wherein the method of claim 22, wherein the method of claim 22, wherein The active immunization mode comprises administering a patient alpha-enolase antigen. (2) The method of claim 22, wherein the passive immunization method comprises directly administering to the patient an α-enolase-specific antibody. 25. The method of claim 24, wherein The anti-system is selected from the group consisting of: a plurality of antibodies, a monoclonal antibody, a human antibody, a chimeric antibody, or an active fragment of the above antibody. ^26. The method of claim 24, Wherein the antibody modulates the activity of the α-enolase. 73 200827718 27. The method of claim 26, wherein the activity comprises a cell transfer ability, a cell proliferative capacity, and/or a cell invasion. 28. The method of claim 24, wherein the enolase-specific antibody reduces and/or prevents the expression of the alpha enolase. 29. - an isolated nucleic acid molecule The nucleotide sequence consists of a sequence: 1 and the sequence of the mouse, or its degenerative variant.