CN1893972A - Listeria-based EphA2 vaccines - Google Patents

Abstract

The present invention relates to methods and compositions designed for the treatment, management, or prevention of cancer, particularly metastatic cancer and cancers of T cell origin, and hyperproliferative diseases involving EphA2-expressing cells. The methods of the invention entail the use of a Listeria-based EphA2 vaccine. The invention also provides pharmaceutical compositions comprising one or more Listeria-based vaccines of the invention either alone in combination with one or more other agents useful for cancer therapy. In certain aspects of the invention, the method entail eliciting both CD4<+> and CD8<+> T-cell responses against EphA2 and/or EphA2-expressing cells.

Description

Based on listerial EphA2 vaccine

The application requires the priority of following application: the U.S. Provisional Application sequence number 60/511 that on October 15th, 2003 submitted, 919, the U.S. Provisional Application sequence number 60/511 of submitting on October 15th, 2003,719, the U.S. Provisional Application sequence number 60/532 of submitting on December 24th, 2003,666, the U.S. Provisional Application sequence number 60/556 of submitting on March 26th, 2004,631, the U.S. Provisional Application (attorney docket 10271-146-888) that the U.S. Provisional Application (attorney docket 10271-144-888) of submitting in October, 2004 and on October 7th, 2004 submit, each application at this by intactly with reference to quoting.

1, technical field

The present invention relates to treat, control or prevent the method and composition of proliferative cell disease. The invention further relates to cause the excessive proliferative cell of antagonism immunoreactive, based on listerial composition and the method for using said composition. Wherein, the present invention includes the vaccine that contains the Listeria (Listeria) of expressing the EphA2 antigenic peptide, and express the immune response of the excessive proliferative cell of EphA2 with this EphA2 vaccine administration to cause antagonism. The present invention also provides vaccine, this vaccine comprise with one or more be used for the treatment of proliferative disorders other agent combination of the present invention one or more based on listerial composition.

2, background technology

2.1. Listeria

Listeria monocytogenes (Listeria monocytogenes) (Listeria) is bacterium in the facultative born of the same parents of Gram-positive, because this bacterium can excite cell-mediated the replying of effective CD4+/CD8+T by MHC I class and II class antigen presentation path, therefore it is developed the vaccine for antigentic specificity, and obtains test as vaccine carrier recently in human clinical's experiment of normal health volunteer.

The Listeria is for many years studied as stimulating the model congenital and antibacterial immunity that adaptability T cell relies on. The ability of Listeria effective stimulus cellular immunity is based on its intracellular life cycle. In case infected the host, this bacterium comprises macrophage and dendritic cells just by phagocyte, absorbs in the phagolysosome rapidly. Most bacterium is degraded subsequently. In the phagosome of the antigen presenting cell that infects, the pathogen proteolysis becomes peptide, and this peptide directly is loaded on the MHC II quasi-molecule. These MHC II-peptide complexes activate CD4+ and " assist " the T cell, thereby stimulate the generation of antibody, and the antigen after the processing is by the present cell surface expression of II class endosome path at this antigen. In described acid cell, some bacterial gene is activated, and comprises cholesterol-dependent cytolysin, LLO, its phagolysosome of can degrading, thus bacterium being discharged in the kytoplasm cell of host cell, breed at this Listeria of survival. Effectively presenting heterologous antigen by MHC I class.path needs the Listeria again to express intrinsic protein. In antigen presenting cell (APC), albumen synthetic by the Listeria and secretion is absorbed by proteosome and degrades. The peptide that produces is transported to endoplasmic reticulum by TAP albumen and is loaded on the MHC I class cell. MHC I-peptide complexes is delivered to cell surface, activates and stimulates the cytotoxic T lymphocyte with homology φt cell receptor to increase and also identify subsequently MHC I-peptide complexes with common stimulation (signal 2) combination of capacity.

2.2. excessive proliferative diseases

2.2.1. cancer

Knurl or tumour are the effluent matter that is produced by unusual uncontrolled Growth of Cells, and it can be optimum or pernicious. Benign tumour generally remains on the part. Malignant tumour is referred to as cancer. Term " pernicious " generally refers to invade and to destroy contiguous human body structure, and diffuse to amphi position point and murderous tumour (about the summary, referring to Robbins and Angell, 1976, Basic Pathology, second edition, W.B.Saunders company, Philadelphia, the 68-122 page or leaf). Cancer can occur in many sites of health and according to its origin different performances be arranged. Cancer cell destroys the body part at its origin place and is diffused into subsequently the other parts of health, and they begin new growth and cause further destruction in these parts.

Have every year and surpass American's cancer stricken of 1,200,000. American cancer be second largest deadly because of, and if according to present trend, expect cancer in 2010 will become first deadly because of. Lung cancer and prostate cancer are the U.S. male sex's maximum cancer killers. Lung cancer and breast cancer are the maximum cancer killers of American Women's. In the U.S., there is a meeting to be diagnosed with cancer certain period in its life among two male sex, there is a meeting to be diagnosed with cancer certain period in its life among three women.

Cancer still can't be cured. Present treatment is selected, and is such as operation, chemotherapy and radiation, usually invalid or produce serious side effect.

2.2.2. metastatic tumor

When tumor cell group has obtained when the ability of growth is settled down in the amphi position point of health and heterogeneous site, usually will produce the cancer of the form that tool life threatens. These metastatic cells are survived by the restriction that breaks through normal inhibition cell and settle down growth to different tissues. For example, if general mammal epithelial cell is transplanted to lung, it generally can not be grown or survive, yet pulmonary metastases is the main cause of breast cancer incidence and the death rate. Recently evidence suggests that the diffusion of metastatic cell in health can occur in primary tumor and occur before the clinical manifestation long time. After detecting and removing primary tumor, these micrometastasis sexual cells can keep dormancy some months or several years. Therefore, the treatment of antagonism metastatic carcinoma and be used for earlier detection and the diagnosis of location metastatic tumor in order to improve design, it is very important to understand better the mechanism that metastatic cell grows in heterogeneous microenvironment and survive.

2.2.3. cancer cell signals

Cancer is a kind of disease of Signal transduction abnormalities. Unusual cell signal has been broken through the grappling dependence limits of cell growth and survival, and (Rhim etc. 1997, Crit Rev in Oncogenesis 8:305; Patarca, 1996, Crit Rev.in Oncogenesis 7:343; Malik etc., 1996, Biochimica et Biophysica Acta 1287:73; Cance etc., 1995, Breast Cancer Res.Treat.35:105). Tyrosine kinase activity is induced by extracellular matrix (ECM) grappling, and in fact, expression or the function of EGFR-TK is enhanced (Rhim etc., 1997, Critical Reviews in Oncogenesis 8:305 usually in malignant cell; Cance etc., 1995, Breast Cancer Res.Treat.35:105; Hunter, 1997, Cell 88:333). In view of the fact of the growth needs tyrosine kinase activity of malignant cell, EGFR-TK becomes the target of new treatment, and (Levitzki etc. 1995, Science 267:1782; Kondapaka etc. 1996, Mol.﹠ Cell Endocrinol.117:53; Fry etc., 1995, Curr. Opin.in BioTechnology 6:662). Unfortunately, the obstacle relevant with tumor cell specific often limits the application of these medicines. Particularly, tyrosine kinase activity is for the function of benign tissue and survival usually most important people such as (, Science 267:1782,1995) Levitzki. For subsidiary toxicity is minimized, at first be identified in the EGFR-TK of selective overexpression in the tumour cell, then extremely important take it as target.

2.2.4. treatment of cancer

The obstacle of research and development antimetastatic agents is for the analytical system that designs and estimate these medicines. The treatment of cancer of most conventional is as target take mushroom cell. Yet cancer cell might not Fast Growth, yet but can survival or growth (Lawrence and Steeg, 1996, World J.Urol.14:124-130) under the condition that does not allow the normal cell survival. Normal cell and malignant cell these basic differences in behavior provide chance for the treatment target. Be diffused into whole body the micrometastasis tumour example highlights externally with in the three-dimensional microenvironment need to assess possible chemotherapeutic agent. Growth or the survival (being monolayer growth) that under cell culture condition commonly used, can measure with the cancer drug analytic approach of multiple standards tumour cell. Yet the cell behavior in two-dimension analysis is the reliably tumour cell behavior in the predictor usually.

At present, treatment of cancer can comprise surgical intervention, chemotherapy, hormone therapy and/or radiotherapy, with the tumour cell of eradicating the patient (referring to, for example, Stockdale, 1998, " Principles of Cancer Patient Management ", Scientific American Medicine, volume 3, Rubenstein and Federman, the 12nd chapter, IV part). Recently, treatment of cancer also can comprise biological therapy or immunization therapy. All these methods have obvious shortcoming for patients. For example, surgical intervention may be not suitable for owing to patient's health condition, and perhaps the patient can not accept. In addition, surgical intervention can not be removed tumor tissues fully. Radiotherapy is only just effective when cambium shows higher radiosensitivity than normal structure, and radiotherapy also can produce serious side effect. Although hormone therapy is that effectively it seldom gives with single agents, after using other treatment to remove most of cancer cell, hormone therapy is usually used in prevention or delays cancer return. Biological therapy/immunization therapy is subject to the number of times restriction, and every kind is treated usually only effective to the cancer of utmost point particular type.

About chemotherapy, there is number of chemical treatment reagent to can be used to treat cancer. The chemotherapy of most of cancer be synthetic by direct inhibition DNA or the biosynthesis that indirectly suppresses deoxidation (ribose) nucleoside triphosphate precursors with prevention dna replication dna and the cell division followed play a role (referring to, for example, the people such as Gilman, 1990, Goodman and Gilman ' s:The Pharmacological Basis of Therapeutics, the 8th edition (Pergamom Press, New York)). These reagent comprise alkylating reagent, such as nitroso ureas, and antimetabolite, such as methotrexate (MTX) and hydroxycarbamide, and other reagent, such as Etoposide (etoposide), campathecins, bleomycin, Doxorubicin, daunorubicins etc. are although they have essential cell cycle specific, but owing to they work to dna replication dna, therefore can be at S phase cell killing. Other reagent, specifically colchicin and vinca alkaloids such as vincaleukoblastinum and vincristine, can disturb the microtubule assembling, thereby mitosis is stagnated. Chemical therapeutic method generally includes the combination that gives chemical treatment reagent and improves result for the treatment of.

Although can use number of chemical treatment reagent, but chemotherapy have a lot of shortcomings (referring to, for example, Stockdale, 1998, " Principles of Cancer Patient Management ", Scientific American Medicine, volume 3, Rubenstein and Federman, the 12nd chapter, the 10th part). Nearly all chemical treatment reagent is all toxic, and the chemotherapy meeting to cause be the side effect of danger close significantly and often, comprise seriously feeling sick bone marrow suppression, immunosupress etc. In addition, even give the combination of chemical treatment reagent, a lot of tumour cells still can have resistance maybe can develop into to chemical treatment reagent and have resistance. In fact, there are those cells of resistance often to be proved to the specific chemical treatment reagent that uses in the methods for the treatment of other drug is also had resistance, even the mechanism of action of those reagent is different from the mechanism of action of used medicine in the specific treatment; This phenomenon is called multidirectional drug resistance or multidrug resistance. Therefore, because drug resistance, kinds cancer is difficult to carry out the chemotherapy of standard.

The so selectable cancer treatment method of significant need is difficult to methods for the treatment of with the cancer of standard cancer treatments method (such as surgical intervention, radiotherapy, chemotherapy, and hormone therapy) treatment in particular for treatment. In addition, it is very rare only to carry out treatment of cancer by a kind of method. Therefore, the new treatment reagent and treatment more effective therapeutic combination cancer, new that need research and development treatment cancer.

2.2.5. other excessive proliferative disorders

2.2.5.1. asthma

Asthma is a kind of illness that intermittent air flue stops up that is characterized as. In western countries, asthma has affected 15% juvenile crowd and the Grown living (Strachan etc., 1994, Arch.Dis.Child 70:174-178) that reaches 7.5%. Most asthma among children and the young man are by the anaphylactogen to sucking, and cause such as the IgE mediation property irritated (atopy) of family dirt mite and cat hair anaphylactogen. Yet not all asthma all is that heredity is hypersensitive, and most atopic individuality is not suffered from asthma. Therefore the factor beyond the atopy be induce this illness necessary (volume such as Fraser, 1994, Synopsis of Diseases of the Chest:635-53 (WB Saunders company, Philadelphia); Djukanovic etc., 1990, Am.Rev.Respri.Dis.142:434-457). Asthma has very strong familial, and is caused by the interaction between inherent cause and the environmental factor. This inherent cause is considered to the variant (" polymorphism ") of normal gene, and the function generation of these variants changes and is easy to occur asthma.

Asthma can be identified with intermittent airflow limitation by periodically panting. The trend of asthma can be quantized by measuring bronchial hyperreactive, wherein make up individual dosage-response curve to bronchoconstriction agent such as histamine or methacholine. This curve is summarized as the dosage that causes air-flow 20% to reduce (PD20) usually, or the slope of a curve (slope) between initial gas flow measurement value and the last dosage.

In the heredity allergic reaction, the B cell produces IgE when replying the anaphylactogen stimulation. These antibody are coated with mast cell and cause a series of cell event by being combined with the high-affinity receptor of IgE, thereby cause the unstable and release inflammatory mediator of cell membrane. This can cause the mucous membrane inflammation, pants, coughs, sneezes and have a stuffy nose.

Atopy can be diagnosed in the following way: the positive skin test of (i) replying common anaphylactogen; (ii) existence of the specific serum IgE of detection anaphylactogen; Or (iii) rising of detection total serum IgE.

2.2.5.2.COPD

Chronic obstructive pulmonary disease (COPD) often is used to describe two kinds of situations of stationarity airways disorders, i.e. chronic bronchitis and emophysematous general name. Chronic bronchitis and pulmonary emphysema the most normally are to be caused by smoking, and about 90% COPD patient is or once was the smoker. Although about 50% smoker has produced chronic bronchitis, only there is 15% smoker the occlusive airflow obstruction to occur. Some animal, particularly horse also can suffer from COPD.

The airflow obstruction relevant with COPD is gradual, may be attended by air flue overreaction and may be partly reversible. Non-specific air flue hyperreactive also may brought into play in the development of COPD and act on, and may be that PFT accelerates depleted tendency.

COPD is dead and disabled major reason. At present it US and European be the fourth-largest deadly because of. Treatment is instructed and is advocated earlier detection and implement the smoking cessation plan, to help to reduce the M ﹠ M of this illness. Yet because many reasons, early stage diagnosis and detection is always very difficult. The time of the development need several years of COPD, and bronchitic acute attack often is not recognized as the early signal of COPD by general doctor. A lot of patients show the feature of various disease conditions (for example chronic bronchitis or asthmatic bronchitis), and this particularly diagnoses in the teiology of this illness disease in early days so that Accurate Diagnosis becomes a kind of challenge. And a lot of patients do not seek medical science and help, until they are subjected to the more serious symptoms relevant with lung failure, and for example expiratory dyspnea, persistent cough and generation phlegm. Therefore, most patients can not get diagnosing or treat until they develop into the more high-stage of this illness.

2.2.5.3. mucoprotein

Mucoprotein is by epithelial cell, comprises the glycoprotein family of the epithelial cells of respiratory tract, intestines and stomach and female genital tract. Mucoprotein makes mucus have viscoplasticity (Thornton etc., 1997, J. Biol.Chem.272:9561-9566). Knownly in human body, express nine kinds of mucin gene: MUC 1, MUC 2, MUC 3, MUC 4, MUC 5AC, MUC 5B, MUC 6, MUC 7 and MUC 8 (Bobek etc., 1993, J.Biol.Chem.268:20563-9; Dusseyn etc., 1997, J.Biol. Chem.272:3168-78; Gendler etc., 1991, Am.Rev.Resp.Dis.144:S42-S47; Gum etc., 1989, J.Biol.Chem.264:6480-6487; Gum etc., 1990, Biochem.Biophys.Res. Comm.171:407-415; Lesuffleur etc., 1995, J.Biol.Chem.270:13665-13673; Meerzaman etc., 1994, J.Biol.Chem.269:12932-12939; Porchet etc., 1991, Biochem.Biophys.Res.Comm.175:414-422; Shankar etc., 1994, Biochem.J. 300:295-298; Toribara etc., 1997, J.Biol.Chem.272:16398-403). Many airway disorders such as chronic bronchitis, chronic obstructive pulmonary disease, bronchietactis, asthma, cystic fibrosis and bacterium infect is characterized as mucoprotein overexpression (Prescott etc., Eur.Respir.J., 1995,8:1333-1338; Kim etc., Eur.Respir.J., 1997,10:1438; Steiger etc., Am J Respir. Cell Mol.Biol., 12:307-314). The mucoprotein excessive secretion causes the mucomembranous cilium damage, thereby air flue mucus is blocked, and this obstruction can promote chronic infection, air flue to stop up, cause sometimes death. For example, COPD, a kind of so that slowly carrying out property and irreversibility airflow limitation are the main cause of death as the illness of feature in developed country. Breathing degenerates comprises that mainly intracavity diameter diminishes, and this is because goblet cell hyperplasia and excessive secretion cause airway walls thickening and mucus to increase. Known EGF (EGF) raises epithelial propagation and mucinous generation/secretion (Takeyama etc., 1999, Proc.Natl. Sci.USA 96:3081-6; Burgel etc., 2001, J.Immunol.167:5948-54). EGF also makes the mucin secretion cell, such as goblet cell propagation and mucoprotein in the human airway epithelial cells is produced increase (Lee etc., 2000, Am.J.Physiol.Lung Cell Mol.Physiol.278:185-92; Takeyama etc., 2001, Am.J.Respir.Crit.Care.Med.163:511-6; Burgel etc., 2000, J Allergy Clin.Immunol.106:705-12). All the time, treat the mucus excessive secretion with two kinds of methods: the physical method that increases scavenger reagent and mucolysis reagent. There is not a kind of method that the patient has been produced positive effect or reduced the mucoprotein obstruction. Therefore, need to reduce the method for the mucoprotein generation illness relevant with the mucoprotein excessive secretion with treatment.

2.2.5.4. ISR

Blood vessel is got involved, and comprises that angioplasty, Stent, resection and transplantation are usually complicated owing to produce unwanted effect. Being exposed to the medical supply that is implanted into or is inserted in the patient body can make bodily tissue produce bad physiological reaction. For example, some conduit or support inserted or implanted and bolt or grumeleuse can be caused in blood vessel, forming. The bad reaction that other blood vessel is got involved comprises endothelial cell proliferation, ISR (being the again obstruction of artery), angiemphraxis, platelet aggregation and the calcification that can cause hyperplasia. The treatment of ISR generally includes the second angioplasty or bypass operation. Specifically, ISR may be because the endothelial cell damage that the blood vessel intervention in treatment ISR process causes causes.

Angioplasty is included in the atherosclerotic injury site of partial blockage foley's tube is inserted artery. The expansion of sacculus is easy to destroy inner membrance and enlarges blocks. About obstruction of 20% to 30% is only at several days or a few Zhou Houzai inaccessible (Eltchaninoff etc., 1998, J.Am Coll.Cardiol.32:980-984). The use of support has reduced the again incidence of obturation, but the percentage that ISR occurs is still very remarkable. The restenosis rate of postangioplasty depends on many factors, comprises the length of patch. According to the risks and assumptions that exists, stenosis rate changes between 10% to 35%. In addition, the angiography that repeats after a year shows only have to seem normal inner chamber in about 30% the blood vessel that lives through this process.

ISR is that the accumulation by extracellular matrix causes, and this extracellular matrix contains the collagen relevant with smooth muscle cell and proteoglycans, finds these matrix in balloon injured or clinical postangioplasty in artery congee sample knurl and the damage of artery hyperplasia. Some delays that the inner chamber relevant with smooth muscle cell proliferation is narrow may be because the new intima smooth muscle cell continues the synthetic substrate material. The matrix that has in vivo various media can change smooth muscle is synthesized.

2.2.5.5. new intima hyperplasia

The new intima hyperplasia is the pathological process of transplanted abdominal sclerosis, narrow and most of vasotransplantation obturation. The new intima hyperplasia is won reaction and operation transplantation is observed at various forms of injury of blood vessel and most of vein transplantation usually after the high pressure arterial circulation.

The smooth muscle cell in vascular wall intermediate layer (being the middle level) is activated, divides, breeds and move internal layer (being theca interna). The short scorching molecule of the unusual new intima cellular expression that generates comprises cell factor, chemokines and adhesion molecule, and they further start cascade event, cause occlusive new intima disease and final graft failure.

Smooth muscle cell proliferation is the critical event in the new intima proliferation response. Making ins all sorts of ways studies, and has confirmed clearly that the blocking-up smooth muscle cell proliferation can make the phenotype of normal blood vessels and function be kept, thereby has reduced new intima hyperplasia and final graft failure.

The existing methods for the treatment of for the treatment of indication as discussed above is also not enough, therefore, need to improve the methods for the treatment of of above-mentioned indication.

2.3.EphA2

EphA2 is the receptor tyrosine kinase of the 130kDa that expresses in becoming the HEP, find that its level in epithelial cell is low and in cell-cell adherence site, obtain enrichment (Zantek etc., 1999, Cell Growth ﹠ Differentiation 10:629; Lindberg etc., 1990, Molecular ﹠ Cellular Biology 10:6316). This Subcellular Localization is very important, because EphA2 is in conjunction with the part (being known as Ephrins A1 to A5) (Eph NK, 1997, the Cell 90:403 that are anchored on the cell membrane; Gale etc., Cell ﹠ Tisue Research 290:227). The elementary result of ligand binding is EphA2 autophosphorylation (Lindberg etc., 1990, the same). But, unlike other receptor tyrosine kinase, in the situation of ligand binding or phosphorylated tyrosine shortage, EphA2 maintenance enzymatic activity (Zantek etc., 1999, the same). EphA2 raises the cell of a large amount of excessive propagation, comprises the invasive cancer cell.

3, summary of the invention

EphA2 overexpression and functional variation occurs in a large amount of malignant cancer. EphA2 is cancer protein and is enough to give the cancer metastasis potentiality. EphA2 also disease relevant with other excessive proliferative cell and that cause with the excessive propagation of cell is relevant. The present invention is based on the inventor's following discovery: individual body and function is expressed the Listeria administration of EphA2 antigenic peptide, useful treatment and the preventive effect of the excessive proliferative disorders relevant with EphA2 overexpression cell that create antagonism. Be not subject to any mechanism or theoretical constraint, believe that treatment and preventive effect are the results who causes immune response with the Listeria administration of expressing the EphA2 antigenic peptide.

Therefore the present invention provides based on listerial EphA2 vaccine and has used their method. Of the present inventionly can cause cellullar immunologic response, HI or both based on listerial EphA2 vaccine. When immune response was cellullar immunologic response, it can be Tc, Th1 or Th2 immune response. In preferred embodiments, this immune response is the Th2 cellullar immunologic response.

In preferred embodiments, one or more antigenic determinants based on listerial EphA2 vaccine expression EphA2 of the present invention, this EphA2 optionally exposes on cancer cell with respect to non-cancer cell (i.e. normal, healthy cell or the not cell of excessive propagation) or increases. In one embodiment, this cancer is the epithelial cell source. In other embodiments, this cancer is cutaneum carcinoma, lung cancer, colon cancer, prostate cancer, breast cancer, oophoroma, cancer of the esophagus, carcinoma of urinary bladder or cancer of pancreas or clear-cell carcinoma or melanoma. In another embodiment, this cancer is the T cell source. In other embodiments, cancer is leukaemia or lymthoma.

In preferred embodiments, method and composition of the present invention is used for the transfer that the tumour of EphA2 is expressed in prevention, treatment or control. In preferred embodiments; the EphA2 that immune response is resisted expresses cell (" target cell ") with respect to the normal health cell overexpression EphA2 of same type, and this can detect with analytical method described herein or well known by persons skilled in the art (for example immunoassay such as ELISA or Western trace, Northern trace or RT-PCR). In preferred embodiments, with respect to the normal health cell of same type, the EphA2 on the target cell seldom with ligand binding, this may be because cell-cells contacting reduces, Subcellular Localization is changed or the quantity of EphA2 increases with respect to part. In another embodiment, cell with respect to the normal health of same type, about 10% or still less, about 15% or still less, about 20% or still less, about 25% or still less, about 30% or still less, about 35% or still less, about 40% or still less, about 45% or still less, about 50% or still less, about 55% or still less, about 60% or still less, about 65% or still less, about 70% or still less, about 75% or still less, about 80% or still less, about 85% or still less, about 90% or still less, about 95% or target cell still less on EphA2 and part (for example EphrinA1) combination, this can detect with analytical method known in the art (for example immunoassay). In another embodiment, cell with respect to the normal health of same type, few 1-10 doubly, 1-8 doubly, 1-5 doubly, 1-4 doubly or 1-2 doubly or EphA2 and part (for example EphrinA1) combination on the target cell of 1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times or 10 times, this can detect with analytical method known in the art (for example immunoassay).

Therefore, the invention provides the immunoreactive method that causes antagonism EphA2 expression cell, described method comprises to cause antagonism EphA2 and expresses the immunoreactive effective dose of cell, uses based on listerial EphA2 vaccine to individual administration.

The invention provides treatment, prevention or control EphA2 and express the method for the excessive proliferative disorders of cell, described method comprises treating or preventing the effective dose of excessive proliferative disorders (for example excessive proliferative disorders of tumour and the excessive proliferative disorders of non-tumour), uses based on listerial EphA2 vaccine to individual administration. The present invention also provide can be used for causing antagonism EphA2 express the immune response of cell and/or be used for the treatment of, prevent or control EphA2 expression cell excessive proliferative disorders based on listerial EphA2 vaccine.

Can comprise Listeria as EphA2 antigenic peptide expression vector based on listerial EphA2 vaccine. In preferred embodiments, the Listeria as EphA2 antigenic peptide expression vector to individual (preferably people) administration is attenuation. For example, may organize at it to the attenuation Listeria of individual (preferably people) administration and weakened aspect taxis (for example inlB mutant) or the diffusivity from cell to cell (for example actA mutant). In specific embodiments, be included in the sudden change (for example delete, add or replace) in one or more internalization element (for example inlA and/or inlB) to the attenuation Listeria as EphA2 antigenic peptide expression vector of individual (preferably people) administration, and this sudden change causes attenuated listeria bacterium. In another embodiment, weakened aspect taxis (for example inlB mutant) and the diffusivity from cell to cell (for example actA mutant) to organizing at it as the attenuation Listeria of EphA2 antigenic peptide expression vector of individual (preferably individual human) administration. In another embodiment, the attenuation Listeria as EphA2 antigenic peptide expression vector of individual (preferably individual human) administration is included in sudden change (for example delete, add or replace) and the sudden change in actA among the internalization element B, and these sudden changes cause attenuated listeria bacterium.

Listeria of the present invention (preferably, the Listeria of attenuation) be preferably constructed to can expression-secretion from listerial EphA2 antigenic peptide. In specific embodiments, the nucleic acid of coding EphA2 antigenic peptide comprises the coding secretion signal, for example SecA secretion signal or Tat signal, nucleotide sequence, this sequence functionally is connected with the nucleotide sequence of the EphA2 antigenic peptide of encoding. In certain embodiments, this burst is the Listeria burst. In other embodiments, this burst is the bacterium burst (being non-Listeria burst) except the burst of Listeria.

The Listeria bacterial strain that is applicable to method and composition of the present invention includes but not limited to Listera grayi (Listeria grayi), listera innocua (Listeria innocua), listeria ivanovii (Listeria ivanovii), listeria monocytogenes (Listeria monocytogenes), Si Shi Listeria (Listeria seeligeri) and Wei Shi Listeria (Listeria welshimeri). The listerial preferred strain that is used for method and composition of the present invention is listeria monocytogenes.

The compositions and methods of the invention can be used for treatment, prevent and/or control excessive proliferative diseases. In certain embodiments, this excessive proliferative diseases is cancer. In certain embodiments, this cancer be the epithelial cell source and/or relate to respect to having the cell of the non-cancer cell overexpression EphA2 of homologue's type with described cancer cell. In specific embodiments, this cancer is cutaneum carcinoma, lung cancer, colon cancer, breast cancer, oophoroma, cancer of the esophagus, prostate cancer, carcinoma of urinary bladder or cancer of pancreas or clear-cell carcinoma or melanoma. In other embodiments, this cancer is the T cell source. In specific embodiments, this cancer is leukaemia or lymthoma. In other embodiments, this excessive proliferative disorders is nontumorous. In specific embodiments, the excessive proliferative disorders of this non-tumour is epithelial cell disorders. The example of the excessive proliferative disorders of non-tumour is asthma, chronic pulmonary obstruction disease, pulmonary fibrosis, bronchus overreaction, psoriasis and seborrhea. In certain embodiments, this excessive proliferative diseases is the endothelial cell illness.

The EphA2 antigenic peptide that is used for method and composition of the present invention can comprise EphA2 or its antigenicity fragment, analog or the derivative of total length. In certain embodiments, the EphA2 antigenic peptide comprises born of the same parents' intracellular domain of ectodomain or the EphA2 of EphA2. In certain embodiments, the EphA2 antigenic peptide lacks the EphA2 membrane spaning domain. In certain embodiments, the EphA2 antigenic peptide comprises the outer and born of the same parents' intracellular domain of born of the same parents of EphA2, and lacks the EphA2 membrane spaning domain. In certain embodiments, the EphA2 antigenic peptide comprises total length EphA2 or its fragment, has by the replacement of lysine to methionine at amino acid residue 646 places of EphA2. In certain embodiments, the EphA2 antigenic peptide comprises the outer and born of the same parents' intracellular domain of born of the same parents of EphA2, lacks the EphA2 membrane spaning domain, and has by the replacement of lysine to methionine at amino acid residue 646 places of EphA2. In certain embodiments, the EphA2 antigenic peptide is chimeric polyeptides, and it comprises at least antigenic portions and the second polypeptide of EphA2.

Express the Listeria of EphA2 antigenic polypeptide and can express one or more EphA2 antigenic peptide. In specific embodiments, 2,3,4,5,6,7,8,9,10,15,20,25 of the Listeria expression of expression EphA2 antigenic peptide or more EphA2 antigenic peptide, or 2-5 is individual, 2-10 is individual, 2-20 is individual, 10-20 is individual or 15-25 EphA2 antigenic peptide. A plurality of EphA2 antigenic peptides can be expressed from single expression construct or a plurality of expression construct. This expression construct can be dissociate or be integrated in the listerial genome. For example, in certain embodiments, the genome of Listeria vaccine strains comprises one or more gene expression frame, in the born of the same parents of their assembly coding EphA2 and ectodomain. In specific embodiments, this one or more expression cassette is integrated in the genome of Listeria.

Vaccine of the present invention can contain one or more Listeria of expressing the EphA2 antigenic peptide. In specific embodiments, vaccine of the present invention contains 2,3,4,5,6,7,8,9,10,15,20,25 or more express the Listeria of EphA2 antigenic peptide, or the Listeria of 2-5,2-10,2-20,10-20 or 15-25 expression EphA2 antigenic peptide.

Method of the present invention comprise based on listerial EphA2 vaccine and one or more other the treatment (for example other anticancer therapy) combination treatment. In certain embodiments, other anticancer therapy is excitability EphA2 antibody, namely is combined with EphA2 and induces the antibody of EphA2 signal and phosphorylation. In other embodiments, this other anticancer therapy is the antiidiotype of anti-EphA2 antibody. In other embodiments, this other anticancer therapy is chemotherapy, biology treatment, immunization therapy, radiotherapy, hormone therapy or operation.

Of the present invention aspect some in, of the present invention based on listerial vaccine and the therapeutic combination administration that improves the EphA2 internalization. In specific embodiments, described reagent is the EphA2 activator, (sees such as Koolpe etc. 2002, J.Biol.Chem.277 (49): 46974-46979) or little molecule such as antibody, peptide. In other particular, this reagent is the phosphatidase of regulating EphA2, for example inhibitor of low-molecular-weight tyrosine phosphatidase (LMW-PTP).

Vaccine of the present invention can pass through in the mucous membrane, nose, administration in the parenteral, muscle, in the intravenous, oral or abdominal cavity. In specific embodiments, vaccine of the present invention injection site administration in disease site passes through for example transplanting or tumour.

In other embodiments, of the present invention based on listerial EphA2 vaccine is used to treat, prevent and/or control is relevant with the excessive propagation of cell non-Cancerous disease or illness, such as but not limited to asthma, chronic obstructive pulmonary disease, ISR (smooth muscle and/or endothelium), psoriasis etc. In preferred embodiments, this excessive proliferative cell is epithelial cell. In preferred embodiments, this excessive proliferative cell overexpression EphA2. In another preferred embodiment, some for example 5% or still less, 10% or still less, 15% or still less, 20% or still less, 25% or still less, 30% or still less, 35% or still less, 40% or still less, 45% or still less, 50% or still less, 55% or still less, 60% or still less, 75% or still less, 85% or still less) EphA2 not with ligand binding, this can detect with analytical method known in the art (for example immunoassay), and this may be because cell-cells contacting reduces, Subcellular Localization is changed or the quantity of EphA2 increases with respect to part.

In other side of the present invention, be used to treat, prevent and/or control with abnormal vascular relevant illness occurs based on listerial EphA2 vaccine. Be used to cause the EphA2 that immune response is expressed at new vessels with antagonism based on listerial EphA2 vaccine. Therefore, the invention provides the method for the treatment of, prevention and/or the control illness relevant with the abnormal vascular generation, the method comprises to the individuality that these needs are arranged treats, prevents and/or the composition of the effective dose of relevant illness occurs for control and abnormal vascular, and said composition comprises the Listeria of expressing the EphA2 antigenic peptide. The example of these diseases includes but not limited to macular degeneration, DRP, retinopathy of prematurity, reangiostenosis, infantile hemangioma, verruca vulgaris, psoriasis, Kaposi sarcoma, neural line fibroma, recessive atrophic type bubble epithelium dissociate disease before disease, rheumatic arthritis, stiff spondylitis, systemic lupus, chronic eczema arthropathy, Lay Te Shi syndrome and house Ge Lunshi syndrome, mullerianosis, the pregnancy period eclampsia, arteriosclerosis and coronary artery disease.

Method and composition of the present invention not only can be used for treating the patient who did not accept treatment, also can be used for treating with present standard and laboratory cancer therapy, such as but not limited to chemotherapy, hormone therapy, biology treatment, radiotherapy and/and operation, be difficult to partially or completely the patient that treats, and improve the effect of these treatments. Particularly, the expression of EphA2 improves the level of cell factor IL-6, and this factor and cancer cell are to different therapeutic schemes, and for example the generation of the resistance of chemotherapy and hormone therapy is relevant. In addition, the EphA2 overexpression can overcome the needs to estrogen receptor activity, thereby causes the tamosifen resistance of breast cancer cell. Therefore, in preferred embodiments, the invention provides treatment, prevention or control and shown that being maybe may be with except treatment and the prevention method that is difficult to the cancer for the treatment of or not replying based on the method the listerial EphA2 vaccine administration of the present invention. In specific embodiments, of the present invention one or more are difficult to the patient that treats or do not reply based on the non-treatment based on EphA2 of listerial EphA2 vaccine (particularly tamoxifen therapy or the resistance treatment relevant with the IL-6 level of rising), thereby make the patient become medicable or reply. Then give effective treatment to the patient who is difficult to treat before this or does not reply.

Method and composition of the present invention not only can be used for treating the patient who did not accept treatment, also can be used for treating with the standard of the excessive proliferative disorders of the non-tumour of present treatment and/or the illness relevant with the abnormal vascular generation and laboratory therapy being difficult to partially or completely the patient that treats. Method and composition of the present invention can be used for treating with the present excessive proliferative disorders for the treatment of tumour and/or with abnormal vascular relevant illness (for example macular degeneration, DRP, retinopathy of prematurity, reangiostenosis, infantile hemangioma, verruca vulgaris, psoriasis, Kaposi sarcoma, neural line fibroma, recessive atrophic type bubble epithelium dissociate disease before disease, rheumatic arthritis, stiff spondylitis, systemic lupus, chronic eczema arthropathy, dish Te Shi syndrome and house Ge Lunshi syndrome, mullerianosis, the pregnancy period eclampsia, arteriosclerosis and coronary artery disease), asthma, chronic pulmonary obstruction venereal disease, pulmonary fibrosis, bronchus overreaction, psoriasis and seborrhea occur) standard and laboratory therapy be difficult to partially or completely the patient that treats.

The present invention also provides the kit that comprises vaccine of the present invention or vaccine component.

3.1. definition

In this manual, the term of use " based on listerial EphA2 vaccine " refers to be fabricated to express the Listeria of EphA2 antigenic peptide, or comprises the composition of this bacterium. When with the effective dose administration, of the present inventionly caused the immune response of resisting the EphA2 on the excessive proliferative cell based on listerial EphA2 vaccine. The listerial bacterial strain that is applicable to vaccine of the present invention includes but not limited to Listera grayi, listera innocua, listeria ivanovii, listeria monocytogenes, Si Shi Listeria and Wei Shi Listeria. In preferred embodiments, the Listeria is listeria monocytogenes.

In this manual, the term of use " EphA2 antigenic peptide " and " EphA2 antigenic polypeptide " refer to the EphA2 polypeptide, preferably SEQ ID NO:2 polypeptide or comprise one or more B cell antigen determinant of EphA2 or fragment, analog or the derivative of T cellular antigens determinant. The EphA2 polypeptide can be from any species. In certain embodiments, the EphA2 polypeptide refers to the rear form of maturation, processing of EphA2. In other embodiments, the EphA2 polypeptide refers to the non-mature form of EphA2.

In document or public database, can find nucleotides and/or the amino acid sequence of EphA2 polypeptide, maybe can use Cloning and sequencing technology definite kernel thuja acid well known by persons skilled in the art and/or amino acid sequence. For example, in the GenBank database, can find the nucleotide sequence (seeing for example registration number BC037166, M59371 and M36395) of people EphA2. In the GenBank database, can find the amino acid sequence (seeing for example registration number NP_004422, AAH37166 and AAA53375) of people EphA2. Other non-limitative example of the amino acid sequence of EhpA2 is enumerated in following table.

Table 1

Species	The GenBank registration number
		Mouse	NP_034269，AAH06954
Rat	XP_345597
		Chicken	BAB63910

In certain embodiments, the EphA2 antigenic peptide is not one or more following peptide: TLADFDPRV (SEQ ID NO:3); VLLLVLAGV (SEQ ID NO:4); VLAGVGFFI (SEQ ID NO:5); IMNDMPIYM (SEQ ID NO:6); SLLGLKDQV (SEQ ID NO:7); WLVPIGQCL (SEQ ID NO:8); LLWGCALAA (SEQ ID NO:9); GLTRTSVTV (SEQ ID NO:10); NLYYAESDL (SEQ ID NO:11); KLNVEERSV (SEQ ID NO:12); IMGQFSHHN (SEQ ID NO:13); YSVCNVMSG (SEQ ID NO:14); MQNIMNDMP (SEQ ID NO:15); EAGIMGQFSHHNIIR (SEQ ID NO:16); PIYMYSVCNVMSG (SEQ ID NO:17); DLMQNIMNDMPIYMYS (SEQ ID NO:18). In certain embodiments, the EphA2 antigenic peptide is not any among the SEQ ID NO:3-12, is not any among the SEQ ID NO:13-15, and/or is not any among the SEQ ID NO:16-18. In another particular, the EphA2 antigenic peptide is not SEQ ID NO:3-18.

In this manual, the protein sample reagent that the term " analog " about protein sample reagent (for example peptide, polypeptide, protein or antibody) that uses refers to have the function similar or identical with the second protein sample reagent (for example EphA2 polypeptide), but it needn't comprise amino acid sequence or the structure similar or identical with this second protein sample reagent. Protein sample reagent with similar amino acid sequence refers to satisfy at least the protein sample reagent with the next item down: (a) have the protein sample reagent with the amino acid sequence at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of the second protein sample reagent, at least 95% or at least 99% identical amino acid sequence; (b) by nucleotide sequence coded protein sample reagent, this nucleotides sequence is listed under the stringent condition nucleotide sequence hybridization with the second protein sample reagent of at least 20 amino acid residues of coding, 30 amino acid residues, 40 amino acid residues, 50 amino acid residues, 60 amino acid residues, 70 amino acid residues, 80 amino acid residues, 90 amino acid residues, 100 amino acid residues, 125 amino acid residues or 150 amino acid residues; And (c) by nucleotide sequence coded protein sample reagent, the nucleotides at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65 % of this nucleotide sequence and coding the second protein sample reagent, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical. Refer to have the secondary similar to this second protein sample reagent to the protein sample reagent that the second protein sample reagent has an analog structure, the protein sample reagent of three grades or quaternary structure. The structure of protein sample reagent can be determined by method known to those skilled in the art, include but not limited to X-radiocrystallography, nuclear magnetic resonance and crystallography electron microscopy. Preferably, this protein sample reagent has the EphA2 activity.

In order to determine the uniformity percentage of two amino acid sequences or two nucleotide sequences, this sequence is compared to realize best comparison purpose (for example, can introduce the interval in the first amino acid or nucleotide sequence compares to carry out the best with the second amino acid or nucleotide sequence). Then relatively at the amino acid position of correspondence or amino acid residue or the nucleotides of nucleotide position. When the correspondence position of the amino acid residue of certain position in the First ray or nucleotides and the second sequence was identical, this two molecule was consistent in this position so. Uniformity percentage between two sequences is the function (being the total number * 100% of the number/position of the consistent lap position of homogeneity %=) of the number of the total consistent position of sequence. In one embodiment, two sequences are with long.

Can determine uniformity percentage between two sequences with mathematical algorithm. Be used for mathematical algorithm preferred of two sequences relatively but nonrestrictive example is Karlin and Altschul, 1990, Proc. Natl Acad Sci.U.S.A.87:2264-2268 and Karlin and Altschul, improved algorithm among 1993, the Proc.Natl Acad Sci.U.S.A.90:5873-5877. This algorithm has been integrated into Altschul etc., in the NBLAST of 1990, J.Mol.Biol.215:403 and the XBLAST program. Can use NBLAST nucleotides parameter configuration, for example with score=100, word long=12 carries out BLAST nucleotides and retrieves to obtain nucleotide sequence with nucleic acid molecules homology of the present invention. Can use XBLAST program parameter configuration, for example with score=50, word long=3 carries out BLAST protein and retrieves to obtain amino acid sequence with protein molecule homology of the present invention. In order to obtain to use such as Altschul etc., the Gapped BLAST described in 1997, the Nucleic Acids Res.25:3389-3402 for being spaced of comparison. Selectively, can use PSI-BLAST to carry out multiple retrieval, this retrieval detects intermolecular remote relation (ibid). When using BLAST, Gapped BLAST and PSI-Blast program, can use the default parameters (such as the parameter of XBLAST and NBLAST) (referring to for example NCBI website) of corresponding program. Another of mathematical algorithm that is used for the sequence comparison is preferred but non-limitative example is Myers and Miller, the algorithm of 1988, CABIOS 4:11-17. This algorithm is integrated in the ALIGN program (2.0 editions), and this program is the part of GCG sequence alignment software kit. When using ALIGN program comparision amino acid sequence, can use PAM120 weight residue table (weight residue table), gap length penalties (gap length penalty) is 12, interval penalties (gap penalty) is 4.

Uniformity percentage between two sequences can with determining with similar technique mentioned above, allow or not allow to use the interval. When calculating uniformity percentage, general pairing of only calculating reality.

In this manual, the term about the nonprotein analog of use " analog " refers to have and the first organic or inorganic molecular mimicry or identical function and the second organic or inorganic molecule similar to this first organic or inorganic molecular structure.

In this manual, the term of use " attenuation " or " attenuation " refer to so that the pathogenic more weak modification in Listeria. The final result of attenuation is when with the Listeria during to individual administration, and the risk of toxicity and other side effect is reduced.

In this manual, the term " derivative " about protein sample reagent (for example protein, polypeptide, peptide and antibody) that uses refers to comprise the protein sample reagent of amino acid sequence, by introducing replacement, deletion and/or the interpolation of amino acid residue, this amino acid sequence is changed. Term " derivative " also refers to the protein sample reagent through modifying as used herein, is about to the covalently bound protein sample reagent to this protein sample reagent of molecule. For example; but be not limited to; the derivative that can prepare protein sample reagent is such as by glycosylation, acetylation, Pegylation, phosphorylation, amidatioon, derived, proteolytic cleavage, prepared with modes such as cell ligand or other albumen are connected by known protection/blocking groups. The derivative of protein sample reagent also can be by using technology well known by persons skilled in the art, includes but not limited to that the metabolism of specificity chemical cracking, acetylation, formylated, tunicamycin is synthetic etc. carry out chemical modification and prepare. In addition, the derivative of protein sample reagent can comprise one or more nonclassical amino acid. The derivative of protein sample reagent with its derived from protein sample reagent have identical function.

In this manual, the term " derivative " about EphA2 protein sample reagent that uses refers to comprise the protein sample reagent of the amino acid sequence of EphA2 polypeptide or EphA2 polypeptide fragment, and this amino acid sequence is changed by replacement, deletion or the interpolation (namely suddenling change) of introducing amino acid residue. Also refer to through modifying about the term " derivative " of EphA2 protein sample reagent as used herein, be about to the molecule of any type covalently bound to this polypeptide the EphA2 polypeptide or the fragment of EphA2 polypeptide. For example; but be not limited to, EphA2 polypeptide or EphA2 polypeptide fragment can be by glycosylations, acetylation, Pegylation, phosphorylation, amidatioon, derived, proteolytic cleavage, modified with modes such as cell ligand or other albumen are connected by known protection/blocking groups. The derivative of EphA2 polypeptide or EphA2 polypeptide fragment also can be by using technology well known by persons skilled in the art, includes but not limited to that the metabolism of specificity chemical cracking, acetylation, formylated, tunicamycin is synthetic etc. carry out chemical modification and modified. In addition, the derivative of EphA2 polypeptide or EphA2 polypeptide fragment can comprise one or more nonclassical amino acid. In one embodiment, polypeptide derivative has the function similar or identical with EphA2 polypeptide described herein or EphA2 polypeptide fragment. In another embodiment, compare with unaltered polypeptide, the activity of the derivative of EphA2 polypeptide or EphA2 polypeptide fragment obtains changing. For example, the derivative of polypeptide or its fragment than EphA2 polypeptide or its fragment different aspect the phosphorylation.

In this manual, the term about nonprotein sample reagent of use " derivative " refers to the second organic or inorganic molecule according to the formation of the first organic or inorganic molecular structures. The derivative of organic molecule includes but not limited to the molecule modified, for example by adding or deletion hydroxyl, methyl, ethyl, carboxyl, nitro or the amino molecule of modifying. Organic molecule also can, for example esterified, alkylation and/or phosphorylation.

In this manual, " the EphrinA1 polypeptide " of use refers to EphrinA1, its analog, derivative or fragment, or comprises the fusion of EphrinA1, its analog, derivative or fragment. The EphrinA1 polypeptide can be from any species. In certain embodiments, term " EphrinA1 polypeptide " refers to the rear form of maturation, processing of EphrinA1. In other embodiments, term " EphrinA1 polypeptide " refers to the immature form of EphrinA1. According to this embodiment, antibody mediated immunity of the present invention is specifically in conjunction with the immature form part corresponding to the EphrinA1 of form after the maturation processing of EphrinA1.

Can in document or public database, find nucleotides and/or the amino acid sequence of EphrinA1 polypeptide, maybe can use Cloning and sequencing technology well known by persons skilled in the art to determine this nucleotides and/or amino acid sequence. For example, can in the GenBank database, can find the nucleotide sequence (seeing for example registration number BC032698) of people EphrinA1. In the GenBank database, can find the amino acid sequence (seeing for example registration number AAH32698) of people EphrinA1. Other non-limitative example of amino acid sequence is enumerated in following table 2.

Table 2

Species	The GenBank registration number
		Mouse	NP_034237
Rat	NP_446051

In specific embodiments, the EphrinA1 polypeptide is the EphrinA1 from any species. In preferred embodiments, the EphrinA1 polypeptide is people EphrinA1.

In this manual, the term " effective dose " that uses refers to the amount for the treatment of (for example prevention or therapeutic agent), this amount is enough to reduce and/or improve seriousness and/or duration or the condition symptoms of illness (for example cancer, the excessive proliferative cell illness of Non-cancerous or the illness relevant with the abnormal vascular generation), prevent the deterioration of described illness, make described illness decline, prevent recurrence, development or the outbreak of one or more symptoms relevant with described illness, or strengthen or improve prevention or the result for the treatment of of another kind for the treatment of (for example prevention or therapeutic agent).

In this manual, the term of use " B cell antigen determinant " refers to animal preferably mammal, most preferably have the part of the EphA2 polypeptide of antigen or immunogen activity in mouse or people. Antigenic determinant with immunogen activity is the part of EphA2 polypeptide, and it causes the antibody response of animal. Antigenic determinant with antigen active is the part of EphA2 polypeptide, and antibody is combined specifically with this partial immunity, this can with any technology known in the art for example immunoassay measure. The antigenicity antigenic determinant need not be immunogenic.

In this manual, the term of use " T cellular antigens determinant " refers to the EphA2 polypeptide, preferably refers at least a portion of the EphA2 polypeptide of SEQ ID NO:2, and this part is identified by φt cell receptor. Term " T cellular antigens determinant " comprises helper cell (Th) antigenic determinant and cytotoxic T cell (Tc) antigenic determinant. Term " helper cell antigenic determinant " comprises Th1 and Th2 antigenic determinant.

In this manual, the term about the EphA2 polypeptide " fragment " that uses comprises EphA2 antigenic peptide or polypeptide, and it comprises the amino acid sequence of at least 5 adjacent amino acid residues of the amino acid sequence of EphA2 polypeptide, at least 10 adjacent amino acid residues, at least 15 adjacent amino acid residues, at least 20 adjacent amino acid residues, at least 25 adjacent amino acid residues, at least 40 adjacent amino acid residues, at least 50 adjacent amino acid residues, at least 60 adjacent amino acid residues, at least 70 adjacent amino acid residues, at least 80 adjacent amino acid residues, at least 90 adjacent amino acid residues, at least 100 adjacent amino acid residues, at least 125 adjacent amino acid residues, at least 150 adjacent amino acid residues, at least 175 adjacent amino acid residues, at least 200 adjacent amino acid residues or at least 250 adjacent amino acid residues.

In this manual, the term of use " fusion " refers to comprise polypeptide or the protein of the amino acid sequence of the amino acid sequence of the first polypeptide or albumen or its fragment, analog or derivative and heterologous polypeptide or albumen. In one embodiment, fusion comprises prevention or the therapeutic agent that merges with heterologous protein, polypeptide or peptide. According to this embodiment, heterologous protein, polypeptide or peptide can dissimilar prevention or the therapeutic agent of yes or no. For example, the two kinds of different albumen with immunoregulatory activity, polypeptide or peptides can merge the formation fusion. In preferred embodiments, with respect to the original polypeptide before merging with heterologous protein, polypeptide or peptide or the activity of albumen, fusion keeps active or has the activity of raising.

In this manual, the term " allos " about nucleotide sequence (for example gene) or amino acid sequence (for example peptide, polypeptide or protein) of use refers to not find and the second nucleotide sequence or the second amino acid sequence nucleotide sequence or the amino acid sequence of different plant species (for example derived from) related nucleotide sequence or amino acid sequence at occurring in nature.

In this manual, the term " excessive proliferative cell illness " that uses, " excessive proliferative cell disease ", " excessive proliferative disorders " and " excessive proliferative diseases " and similar term refer to such illness, and the excessive propagation of cell wherein or any type of excessive cell accumulation cause or cause pathological state and the symptom of this illness. In certain embodiments, the epithelial cell that is characterized as excessive propagation of excessive proliferative cell illness. In other embodiments, the endothelial cell that is characterized as excessive propagation of excessive proliferative cell illness. In other embodiments, the fibroblast that is characterized as excessive propagation of excessive proliferative cell illness. In certain embodiments, excessive proliferative cell illness is not tumorous. The example of the excessive proliferative cell illness of Non-cancerous is asthma, chronic pulmonary obstruction disease, fibrillatable (for example lung, liver and kidney fibrosis), bronchus overreaction, psoriasis and seborrhea. In preferred embodiments, the feature of excessive proliferative cell illness is the excessive proliferative cell of expressing (preferably overexpression) EphA2.

In this manual, the term that uses " with the combination of EphA2 immunologic opsonin ground " and similar terms refer to be combined with EphA2 acceptor or its one or more fragments specific, and peptide, polypeptide, albumen, fusion and antibody or its fragment of not being combined with other acceptor or its fragments specific. Term " with the combination of EphrinA1 immunologic opsonin ground " and similar terms refer to be combined with EphrinA1 or its one or more fragments specific, and peptide, polypeptide, albumen, fusion and antibody or its fragment of not being combined with other part or its fragments specific. May be combined with lower affinity with other peptide, polypeptide or albumen with peptide, polypeptide, albumen or antibody that EphA2 or EphrinA1 or their fragment immunologic opsonin are combined, this can pass through, and for example immunoassay or other detection method known in the art are measured. With the antibody of EphA2 or the combination of EphrinA1 immunologic opsonin or fragment may with relevant antigenic cross-reaction. Preferably, antibody or its fragment of being combined with EphA2 or EphrinA1 immunologic opsonin can be passed through, and for example immunoassay or other technology well known by persons skilled in the art are differentiated. When the combination of antibody or its fragment and EphA2 or EphrinA1 when having higher affinity than the combination with any cross-reactive antigen, this antibody or its fragment and EphA2 or EphrinA1 specific binding, this can measure by using laboratory technique such as radiommunoassay (RIAs) and Enzyme Linked Immunoadsorbent Assay (ELISA). Compile 1989, Fundamental Immunology, the 2nd edition, Raven publishing house, New York, 332-336 page or leaf about the specific discussion of antibody referring to for example Paul. The antibody of being combined with EphA2 or EphrinA1 immunologic opsonin in preferred embodiments, is not combined with other antigen or cross reaction is occured. The antibody of being combined with EphA2 or EphrinA1 fusion in another embodiment, the specifically EphA2 in fusion or EphrinA1 part is combined.

Antibody of the present invention includes but not limited to antibody (comprising bispecific antibody), people's antibody, humanized antibody, chimeric antibody, interior antibody (intrabodies), scFv s (scFv) (such as comprising that monospecific is connected with bispecific), Fab fragment, F (the ab ') fragment of antibody, the polyspecific of synthetic antibody, monoclonal antibody, restructuring preparation, the antigenic determinant binding fragment of Fvs (sdFv), antiidiotype (anti-Id) antibody and above any antibody that disulfide bond connects. Particularly, antibody of the present invention comprises the immunocompetence part of immunoglobulin molecules and immunoglobulin molecules, namely comprises the molecule with the antigen binding site of EphA2 antigen or EphrinA1 antigen immune specific binding (for example anti-EphA2 antibody or anti-EphrinA1 antibody one or more determine complementary district (CDR)). Antibody of the present invention can be any type (for example IgG, IgE, IgM, IgD, IgA and IgY), classification (IgG for example₁、IgG ₂、IgG ₃、IgG ₄、IgA ₁And IgA₂) or the immunoglobulin molecules of subclass.

In this manual, use about organic or inorganic molecule (no matter it is little molecule or large molecule) and the term " separation " of nonprotein sample reagent or nucleic acid refers to be substantially free of the organic or inorganic molecule of different organic or inorganic molecules. Preferably, 60% of the organic or inorganic molecule, 65%, 70 %, 75%, 80%, 85%, 90%, 95% or 99% do not contain the different organic or inorganic molecule of the second. In preferred embodiments, organic and/or inorganic molecule separates.

In this manual, the term " separation " about protein sample reagent (for example peptide, polypeptide, fusion or antibody) that uses is that finger protein sample reagent is substantially devoid of from the derived cell of this reagent or cell material or the contaminating protein of tissue source, or is substantially free of precursor or other chemicals when chemical synthesis. Language " is substantially devoid of cell material " and comprises the preparation of protein sample reagent, and wherein this protein sample reagent separates with the cell component that separation or reorganization is prepared the cell of this reagent. Therefore, the protein sample reagent that is substantially devoid of cell material comprises having the preparation that (dry weight) is less than the protein sample reagent of about heterologous protein of 30%, 20%, 10% or 5%, polypeptide, peptide or antibody (also being known as " contaminating protein "). When this protein sample reagent of restructuring preparation, it also preferably is substantially devoid of culture medium, and the culture medium that namely exists is less than about 20%, 10% or 5% of protein sample reagent formulation volume. When chemical synthesis prepared this protein sample reagent, it preferably was substantially devoid of precursor or other chemicals, i.e. this protein sample reagent precursor or other chemical separation synthetic with participating in this protein sample reagent. Therefore, the preparation of this protein sample reagent has (dry weight) less than precursor or the compound of about 30%, 20%, 10%, 5 % except interested protein sample reagent. In specific embodiments, protein sample reagent disclosed herein separates.

In this manual, the term about nucleic acid molecules of use " separation " refers to separate the nucleic acid molecules from being present in other nucleic acid molecules of natural origin. In addition, " separation " nucleic acid molecules for example cDNA molecule preferably is substantially devoid of other cell material, or does not contain the culture medium when preparing with recombinant technique, or precursor or other chemicals when being substantially free of useful chemical synthesis. In specific embodiments, nucleic acid molecules separates.

In this manual, the term of use " disease " and " illness " are used alternatingly to represent a kind of state.

In this manual, the term of use " combination " refers to use multiple treatment (for example preventing and/or treating property reagent). The use of term " combination " do not limit to suffer from excessive proliferative cell illness particularly the individuality of cancer treat the order of (for example preventing and/or treating property reagent). the first treatment (for example preventing and/or treating property reagent) can carried out the second treatment (for example preventing and/or treating property reagent) (for example 1 minute before, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 the week before), simultaneously, or (for example 1 minute afterwards, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 the week after) give to suffer from, just suffering from or doubtfully suffering from the particularly individuality of cancer of excessive proliferative cell illness. Successively and within the time interval, give individual this treatment (for example preventing and/or treating property reagent), thereby reagent of the present invention can be played a role with other reagent, to provide than the more benefit of other administering mode. Any other treatment (for example preventing and/or treating property reagent) can give with any order with any other treatment (for example preventing and/or treating property reagent).

In this manual, the phrase that uses " low tolerance " refers to a state, the patient bears the side effect that treatment brings under this state, therefore because ill effect and/or from the injury of side effect surpassed treatment benefit and so that the patient can not from treatment, benefit and/or can not proceed treatment.

In this manual, the term of use " control " refers to that individuality obtains useful effect from the treatment (for example preventing and/or treating property reagent) that gives, but this treatment can not be cured this disease. In certain embodiments, individuality is carried out one or more treatments (for example preventing and/or treating property reagent) prevent disease thus with " control " disease carrying out or deterioration.

In this manual, the term " tumour " that uses refers to relate to the disease of cell, these cells have the potentiality that are transferred to the far-end site and show the phenotypic characteristic that is different from non-tumor cell, for example, three-dimensional substrate for example soft agar form group or at basilar memebrane or extracellular matrix formulation example such as the MATRIGEL of three-dimensional^TMMiddle formation tubulose network or network sample matrix. Non-tumor cell does not form group and form obvious ball spline structure in three-dimensional substrates film or extracellular matrix preparation in soft agar. Although have different mechanism, tumour cell obtains distinctive one group of functional capabilities in its evolution. These abilities comprise hide that apoptosis, growth signals are self-sustaining, insensitive for anti-growth signals, tissue invasion/shift, infinite copy potential and the blood vessel that continues occur. Therefore, " non-tumour " refers to not relate to symptom, disease or the illness of cancer cell.

In this manual, the phrase that uses " do not reply/be difficult to treat " for describing the patient who carried out treatment with one or more present adoptable treatments (for example treatment of cancer), present adoptable treatment such as chemotherapy, radiotherapy, operation, hormone therapy and/or biology treatment/immunization therapy, particularly for the standard care scheme of particular cancers, wherein this treatment is not enough to clinically treat the patient and makes the extra effective treatment of these needs of patients, and is namely insensitive to treating. This phrase also can be described treatment is produced the patient who replys but suffer side effect, recurrence, generation resistance etc. In various embodiments, " do not reply/be difficult to treat " refers to that at least some signal portions of cancer cell are not killed or their cell division is not stopped. Can be by any method known in the art, in vivo or the vitro detection treatment to the validity of cancer cell, whether determine cancer cell " do not reply/be difficult to treat ", in this process, use the implication of " being difficult to treat " that this area accepts. In various embodiments, cancer is " do not reply/be difficult to and treat ", and wherein the number of cancer cell is not significant during treating reduces or increase.

In this manual, the term about the EphA2 overexpression " overexpression " that uses refers to encode the gene of EphA2 with the horizontal expression more than the expression of normal cell, and this can measure by or well known by persons skilled in the art detection method (be immunoassay such as ELISA or Western blotting, Northern blotting or RT-PCR) illustrated at this.

In this manual, the term of use " reinforcement " but refer under common perhaps dosage to improve the effect for the treatment of.

In this manual, the term of use " prevention " refers to treat (for example prevention or therapeutic agent) or therapeutic combination is shown effect, recurred or spread to prevent disease in individual body.

In this manual, the term " preventative reagent " that uses refers to can be used for to prevent the illness relevant with the EphA2 overexpression, with abnormal vascular any reagent of outbreak, recurrence or the diffusion of relevant illness and/or excessive proliferative cell disease, particularly cancer occurs. In certain embodiments, term " preventative reagent " refers to of the present invention based on listerial EphA2 vaccine. At some in other the embodiment, term " preventative reagent " refers to except based on the treatment the listerial EphA2 vaccine, for example cancer chemotherapy, radiotherapy, hormone therapy, biology treatment (for example immunization therapy). In other embodiments, multiple capable of being combined the giving of preventative reagent.

In this manual, " the prevention effective dose " used refers to be enough to prevent the amount for the treatment of (for example preventative reagent) of outbreak, recurrence or the diffusion of illness (for example with abnormal vascular relevant illness and excessive proliferative cell disease, preferably cancer occuring). The prevention effective dose can refer to be enough to prevent the illness in the individual body (for example with abnormal vascular relevant illness and excessive proliferative cell disease to occur, the amount of the treatment (for example preventative reagent) of cancer preferably) outbreak, recurrence or diffusion, individuality includes but not limited to be easy to suffer from the individuality of excessive proliferative cell disease, for example those hereditary individualities of easy cancer strickens or being exposed to before this carcinogen. The prevention effective dose can also refer to provide the amount of the treatment (for example preventative reagent) of prevention benefit in the prevention of illness (for example with abnormal vascular relevant illness and excessive proliferative cell disease occuring). Further, refer to the amount of independent treatment (for example preventative reagent) about the prevention effective dose for the treatment of (for example preventative reagent), or with the amount of other treatment (for example reagent) combination, this amount provides the prevention benefit in the prevention of illness (for example illness and the excessive proliferative cell disease relevant with the abnormal vascular generation). When being combined with the amount of EphA2 vaccine of the present invention, this term can comprise the raising whole prevention or strengthen the preventive effect of another treatment (for example preventative reagent) or with another treatment (for example preventative reagent) synergistic amount occurs.

In this manual, " scheme " of use comprises administration time and dosage regimen.

In this manual, the phrase of use " side effect " comprises the unwanted or bad effect of prevention or therapeutic agent. Ill effect is always unwanted, but unwanted effect must not be bad. The ill effect for the treatment of (for example prevention or therapeutic agent) can be that be harmful to or discomfort or risky. The side effect of chemotherapy includes but not limited to gastrointestinal toxicity, such as but not limited to early stage and diarrhoea and the flatulence that occurs late period, feel sick, vomiting, apocleisis, white blood cell minimizing, anaemia, neutrophils minimizing, weak, eilema, fever, pain, lose weight, dehydration, alopecia, expiratory dyspnea, insomnia, dizzy, catarrh, xerostomia, kidney failure and constipation, N﹠M effect, short-term or permanent kidney and trauma of bladder, parainfluenza symptom, body fluid delay and short-term or forever sterile. The side effect of radiotherapy includes but not limited to the forfeiture of fatigue, dry and appetite. The side effect of biology treatment/immunization therapy includes but not limited to dermatitis or the swelling of administration site, and the parainfluenza symptom is such as fever, cold and fatigue, alimentary canal problem and allergic reaction. The side effect of hormone therapy includes but not limited to feel sick, the fluctuation of forfeiture, eyes problem, headache and the body weight of fertility Issue, depression, appetite. Other unwanted effect that the patient can stand usually has a lot and is known in the art. In Physicians ' Desk Reference (the 56th edition, 2002, the 57 editions, 2003 and the 58th editions, 2004), many explanations are arranged.

In this manual, the term of use " individuality " and " patient " commutative use. In this manual, the individuality of use is mammal preferably, such as non-human primate (such as ox, pig, horse, cat, dog, mouse etc.) and primate (for example monkey and people), most preferably is the people. In specific embodiment, this individuality is the non-human animal. In another embodiment, this individuality is domestic animal (for example horse, pig, sheep or ox) or pet (for example dog, cat, rabbit or bird). In another embodiment, this individuality is the animal except laboratory animal or animal model (for example mouse, rat, cavy or monkey). In preferred embodiments, this individuality is the people. In another preferred embodiment, this individuality is the not impaired or immune not repressed people of immunity. In another preferred embodiment, this individuality is that the average absolute LC is about 500 cells/mm³, about 600 cells/mm³, about 650 cells/mm³, about 700 cells/mm³, about 750 cells/mm³, about 800 cells/mm³, about 850 cells/mm³, about 900 cells/mm³, about 950 cells/mm³, about 1000 cells/mm³, about 1050 cells/mm³, about 1100 cells/mm³Or about 1150 cells/mm³Or about 1200 cells/mm³The people.

In this manual, the term " treatment " that uses refers to give one or more treatments (for example therapeutic agent) to eradicate, to alleviate or improve illness or its symptom, eradicates especially, removes, improves or control primary, regionality or metastatic carcinoma tissue. In certain embodiments, this term is to point to the individuality of suffering from this disease to give one or more treatments (for example therapeutic agent) so that the diffusion of cancer minimizes or delays.

In this manual, the term of use " therapeutic agent " refers to can be used for preventing, treating or control any reagent of disease (for example illness relevant with the EphA2 overexpression and/or excessive proliferative cell disease, particularly cancer). In certain embodiments, term " therapeutic agent " refers to of the present invention based on listerial EphA2 vaccine. In some other embodiment, term " therapeutic agent " refers to except based on the treatment the listerial EphA2 vaccine, for example cancer chemotherapy, radiotherapy, hormone therapy, biology treatment (for example immunization therapy). In other embodiments, can be used in combination multiple treatment (for example therapeutic agent).

In this manual, " the treatment effective dose " used refers to be enough to the amount of the treatment (for example therapeutic agent) for the treatment of or control illness (for example the illness relevant with the EphA2 overexpression, relevant illness and/or excessive proliferative cell disease occur with abnormal vascular), and preferably, be enough to destroy, change, control or remove the amount of primary, regionality or metastatic carcinoma tissue. The treatment effective dose can refer to be enough to make the outbreak of illness (being excessive proliferative cell disease) to delay or the amount of minimized treatment (for example therapeutic agent), the diffusion of cancer is delayed or minimizes. The treatment effective dose can also refer to provide the amount of the treatment (for example therapeutic agent) for the treatment of benefit in the treatment of illness (for example cancer) or control. Further, refer to the amount of independent treatment (for example therapeutic agent) about the treatment effective dose for the treatment of (for example therapeutic agent), or with the amount of other treatment (for example reagent) combination, this amount provides the treatment benefit in the treatment of illness (for example excessive proliferative cell disease, such as cancer) or control. When being combined with the amount of EphA2 vaccine of the present invention, this term can comprise and improves wholistic therapy, reduces or avoid unwanted effect or strengthen the result for the treatment of of another treatment (for example therapeutic agent) or with another treatment (for example therapeutic agent) synergistic amount occurs.

In this manual, the term of use " treatment " refers to can be used for any step, scheme and/or the reagent of prevention, treatment or control illness (for example excessive proliferative cell illness, illness and/or the Non-cancerous excessive proliferative cell illness relevant with the abnormal vascular generation) or its symptom. In certain embodiments, term " treatment " refers to those skilled in the art, such as healthcare givers known the biology treatment that is used for the treatment of, controls, prevents or improve illness (for example excessive proliferative cell illness of excessive proliferative cell illness and/or Non-cancerous) or its one or more symptoms, supportive treatment and/or other treatment.

In this manual, the combination for the treatment of (for example preventing or therapeutic agent) " occurs " to refer to synergistic in the term that uses, and this combination has the effect with better effects if of adding than any two or more single treatments (for example one or more preventions or therapeutic agent). The synergy of therapeutic combination (for example combination of prevention or therapeutic agent) allows one or more treatments (for example one or more treatments or preventative reagent) to use with lower dosage, and/or gives described treatment to the individuality of suffering from Non-cancerous excessive propagation epithelium illness and/or endothelial cell illness with lower frequency. Use the more treatment of low dosage (for example prevention or therapeutic agent) and/or give described treatment with lower frequency and reduced and give described treatment relevant toxicity to individuality, and can not reduce the effect of described Prevention or treatment illness (for example excessive increment cell illness). In addition, synergy can improve the effect of described treatment (for example prevention or therapeutic agent) prevention or treatment illness (for example with abnormal vascular relevant illness and excessive proliferative cell illness occuring). Bad or the unwanted side effect that synergy can be avoided or reduction is relevant with the use of any single treatment of at last, treatment (for example prevention or therapeutic agent) combination.

In this manual, the term of use " T cell malignancies " refers to the excessive proliferative disorders of any T cell lymph, comprises thymus gland and rear thymus gland (post-thymic) malignant tumour. The T cell malignancies comprises the tumour of T cell source. The T cell malignancies refers to the tumour in lymph CFU-GM, thymocyte, T cell, NK cell or antigen presenting cell source. The T cell malignancies includes but not limited to leukaemia, include but not limited to lymphoblast leukaemia, thymoma, acute lymphoblast leukaemia and lymthoma, comprise hodgkin's and Non-Hodgkin lymphoma, prerequisite is that the T cell malignancies is not the T cell malignancies of skin, is not skin sexual cell lymthoma especially. In preferred embodiments, the T cell malignancies is systematic, non-skin T cell malignancies.

3.2. sequence

Below be the general introduction of the sequence listed in the appended sequence table, this sequence table is intactly drawn at this and is reference:

SEQ ID NO：1

People EphA2 cDNA (total length)

SEQ ID NO：2

People EphA2 polypeptide (total length)

SEQ ID NO：3-18

People EphA2 peptide

SEQ ID NO：19

Construct: LLOss-PEST-hEphA2

The PEST of natural LLO signal peptide+merge with total length people EphA2

Without codon optimized

Without antigen determinant label (myc or the FLAG that for example are used for this construct)

Show fusion coding subsequence

SEQ ID NO：20

Construct: LLOss-PEST-hEphA2

The PEST of natural LLO signal peptide+merge with total length people EphA2

Without codon optimized

Without antigen determinant label (myc or the FLAG that for example are used for this construct)

The fusion that shows prediction

SEQ ID NO：21

EphA2 EX2 domain

The natural nucleus glycoside acid sequence

SEQ ID NO：22

EphA2 EX2 domain

The nucleotide sequence of the optimization codon that uses in the Listeria

SEQ ID NO：23

EphA2 EX2 domain

Primary amino acid sequence

SEQ ID NO：24

Construct: LLOss-PEST-EX2_hEphA2

The PEST of natural LLO signal peptide+merge with the external structure territory of people EphA2

Without codon optimized

Without antigen determinant label (myc or the FLAG that for example are used for this construct)

SEQ ID NO：25

Construct: LLOss-PEST-EX2_hEphA2

The PEST of natural LLO signal peptide+merge with the external structure territory of people EphA2

Without codon optimized

Without antigen determinant label (myc or the FLAG that for example are used for this construct)

The fusion that shows prediction

SEQ ID NO：26

Natural LLOss-PEST-FLAG-EX2_EphA2-myc-is codon optimized

(natural listeria monocytogenes LLO signal peptide+PEST-codon optimized-FLAG-EX-2 EphA2-Myc)

Nucleotide sequence (comprising the hly promoter)

SEQ ID NO：27

Natural LLOss-PEST-FLAG-EX2_EphA2-myc-is codon optimized

(natural listeria monocytogenes LLO signal peptide+PEST-codon optimized-FLAG-EX-2 EphA2-Myc)

Primary amino acid sequence

SEQ ID NO：28

Codon optimized LLOss-PEST-FLAG-EX2_EphA2-myc-is codon optimized

(codon optimized listeria monocytogenes LLO signal peptide+PEST-codon optimized-FLAG-EX-2 EphA2-Myc)

Nucleotide sequence (comprising the hly promoter)

SEQ ID NO：29

Codon optimized LLOss-PEST-FLAG-EX2_EphA2-myc-is codon optimized

(codon optimized listeria monocytogenes LLO signal peptide+PEST-codon optimized-FLAG-EX-2 EphA2-Myc)

Primary amino acid sequence

SEQ ID NO：30

PhoD-FLAG-EX2_EphA2-myc-is codon optimized

(codon optimized bacillus subtilis PhoD Tat signal peptide-FLAG-EX-2 EphA2-Myc)

Nucleotide sequence (comprising the hly promoter)

SEQ ID NO：31

PhoD-FLAG-EX2_EphA2-myc-is codon optimized

(codon optimized bacillus subtilis phoD Tat signal peptide-FLAG-EX-2 EphA2-Myc)

Amino acid sequence

SEQ ID NO：32

EphA2 CO domain

The natural nucleus glycoside acid sequence

SEQ ID NO：33

EphA2 CO domain

The nucleotide sequence of the optimization codon that uses in the Listeria

SEQ ID NO：34

EphA2 CO domain

Primary amino acid sequence

SEQ ID NO：35

Construct: LLOss-PEST-CO-huEphA2

The PEST of natural LLO signal peptide+merge with the cytoplasmic structure territory of people EphA2

Without codon optimized

Without antigen determinant label (myc or the FLAG that for example are used for this construct)

Show the fusion coded sequence

SEQ ID NO：36

Construct: LLOss-PEST-CO-huEphA2

The PEST of natural LLO signal peptide+merge with the cytoplasmic structure territory of people EphA2

Without codon optimized

Without antigen determinant label (myc or the FLAG that for example are used for this construct)

The fusion that shows prediction

SEQ ID NO：37

Natural LLOss-PEST-FLAG-CO_EphA2-myc-is codon optimized

(natural listeria monocytogenes LLO signal peptide+PEST-is codon optimized-FLAG-CO_EphA2-Myc)

Nucleotide sequence (comprising the hly promoter)

SEQ ID NO：38

Natural LLOss-PEST-FLAG-CO_EphA2-myc-is codon optimized

(natural listeria monocytogenes LLO signal peptide+PEST-is codon optimized-FLAG-CO_EphA2-Myc)

Primary amino acid sequence

SEQ ID NO：39

Codon optimized LLOss-PEST-FLAG-CO_EphA2-myc-is codon optimized

(codon optimized listeria monocytogenes LLO signal peptide+PEST-is codon optimized-FLAG-CO_EphA2-Myc)

Nucleotide sequence (comprising the hly promoter)

SEQ ID NO：40

Codon optimized LLOss-PEST-FLAG-CO_EphA2-myc-is codon optimized

(codon optimized listeria monocytogenes LLO signal peptide+PEST-is codon optimized-FLAG-CO_EphA2-Myc)

Primary amino acid sequence

SEQ ID NO：41

PhoD-FLAG-CO_EphA2-myc-is codon optimized

(codon optimized bacillus subtilis PhoD Tat signal peptide-FLAG-CO_EphA2-Myc)

Nucleotide sequence (comprising the hly promoter)

SEQ ID NO：42

PhoD-FLAG-CO_EphA2-myc-is codon optimized

(codon optimized bacillus subtilis PhoD Tat signal peptide-FLAG-CO_EphA2-Myc)

Amino acid sequence

SEQ ID NO：43

Construct: pAM401-MCS

Comprise the plasmid pAM401 from the MCS (MCS) of pPL2 carrier

Little Aat II MCS fragment from pPL2 is inserted between the flat terminal XbaI and NruI site of pAM401

Show complete pAM401-MCS plasmid sequence.

4, brief description of drawings

Fig. 1. life cycle in Liszt's mycetocyte, antigen presenting cell activation and antigen presentation;

Fig. 2. the Western engram analysis of the listerial secretory protein of restructuring, this bacterium natural EphA2 CO domain sequence of encoding;

Fig. 3. the Western engram analysis of the listerial secretory protein of restructuring, this bacterium coding, natural or codon optimized LLO secA1 signal peptide fusion with codon optimized EphA2 EX2 domain sequence signal peptide;

Fig. 4. the Western engram analysis of the listerial secretory protein of restructuring, this bacterium coding and codon optimized EphA2 CO domain sequence LLO secA2 signal peptide that merge, natural or codon optimized or codon optimized Tat signal peptide;

The Flow Cytometry Analysis that people EphA2 in Fig. 5 .CT2 mouse cancer cell expresses. Adopt standard technique to carry out unicellular FACS classification analysis with the CT26 cell clone of identification high level expression people EphA2;

Fig. 6. the Western engram analysis that compiles the group of the CT26 mouse colonic cell of high level expression people EphA2 albumen;

Fig. 7. express the flow cytometry of the B16F10 cell of huEphA2;

Fig. 8. with behind pCDNA4 plasmid DNA transfection 293 cells of coding total length natural EphA2 sequence 48 hours, the Western engram analysis of the cell pyrolysis liquid of this 293 cell;

Fig. 9 A-9B. carries out therapeutic immunization with the Listeria positive control of expressing AH1-A5 in the CT26 tumor model;

Figure 10 A-10B. is with the Listeria of the ECD that the expresses hEphA2 CT26-hEphA2 tumor growth (Figure 10 A) that carried out preventative immunosupress and improved survival rate (Figure 10 B);

Figure 11 A-11D. contrasts the intravenous administration of (L4029) in L4029EphA2-exFlag, Listeria or contains Listeria positive control (L4029-AH1) (the 100 μ l volumes 5 * 10 of AH1 albumen⁵Individual cell) the preventative research behind the subcutaneous or intravenous administration. Figure 11 A has shown the gross tumor volume of the mouse that has inoculated CT26 cell, carrier (HBSS), Listeria (L4029) or Listeria positive (L4029-AH1) contrast of expressing the ECD of huEphA2. Figure 11 B has shown that the CT26 cell (L4029-EphA2 exFlag) of the ECD that has inoculated expression huEphA2 is with respect to the mean tumour volume of the mouse that has inoculated Listeria (L4029) contrast. Figure 11 C has shown the preventative result of study in subcutaneous model, has measured the survival percentage of the mouse behind the CT26 tumor cell inoculation. Figure 11 D has shown the preventative result of study in the lung metastasis model, has measured the survival percentage of the mouse behind tumor cell inoculation;

Figure 12. carry out preventative immunity with the Listeria of the ECD that expresses hEphA2 and improved the survival rate of suffering from after the RenCa-hEphA2 tumour;

Figure 13 A-13C. Figure 13 A-13C has shown the typical treatment Journal of Sex Research result of the animal that has inoculated the CT26 mouse colonic cell, this cancer cell transfection the contrast of people EphA2 (L4029-EphA2 exFlag), Listeria (L4029-contrast) or carrier (HBSS). In Figure 13 A, after inoculation with some interval measurement gross tumor volumes. Figure 13 B has shown the mean tumour volume with the mouse of the CT26 cell inoculation of the ECD that contains Listeria contrast or huEphA2. Figure 13 C has presented the therapeutic studies result who uses the lung metastasis model, measures the survival percentage with the postvaccinal mouse of listerial CT26 cell of the ECD that contains the contrast of HBSS or Listeria or expression huEphA2;

Figure 14 A-F. Figure 14 A. carries out the growth that therapeutic immunization has suppressed the CT26-hEphA2 tumour of generation with the Listeria of the ICD that expresses hEphA2 to the Balb/C mouse. Figure 14 B. carries out for a long time survival of immunity acquisition with the recombinant listeria bacterium of coding EphA2 CO domain to the Balb/C mouse that suffers from CT26.24 (huEphA2+) lung neoplasm. Figure 14 C. suffers from CT26.24 (huEphA2+) lung neoplasm Balb/c mouse with the recombinant listeria bacterium immunity of coding OVA.AH1 or OVA.AH1-A5 and obtains long-time survival. Figure 14 D. has improved survival with the Balb/C mouse that the recombinant listeria bacterium immunity of coding password EphA2 CO domain sequence that optimize or natural suffers from CT26.24 (huEphA2+) lung neoplasm. The recombinant listeria bacterium immunity of the codon optimized secA1 signal peptide that Figure 14 E. merges with coding and codon optimized EphA2 EX2 domain sequence suffers from the Balb/C mouse of CT26.24 (huEphA2+) lung neoplasm and has improved survival. The DNA of the total length EphA2 that need not encode carries out for a long time survival of immunity acquisition to the Balb/C mouse that suffers from CT26.24 (huEphA2+) lung neoplasm to Figure 14 F. with the recombinant listeria bacterium of coding EphA2 CO domain;

Figure 15. the CT26-hEphA2 tumor growth of long term inhibition when again being on the hazard;

Figure 16. carry out immunity with the Listeria of expressing hEphA2 and caused specific CD8+T cell response;

Figure 17 .CD4+ and CD8+T cell response all are that best hEphA2 guided bone antitumor action is necessary;

Figure 18 A-B. carries out the permeability that the therapeutic inoculation has improved the CD45+ tumour with the Listeria of expressing people EphA2 ICD. Figure 18 A has illustrated the tumor biopsy figure with biotinylation rat anti-mouse CD45/B200 dyeing. Figure 18 B is the block diagram that view data is normalized to gross tumor volume.

5, detailed description of the present invention

The present invention partly is based on the inventor's following discovery: comprise listerial can the creating antagonism based on listerial vaccine that is built into expression EphA2 antigenic peptide and express useful treatment and the preventive effect of the relevant excessive proliferative diseases of cell with EphA2.

The invention provides prevention, treatment, inhibition and the control illness relevant with the EphA2 overexpression, with abnormal vascular relevant illness and/or the method and composition of excessive proliferative cell illness occur. Concrete aspect of the present invention relates to the method and composition of inclusion compound, when to suffering from when expressing the individual administration of the relevant excessive proliferative cell illness of cell with EphA2, this compound can cause or mediate the immune response of antagonism EphA2, thereby the growth that makes this EphA2 relevant with excessive proliferative cell illness express cell is suppressed. The invention further relates to method and composition, particularly human breast carcinoma, oophoroma, cancer of the esophagus, lung cancer, cutaneum carcinoma, prostate cancer, carcinoma of urinary bladder and cancer of pancreas and clear-cell carcinoma and the melanoma for the treatment of, inhibition or the source metastasis of cancer of control epithelial cell. The invention further relates to method and composition, particularly leukaemia and the lymthoma of prevention, treatment, inhibition or control T cell source cancer. In addition, the compositions and methods of the invention comprise the active component with other type based on the combination of listerial EphA2 vaccine of the present invention. In certain embodiments, composition of the present invention is used to treatment, prevents or controls other nontumorous excessive proliferative cell illness, such as but not limited to asthma, psoriasis, ISR, COPD etc.

The present invention also relates to treat, suppress and control the method that is difficult to partially or completely the cancer for the treatment of and other excessive proliferative cell illness with treatment present or standard (for example treatment of cancer, for example chemotherapy, radiotherapy, hormone therapy and biology/immunization therapy).

5.1. based on listerial vaccine

The invention provides and be built into the Listeria of expressing the EphA2 antigenic peptide and this Listeria in control, treatment or the prevention disease relevant with the EphA2 overexpression and/or the purposes in the excessive proliferative cell illness.

Can comprise the Listeria bacterial strain that one or more express the EphA2 antigenic peptide based on listerial EphA2 vaccine. In other embodiments, can comprise the Listeria bacterial strain that is built into one or more EphA2 antigenic peptides of expression based on listerial EphA2 vaccine.

In preferred embodiments, of the present inventionly comprise listeria monocytogenes based on listerial EphA2 vaccine and belong to.

5.1.1. attenuation

In order in the treatment of humans and animals, to use safely the Listeria, preferably, with the pathogenic perform toxic attenuation of this bacterium. Final result reduces to the patient to use this Listeria and cause the risk of toxic shock or other side effect. Can isolate by many technology the Listeria of this attenuation. These methods comprise the new bacterial strain of the antibiotics sensitivity bacterial strain that uses microorganism, the gene mutation of microorganism, the microbial mutation body of selecting the disappearance virulence factor and the reformed microorganism of structure cell membrane lipopolysaccharides.

In certain embodiments, can suddenly change to delete or destroy by for example homologous recombination technique and chemical mutation or transposon the dna sequence dna of coding virulence factor, with with attenuated listeria bacterium, these virulence factors are guaranteed the Listeria at host cell, particularly survive in macrophage and neutrophil cell. Have a lot in these virulence factors of studying, but not all relevant with the survival in macrophage, this survival makes these factors at pressure, acidization for example, effect under in macrophage, express specifically, or be used for the host cell of inducing specific and reply, for example giant cell drink effect, Fields etc., 1986, Proc.Natl.Acad.Sci.USA 83:5189-5193. The embodiment of virulent gene includes but not limited to hly, plcA, plcB, mpl, actA, inlA and inlB, also sees Autret etc., 2001, Infection and Immunity 69:2054-2065.

In specific embodiments, listerial taxis (for example inlB mutant) and/or they ability (for example actA mutant) from cell to cellular invasion of organizing is weakened. In another embodiment, the Listeria comprises the sudden change (for example delete, add or replace) of one or more internalization element (for example inlA and/or inlB). In another embodiment, the Listeria comprises sudden change (for example delete, add or replace) in actA.

As the method for guaranteeing the attenuation phenotype and avoiding taking a turn for the worse to non-attenuation phenotype, can make up the Listeria and make its in many ways attenuation, for example the sudden change (for example inlB mutant) of taxis and the sudden change (for example actA mutant) of the ability of impact from cell to cellular invasion are organized in impact. In preferred embodiments, this Listeria comprises the sudden change among sudden change among the internalization element B (deletion, add or replace) and the actA.

5.1.2. expression system

The EphA2 antigenic peptide preferably uses the allogeneic gene expression frame table to reach in the Listeria. The allogeneic gene expression frame typically is comprised of the element of arranging in the following order: (1) prokaryotic promoter; (2) SD (Shine-Dalgarno) sequence; (3) secretion signal (signal peptide); (4) heterologous gene. Selectively, this allogeneic gene expression frame can also comprise transcription terminator, and its structure can stably be incorporated in the bacterial chromosome. Although optional, in the allogeneic gene expression frame, comprise transcription terminator and can prevent from reading over (read-through) and transcribe the polarization effect is regulated in the expression of adjacent gene as being arranged in last element.

Be incorporated into above-mentioned expression vector based on listerial EphA2 vaccine and be preferably designed so that and make the Listeria produce the EphA2 peptide, and alternatively, the second tumour antigen is by this Listeria secretion. Many bacterium secretion signals are well known in the art, can be used for the compositions and methods of the invention. The example of the secretion signal that can use with the Listeria is SecA, as described in 5.2.1.4 part hereinafter.

The promoter that drives the expression of EphA2 antigenic peptide can be the formation type, and wherein this peptide is by continuous expression; Induction type, wherein this peptide only just is expressed when inducing molecule exists; Or cell type-specific, wherein peptide or enzyme are only expressed in some cell type.

The preferred embodiment of the component of EphA2 antigenic peptide expression system below is provided, and this system will be combined with the nucleic acid of the coding EphA2 antigenic peptide described in 5.2 parts.

5.1.2.1. construct skeleton

Those of ordinary skill in the art will recognize, can obtain the various plasmid construction body skeletons that are suitable for assembling the allogeneic gene expression frame. Select concrete plasmid construction body skeleton according to the expression of the allogeneic gene expression frame of bacterial chromosome or extrachromosome episome is whether desirable.

As non-limitative example, use integration vector the allogeneic gene expression frame to be integrated in the bacterial chromosome of listeria monocytogenes (Listeria), this integration vector comprises the expression cassette of Liszt bacteriophage (listeriophage) integrase, is incorporated in the chromosome of Listeria this integrase catalytic carrier sequence-specific. For example, the integration vector that is known as pPL1 and pPL2 impels heterologous protein (for example EphA2 antigenic peptide) expression cassette at the Toxic Free Area of bacterial genomes stable single copy integration to occur, this has explanation (Lauer etc. in the literature, 2002, J.Bacteriol.184:4177-4178). Integration vector is stable as plasmid in Escherichia coli, and it is incorporated in the desirable Listeria background by joint. Each carrier lacks Listeria specificity replication origin and the bacteriophage integrase of encoding, thereby this carrier only is only stable after being integrated into chromosomal phage attachment site. From desirable plasmid construction body, the time in about 1 week of process need of the recombinant listeria bacterium bacterial strain of preparation expression ideal protein. PPL1 and pPL2 integration vector are based on respectively U153 and PSA Liszt bacteriophage. The pPL1 carrier is incorporated in the ORFs of comK gene, and pPL2 is incorporated in the tRNAArg gene by this way: in case the native sequences of this gene of successful integration just obtains recovery, thereby keep its complete natural expressive function. Contain the plasmid of heterologous protein (for example EphA2 antigenic peptide) expression cassette for the ease of structure, pPL1 and pPL2 integration vector contain the MCS sequence.

Selectively, can EphA2 antigenic peptide expression cassette be integrated in the chromosome of Listeria by allele exchange process well known by persons skilled in the art. In concrete combination, become at the gene integration of the antibiotic resistance albumen of not wishing to encode in the situation of a part of the construct that contains the allogeneic gene expression frame, the allele exchange process is desirable. For example, pKSV7 carrier (Camilli etc., 1993, Mol.Microbiol 8:143-157) contains responsive to temperature type Listeria Gram-positive replication origin, this replication origin is used for selecting recombinant clone under limit temperature, and this clone's expression pKSV7 plasmid is recombinated in the chromosome of Listeria. Comprise the plasmid of heterologous protein (for example EphA2 antigenic peptide) expression cassette and chloramphenicol resistance gene for the ease of structure, pKSV7 allele exchange plasmid vector comprises the MCS sequence. In order to be inserted in the listerial chromosome, the best flank of allos EphA2 antigenic peptide expression cassette construct connects the chromosomal DNA sequence of about 1kb, the accurate site that these sequences are integrated corresponding to ideal. PKSV7 heterologous protein (for example EphA2 antigenic peptide) expression cassette plasmid preferably imports in the desirable strain by electroporation according to the standard method of gram-positive bacteria electroporation. In brief, by under coated plate and the allowance temperature at 30 ℃ on the BHI agar medium that contains chloramphenicol (10 μ g/ml), cultivating the bacterium of selecting through pKSV7 heterologous protein (for example EphA2 antigenic peptide) expression cassette plasmid electroporation. In containing the culture medium of chloramphenicol, under 41 ℃ limit temperature, many generations go down to posterity the chromosomal single cross of selecting bacteria to change integration several single clones. At last, by not containing excision and the removal (double crossing over) of under the allowance temperature at 30 ℃ several single clones being gone down to posterity to finish plasmid on the BHI culture medium of chloramphenicol many generations. Pass through PCR, utilize the zone the bacterial chromosome target sequence that primer do not comprise the pKSV7 plamid vector construction body that increased in heterologous protein (for example EphA2 antigenic peptide) expression cassette, can verify heterologous protein (for example EphA2 antigenic peptide) integration of expression cassette in bacterial chromosome.

In other combination, expressing heterologous albumen from stable plasmid episome (for example EphA2 antigenic peptide) is desirable. Going down to posterity to keep the plasmid episome by many generations then needs co expression that the albumen of selective advantage is provided for the bacterium that contains this plasmid. As non-limitative example, albumen with heterologous protein (for example EphA2 antigenic peptide) combination coexpression from plasmid can be antibiotic resistance albumen, chloramphenicol for example, maybe can be bacterioprotein (expressing in the chromosome of wild-type bacterium), it also can give selective advantage. The non-limitative example of bacterioprotein comprises that purine or amino acid bio synthesize required enzyme (selecting) or give the needed transcription factor (Gunn etc. of gene expression of selective advantage in external or the body in the predetermined culture medium that lacks related amino acid or other necessary precursor macromolecule, 2001, J.Immuol.167:6471-6479). As non-limitative example, pAM401 is the suitable carrier (Wirth etc., 1986, J Bacteriol 165:831-836) that the episome of the heterologous protein (such as the EphA2 antigenic peptide) selected is expressed in different gram-positive bacterias.

5.1.2.2.SD (Shine-Dalgarno) sequence

3 ' end in promoter contains poly-purine SD sequence, and this sequence is that 30S ribosomes subunit (by 16S rRNA) is combined and the necessary element of translation initiation with heterologous gene rna transcription thing. The SD sequence typically has following identical sequence (SEQ ID NO:66): 5 '-NAGGAGGU-N5-10-AUG (initiation codon)-3 '. The variant that has poly-purine SD sequence. Known ground, the Listeria hly gene of coding Liszt bacteriolysin O (LLO) has following SD sequence (SEQ ID NO:67): AAGGAGAGTGAAACCCATG (the underscore place is the SD sequence, overstriking be translation initiation codon).

5.1.2.3. codon optimized

In certain embodiments, for the translation efficiency of the selected heterologous protein of optimization, use ideally the codon of Listeria preference. Being used for the preference codon of bacterial expression can be such as Nakamura etc., and the description among 2000, the Nucl.Acids Res.28:292 is determined. In certain embodiments, codon optimized expression EphA2 antigenic peptide is desirable from listeria monocytogenes.

Following table 3 has shown each amino acid whose optimization codon that listeria monocytogenes uses.

Amino acid	The single-letter coding	The Listeria codon of optimizing
			Alanine	A	GCA
Arginine	R	CGU
			Asparagine	N	AAU
Aspartic acid	D	GAU
			Cysteine	C	UGU
Glutamine	Q	CAA
			Glutamic acid	E	GAA
Glycine	G	GGU

Histidine	H	CAU
			Isoleucine	I	AUU
Leucine	L	UUA
			Lysine	K	AAA
Methionine	M	AUG
			Phenylalanine	F	UUU
Proline	P	CCA
			Serine	S	AGU
Threonine	T	ACA
			Tryptophan	W	UGG
Tyrosine	Y	UAU
			Valine	V	GUU

Table 3: Listeria codon preference: will be for the codon of Optimal Expression

5.1.2.4. signal peptide

Bacterium utilizes different PE paths, comprises secA1 and two Arg displacements (Tat), and they are positioned at the N end of front albumen. Most secretory protein utilizes the Sec path, and wherein albumen is shifted by the protein s ec hole that is embedded in the bacterial cell membrane with not folding conformation. On the contrary, utilize the protein of Tat path with folding conformation secretion.

Encode the corresponding signal peptide of these PE paths (include but not limited to describe in the signal peptide described in the 5.1.2.4. part and the 5.2.1. part signal and guiding peptide) nucleotide sequence can with desirable heterologous protein coded sequence with the frame gene fusion. Signal peptide is preferably in its carboxyl terminal and comprises signal peptidase, to incite somebody to action really desirable protein delivery (Sharkov and Cai, 2002, J.Biol. Chem.277:5796-5803 in born of the same parents' external environment; Nielsen etc., 1997, Protein Engineering 10:1-6). Can service routine such as SignalP 3.0 (Bendtsen etc., 2004, J.Mol.Biol.340:783-795) predicted signal peptide cracking site. Signal peptide not only can derived from different secretion paths, can also belong to derived from different bacteriums. Signal peptide has common structure and forms, and have the C stub area (C-structure territory) that charged N holds (N domain), hydrophobic core district (H domain) and has more polarity, but they does not have sequence conservation. The C-structure territory of signal peptide is with I type signal peptidase (SPase I) cracking site, with respect to the position 1 and 3 of cracking site with identical sequence A-X-A. The signal peptide that has average 28 residues by the albumen of sec path secretion. The signal peptide that links to each other with the albumen of secreting by the Tat path has the composition of three parts similar to the Sec signal peptide, but it is characterized in that having RR motif (R-R-X-#-#, wherein # is hydrophobic residue) at N domain/H domain intersection. Bacterium Tat signal peptide is on average grown 14 amino acid than sec signal peptide. Hay bacillus secretory protein (secretome) can comprise nearly 69 albumen of inferring that utilize Tat secretion path, and wherein 14 comprise SPase I cracking site (Jongbloed etc., 2002, J.Biol.Chem.277:44068-44078; Thalsma etc., 2000, Microbiol.Mol.Biol.Rev.64:515-547). Be the non-limitative example of signal peptide in the following table 4, these signal Toplink are used in the fusion combination of selected heterologous gene, and encoding proteins is secreted from bacterium.

The secretion path	Signal peptide amino acid sequence (NH2-CO 2)	The signal peptidase site (restriction enzyme site is expressed as ')	Gene	Kind/belong to	SEQ   ID   NO：
						secA1	MKKIMLVFITLIL   VSLPIAQQTEAK   D	TEA’KD(SEQ ID  NO：70)	hly(LLO)	Listeria monocytogenes	44
Tat	MTDKKSENQTE   KTETKENKGMT   RREMLKLSAVA   GTGIAVGATGLG   TILNVVDQVDKA   LT	DKA’LT(SEQ ID  NO：71)	lmo0367	Listeria monocytogenes	45
							MAYDSRFDEWV   QKLKEESFQNNT   FDRRKFIQGAGK   IAGLSLGLTIAQS   VGAFG	VAG’FG(SEQ ID  NO：72)	PhoD (alkaline phospholipase)	Bacillus subtilis	46

Table 4: be used for the bacterial expression of EphA2 and the burst of secretion

Various albumen by the secretion of Tat path in belonging to, various bacteriums are arranged. In certain embodiments, selection and the ideal sequence corresponding Tat signal peptide of these albumen and coding EphA2 antigenic peptide be with the frame gene fusion, with the Tat signal peptide of promotion functions connection-EphA2 albumen chimera by the secretion of Tat path. Non-limitative example from the albumen of bacillus subtilis and Listeria (listera innocua and listeria monocytogenes) below is provided, and these albumen be it is predicted and utilized the secretion of Tat path.

The bacillus subtilis mycoprotein that passes through the Tat secretion of inferring

＞gi/2635523/emb/CAB15017.1/ is to two (component inductor histidine kinase (YtsA) (bacillus subtilis) is similar

＞gi/2632548/emb/CAB12056.1/ phosphodiesterase/alkaline phospholipase D (bacillus subtilis)

＞gi/2632573/emb/CAB12081.1/ is similar to putative protein (bacillus subtilis)

＞gi/2633776/emb/CAB13278.1/ is similar to putative protein (bacillus subtilis)

＞gi/2634674/emb/CAB14172.1/menaquinol: cytochrome c oxidoreducing enzyme (iron (sulphur subunit) (bacillus subtilis)

＞gi/2635595/emb/CAB15089.1/yubF (bacillus subtilis)

＞gi/2636361/emb/CAB15852.1/ replaces the gene name: ipa (29d～similar to putative protein (bacillus subtilis)

The Listeria albumen that passes through the Tat secretion of inferring

The conservative putative protein (listera innocua) of＞gi/16799463/ref/NP_469731.1/ and bacillus subtilis YwbN protein similar

(((carrier protein synzyme (listera innocua) is similar for acyl group for the carbonyl acyl group for＞gi/16801368/ref/NP_471636.1/ and 3

Listeria monocytogenes EGD (e)

＞gi/16802412/ref/NP_463897.1/ with conservative putative protein (the listeria monocytogenes EGD (e) of bacillus subtilis YwbN protein similar

Microorganism utilizes the expression of the preference of codon being regulated concrete endogenous gene. Therefore, the signal peptide that is used for the selected heterologous protein of secretion may not comprise the codon that uses the preference codon, thereby protein is become with non-optimum hydration. In certain embodiments, the signal peptide sequence that merges with frame with the gene of the selected heterologous protein of coding is the selected employed optimization codon of bacterium. In certain embodiments, for expression and secretion from the listeria monocytogenes of restructuring, the nucleotide sequence of selected signal peptide is the above codon of table 4 optimized expression in listeria monocytogenes of basis.

5.1.2.5. transcription terminator

In certain embodiments, transcription terminator can insert in the heterologous protein expression frame, in the downstream of the C end of translation stop codon of heterologous protein. Can put into suitable sequential element well known by persons skilled in the art in the heterologous protein expression frame, this element promotes rho dependence or the dependent tanscription termination of non-rho.

5.2.EphA2 antigenic peptide

As mentioned above, the present invention relates to listerial application, this bacterium is built into expresses the EphA2 antigenic peptide. Be not subjected to the constraint of any mechanism, in a single day this Listeria awards the individuality of suffering from the disease relevant with the EphA2 overexpression, and it can cause the immune response of antagonism EphA2, thus the cell of the endogenous EphA2 that creates antagonism or HI.

In principle, the EphA2 antigenic peptide (sometimes being called " EphA2 antigenic polypeptide ") that is used for method and composition of the present invention can be any EphA2 antigenic peptide, and this peptide can cause the antagonism EphA2 relevant with excessive proliferative disorders and express the immune response of cell. Therefore, the EphA2 antigenic peptide can be the EphA2 polypeptide, the EphA2 polypeptide of SEQ ID NO:2 preferably, or the fragment of EphA2 polypeptide or derivative, this fragment or derivative (1) show the antigenicity (be combined with EphA2 or compete with the ability in conjunction with anti-EphA2 antibody) of EphA2, (2) show the immunogenicity (producing the ability of the antibody be combined with EphA2) of EphA2, and/or (3) comprise the T cellular antigens determinant of one or more EphA2.

In certain embodiments, the EphA2 antigenic peptide is by the following nucleotide sequence coded sequence that provides or its fragment or derivative:

Genbank registration number NM_004431 people

Genbank registration number NM_010139 mouse

Genbank registration number AB038986 chicken (part)

In certain embodiments, the EphA2 antigenic peptide is the people EphA2 (SEQ ID NO:2) of total length.

In other embodiments, the EphA2 antigenic peptide comprises born of the same parents' intracellular domain (residue 22 to 554 of SEQ ID NO:2) of EphA2.

In other embodiments, the EphA2 antigenic peptide comprises born of the same parents' intracellular domain (residue 558 to 976 of SEQ ID NO:2) of EphA2.

In certain embodiments, the EphA2 antigenic peptide comprise total length people EphA2 more than one domain. In specific embodiments, the EphA2 antigenic peptide comprises ectodomain and the born of the same parents' endoplasm domain that links together. According to this embodiment, the membrane spaning domain of EphA2 is deleted.

In certain embodiments of the invention, the tyrosine kinase activity of EphA2 is removed. Therefore, EphA2 can comprise deletion, interpolation or the replacement of amino acid residue, so that tyrosine kinase activity is removed. In preferred embodiments, 646 places exist by the replacement of lysine to methionine in the position.

In preferred embodiments, the membrane spaning domain of deletion EphA2, EphA2 antigenic peptide comprise the outer and cytoplasmic structure territory of the born of the same parents of EphA2 and 646 places have by the replacement of lysine to methionine in the position.

In certain embodiments, above-mentioned peptide is corresponding or comprise the EphA2 antigenic determinant, and this antigenic determinant exposes in cancer cell but conductively-closed in non-cancer cell. In preferred embodiments, the EphA2 antigenic peptide is preferably included on the cancer cell and the EphA2 antigenic determinant (" the EphA2 antigenic determinant peptide of exposure ") that optionally do not expose or increase on non-cancer cell.

The present invention further is included in the compositions and methods of the invention and uses a plurality of EphA2 antigenic peptides, and for example 2,3,4,5,6 or more EphA2 antigenic peptide.

The fragment that is used for the EphA2 of method and composition of the present invention can be included in deletion, interpolation or the replacement by the amino acid residue in the amino acid sequence of EphA2 gene code. Preferably, sudden change causes reticent the change, produces thus the EphA2 gene outcome of function equivalence. EphA2 gene outcome with " function equivalent " expression sudden change has the function identical with wild type EphA2 gene outcome, for example comprises one or more antigenic determinant of EphA2.

EphA2 antigenic peptide sequence preference ground comprises amino acid sequence, and this sequence and people EphA2 show about at least 65% sequence similarity, more preferably show about at least 70% sequence similarity and more preferably show about at least 75% sequence similarity with people EphA2 with people EphA2. In other embodiments, the EphA2 peptide sequence preferably comprises amino acid sequence, this sequence and people EphA2 show about at least 85% sequence similarity, more preferably show about at least 90 % sequence similarities with people EphA2, and most preferably show about at least 95% sequence similarity with people EphA2.

Other polypeptide that is fit to this method is the polypeptide by the nucleic acid coding of following 5.2 parts description.

Can determine uniformity percentage between two sequences with mathematical algorithm. Be used for mathematical algorithm preferred of two sequences relatively but nonrestrictive example is Karlin and Altschul, 1990, Proc. Natl Acad Sci.U.S.A.87:2264-2268 and Karlin and Altschul, improved algorithm among 1993, the Proc.Natl Acad Sci.U.S.A.90:5873-5877. This algorithm has been integrated into Altschul etc., in the NBLAST of 1990, J.Mol.Biol.215:403-410 and the XBLAST program. Can use the NBLAST parameter, with score=100, word long=12 carries out the retrieval of BLAST nucleotides, with the nucleotide sequence of acquisition with nucleic acid molecules homology of the present invention. Can use the XBLAST program, with score=50, word long=3 carries out the retrieval of BLAST protein, with the amino acid sequence of acquisition with protein molecule homology of the present invention. In order to obtain to use Gapped BLAST, such as Altschul etc., described in 1997, the Nucleic Acids Res.25:3389-3402 for being spaced of comparison. Selectively, can use PSI-BLAST to carry out multiple retrieval, this retrieval detects intermolecular remote relation (going out the same). When using BLAST, Gapped BLAST and PSI-Blast program, can use the default parameters (such as the parameter of XBLAST and NBLAST) of corresponding program.

Another of mathematical algorithm that is used for the sequence comparison is preferred, non-limitative example is Myers and Miller, the algorithm of 1988, Comput Appl Biosci 4:11-17. This algorithm has been incorporated in the ALIGN program (2.0 editions), and this program is the part of GCG sequence alignment software kit. When using ALIGN program comparision amino acid sequence, can use PAM120 weight residue table, the gap length penalties is 12, the interval penalties is 4. Other algorithm of sequence analysis is known in the art, comprises such as Torellis and Robotti ADVANCE and the ADAM described in 1994, the Comput Appl Biosci 10:3-5; And Pearson and Lipman, the FASTA described in 1988, the Proc Natl Acad Sci USA 85:2444-8. In FASTA, ktup is that the susceptibility of setting search and the control of speed are selected. If ktup=2, the residue of then checking to be compared in two sequences that are compared is to seeking similar area; If ktup=1 then checks the single amino acid that is compared. Ktup can be set as 2 or 1 for protein sequence, or can be made as 1 to 6 for dna sequence dna. If do not indicate, the default value of ktup is 2 for protein and is 6 for DNA. About further specifying of FASTA parameter, referring to http://bioweb.pasteur.fr/docs/man/man/fasta.1.html#sect2.

Uniformity percentage between two sequences can be determined with similar technique mentioned above, allows or do not allow to use the interval. When calculating uniformity percentage, only calculate actual pairing. But, when evaluation and EphA2 sequence disclosed herein have the sequence of low uniformity percentage, should consider conservative the replacement.

In specific embodiment, the present invention uses and comprises at least EphA2 polypeptide, 10,20,30,40,50,75,100 or 200 amino acid whose EphA2 antigenic peptides of preferred SEQ ID NO:2. In preferred embodiments, the present invention uses and comprises at least EphA2 polypeptide, the EphA2 antigenic peptide of 10,20,30,40,50,75,100 or 200 continuous amino acids of preferred SEQ ID NO:2. In preferred embodiments, this peptide species comprises all or part of of ectodomain of the EphA2 polypeptide of SEQ ID NO:2.

5.2.1. fusion

In certain embodiments of the invention, expressing based on listerial EphA2 vaccine is the EphA2 antigenic peptide of fusion. Therefore, the present invention comprises composition and method, and wherein the EphA2 antigenic peptide is fusion, and this fusion comprises and the allos component, for example whole or fragment or the derivative of the EphA2 of heterologous peptides operability connection. The allos component can include but not limited to be conducive to the separation of fusion and the sequence of purifying. The allos component also can comprise the sequence of giving EphA2 antigenic peptide stability. These merge component is well known to those skilled in the art.

The present invention includes the application of fusion, this fusion comprises EphA2 polypeptide (for example polypeptide of SEQ ID NO:2 or its fragment) and heterologous polypeptide (being polypeptide or its fragment, the preferably fragment of at least 10 of this polypeptide, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 continuous amino acids). Fusion can be directly, also can realize by the linker sequence. Heterologous peptides can merge at the N of EphA2 antigenic peptide end or C terminal. Selectively, but the heterologous peptides flank connects the EphA2 peptide sequence.

Fusion can be included in the terminal EphA2 antigenic peptide that merges with the allos burst of its N. Various bursts are commercially available. Except the described burst of 5.1.2.4. part above, the protokaryon allos burst that can be used for method of the present invention includes but not limited to the phoA secretion signal (volume such as Sambrook, 1989, Molecular Cloning:A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory publishing house, Cold Spring Harbor, NY) and albumin A secretion signal (Pharmacia Biotech, Piscataway, NJ).

The EphA2 antigenic peptide can merge with sequence label, and this label is six histidine peptides for example, and such as the label that provides at pQE carrier (QIAGEN company, Chatsworth, CA) etc., wherein many is commercial obtainable, to be used for method of the present invention. Such as Gentz etc., described in 1989, the Proc.Natl.Acad.Sci. USA 86:821-824, provide six histidines to be convenient to purified fusion protein. Other example of peptide tag is erythrocyte agglutination element " HA " label, it is corresponding to the antigenic determinant (Wilson etc. that derive from influenza erythrocyte agglutination fibroin, 1984, Cell, 37:767), and " flag " label (Knappik etc., 1994, Biotechniques, 17 (4): 754-761). These labels are particularly useful to the EphA2 antigenic peptide of purification of Recombinant preparation.

Utilization has the easily any fusion of purifying of specificity or antibody selective to the fusion of expressing. Such as the fusion (Janknecht etc., 1991, Proc.Natl.Acad.Sci.USA 88:8972) by the easily non-sex change in the human cell line, expressed of purifying of the system of the descriptions such as Janknecht. In this system, interested gene is inserted in the vaccinia recombination plasmid, thereby the ORFs of this gene merges with the amino terminal label translation property ground that is comprised of six histidine residues. With the extract application of sample of the cell of recombinant vaccinia virus infection to Ni²⁺On-nitrile acetic acid the agarose column, the buffer solution that is contained imidazoles with histidine-tagged albumen is wash-out optionally.

In order to use and method of the present invention, also the carboxyl terminal of affinity tag at its amino terminal and EphA2 antigenic peptide can be merged. Not critical in the accurate site that carboxyl terminal merges. Can determine optimum site by the normal experiment method. In order to use and method and composition of the present invention, the amino terminal of affinity tag at its carboxyl terminal and EphA2 antigenic peptide can also be merged.

Also can use various affinity tag known in the art, (also can be referring to Petty such as but not limited to constant region for immunoglobulin, 1996, Metal-chelate affinity chromatography, see Current Protocols in Molecular Biology, the 2nd volume, the volumes such as Ausubel, Greene Publish.Assoc. ﹠ Wiley Interscience), glutathione s-transferase (GST; Smith, 1993, Methods Mol. Cell Bio.4:220-229), Escherichia coli maltose-binding protein (Guan etc., and various cellulose binding domain (U.S. Patent number 5 1987, Gene 67:21-30),, 496,934,5,202,247,5,137,819, Tomme etc., 1994, Protein Eng.7:117-123) etc. Other affinity tag is identified by specific binding component, thereby is conducive to be fixed on the solid support this by affine combination and separates in conjunction with component. Some affinity tag also can make the EphA2 antigenic peptide produce new structure attribute, for example form polymeric ability. These affinity tag are usually derived from common protein as existing with aggressiveness. Ectodomain (the Shiue etc. of affinity tag such as CD8,1988, Med.168:1993-2005) or CD28 (Lee etc. J.Exp., 1990, J Immunol.145:344-352) or comprise the fragment that interchain disulfide bond forms the immunoglobulin molecules in site, may cause polymer to form.

As it will be appreciated by those skilled in the art that many methods can be used for obtaining the code area of above-mentioned affinity tag, include but not limited to dna clone, DNA cloning and synthetic method. The detection that is used for them is commercial obtainable with some affinity tag of separating and reagent.

Can use various homing sequence known in the art so that the EphA2 antigenic peptide is effectively secreted out (von Heijne, J.Mol.Biol.184:99-105) from bacterial cell such as Listeria. Except the burst of describing in mentioned above and the 5.1.2.4. part, the suitable homing sequence that guiding EphA2 antigenic peptide is expressed in bacterial cell includes but not limited to e. coli protein OmpA (Hobom etc., 1995, Dev.Biol.Stand.84:255-262), Pho A (Oka etc., 1985, Proc.Natl.Acad.Sci 82:7212-16), OmpT (Johnson etc., 1996, Protein Expression 7:104-113), LamB and OmpF (Hoffman ﹠ Wright, Proc.Natl.Acad. Sci.USA 82:5107-5111), beta-lactamase (Kodonaga etc., 1984, J.Biol.Chem.259:2149-54), enterotoxin (Morioka-Fujimoto etc., 1991, J.Biol.Chem.266:1728-32), with Staphylococcus aureus a-protein (Abrahmsen etc., 1986, Nucleic Acids Res.14:7487-7500), interior glycogenase (Lo etc. with bacillus subtilis, Appl.Environ.Microbiol.54:2287-2292) homing sequence, and artificial and synthetic burst (MacIntyre etc., 1990, Mol.Gen.Genet.221:466-74; Kaiser etc., 1987, Science, 235:312-317).

In certain embodiments, merge component and comprise and the corresponding non-EphA2 polypeptide of cell type related antigen, this cell type is that the type resisted is wished in treatment or epidemic prevention. For example, non-EphA2 polypeptide can comprise the antigenic determinant of tumor associated antigen, such as but not limited to MAGE-1, MAGE-2, MAGE-3, gp100, TRP-2, tyrosinase, MART-1, β-HCG, CEA, Ras, β-linkage protein (β-catenin), gp43, GAGE-1, GAGE-2,2-Acetamido-2-deoxy-D-glucose transferase-V, p15, β-linkage protein, BAGE-1, PSA, MUM-1, CDK4, HER-2/neu, human papilloma virus-E6, human papilloma virus-E7 and MUC-1,2,3.

The polynucleotide of encoding fusion protein can prepare by the recombinant DNA technology of standard, and for example the nucleic acid molecules of encoding fusion protein can be synthetic by routine techniques, comprises automatic dna synthesizer. Selectively, can use the anchor primer that complementary cohesive end is provided between two adjacent genetic fragments to carry out the pcr amplification of genetic fragment, can and increase again and produce chimeric gene order and (see for example Current Protocols in Molecular Biology the annealing of this genetic fragment subsequently, the volumes such as Ausubel, John Wiley ﹠ Sons, 1992).

The nucleotide sequence of encoding fusion protein can be inserted into suitable expression vector, namely comprises in the carrier of the necessary element of transcribing and translating of the albumen coded sequence that is inserted into. The expression of fusion can be by consisting of type, induction type or tissue specificity or optionally promoter adjusting. The technical staff can understand, and fusion can promote dissolving and/or expression, and can prolong the EphA2 antigenic peptide half-life in vivo, therefore can be used for method of the present invention. EphA2 antigenic peptide or its fragments of peptides or fusion can be used for detecting or measure calibration and the standardization of any detection method or these detection methods of EphA2 antigenic peptide.

Method of the present invention comprises the application of EphA2 antigenic peptide or its fragments of peptides, and these peptides can use technology well known in the art to prepare by recombinant DNA technology. Therefore, the method that the nucleic acid that comprises EphA2 antigenic gene sequence by expression prepares EphA2 antigenic peptide of the present invention obtains describing at this. Can come construction of expression vector with method well known to those skilled in the art, this expression vector comprises EphA2 antigenic peptide coded sequence for example (nucleic acid of the whole or antigenic portions of the SEQ ID NO:2 polypeptide that includes but not limited to encode) and suitable transcribes and translate control signal. These methods comprise for example interior Genetic Recombination of extracorporeal recombinant DNA technology, synthetic technology and body. See such as at Sambrook etc., 1989, the same, and Ausubel etc., 1989, technology same as above. Selectively, can the encode RNA of EphA2 antigenic peptide sequence can come chemical synthesis (to see for example in Oligonucleotide Synthesis, 1984 with for example synthesizer, Gait, M.J. compile IRL publishing house, the technology described in the Oxford).

In certain embodiments, the EphA2 antigenic peptide is connected with the internalization signal peptide is functional, and this signal peptide is also referred to as " nexin transduction domain ", and it is absorbed in the nucleus. In certain embodiments, this internalization signal is that feeler foot (antennapedia) is (referring to Prochiantz, 1996, Curr. Opin.Neurobiol.6:629-634, Hox A5 (Chatelin etc., 1996, Mech.Dev.55:111-117), HIV TAT albumen (Vives etc., 1997, J.Biol.Chem.272:16010-16017) or VP22 (Phelan etc., 1998, internalization signal Nat.Biotechnol.16:440-443).

5.2.2. the polynucleotide of coding EphA2 antigenic peptide

The present invention also comprises the application based on listerial vaccine, this vaccine comprises or contains polynucleotide, this polynucleotide hereinafter strict, the moderate of defined height is strict or than under the strict hybridization conditions of low degree, with the polynucleotide hybridization of coding EphA2 antigenic peptide of the present invention.

As an example and non-limiting, for the hybridization zone more than 90 nucleotides, use the step of low stringent condition following (also can be referring to Shilo and Weinberg, Proc.Natl.Acad.Sci.USA 78:6789-6792). In the solution of the salmon sperm dna that contains 35% formamide, 5 * SSC, 50mM Tris-HCl (pH7.5), 5mM EDTA, 0.1%PVP, 0.1%Ficoll, 1%BSA and 500 μ g/ml sex change, will comprise the filter of DNA 40 ℃ of preliminary treatment 6 hours. In having the same solution of following change, hybridize: 0.02%PVP, 0.02%Ficoll, 0.2%BSA, 100 μ g/ml salmon sperm dnas, 10% (mass/volume) dextran sulfate, and use 5～20 * 10⁶cpm  ³²The probe of P-mark. 40 ℃ of lower cultivations were also washed 1.5 hours under 55 ℃ in the solution that contains 2 * SSC, 25mM Tris-HCl (pH7.4), 5mM EDTA and 0.1%SDS in 18-20 hour subsequently in hybridization mixture. Wash solution was placed 1.5 hours with the fresh solution displacement and at 60 ℃ again. Filter is blotted and expose for autoradiograph. If necessary, under 65-68 ℃, filter washed for the third time and again be exposed to film. Operable low other strict condition is (for example being used for the condition that interspecific hybridization is used) well known in the art.

Equally, as an example and non-limiting, for the hybridization zone more than 90 nucleotides, use the step of highly strict condition as follows. Under 65 ℃, the filter that will comprise DNA carries out prehybridization 8 hours to spending the night in the buffer solution of the salmon sperm dna that contains 6 * SSC, 50mM Tris-HCl (pH7.5), 1mM EDTA, 0.02%PVP, 0.02%Ficoll, 0.02%BSA and 500 μ g/ml sex change. Containing 100 μ g/ml denatured salmon sperm dnas and 5～20 * 10⁶cpm  ³²Under 65 ℃ filter was hybridized 48 hours in the prehybridization mixture of the probe of P-mark. In the solution that contains 2 * SSC, 0.01%PVP, 0.01%Ficoll and 0.01%BSA 37 ℃ with filter washing 1 hour. After this in 0.1 * SSC 50 ℃ the washing 45 minutes, then autoradiograph.

Other strict condition of spendable height depends on the character (such as length, GC content etc.) of nucleic acid and the purpose (detection, amplification etc.) of hybridization, and this is known in the art. For example, the nucleic acid of about 15-40 base and the strict hybridization of complementary series are carried out under the following conditions in polymerase chain reaction (PCR): the buffer concentration of the salinity of 50mM KCl, 10mM Tris-HCl, the Mg of 1.5mM²⁺The pH of concentration, 7-7.5 and 55-60 ℃ annealing temperature.

The selection of the appropraite condition that moderate is strict also is well known in the artly (to see such as Sambrook etc., 1989, Molecular Cloning, A Laboratory Manual, the 2nd edition, Cold Spring Harbor Laboratory publishing house, Cold Spring Harbor, New York; Also see the volumes such as Ausubel, Current Protocols in Molecular Biologyseries of laboratory technique manuals,  1987-1997, Current Protocols,  1994-1997 John Wiley and Sons company).

Being used for nucleic acid of the present invention can be by any method preparation known in the art. For example, if the nucleotide sequence of EphA2 antigenic peptide is known, the nucleotide sequence of encoded peptide can be from the assembling of the oligonucleotides of chemical synthesis (such as at Kutmeier etc. so, described in 1994, the BioTechniques 17:242), wherein, in brief, comprise the synthetic overlapping oligonucleotides of having of some peptide-coding sequence that contains, with these oligonucleotides annealing and connection, by PCR the oligonucleotides that connects is increased subsequently.

Selectively, the polynucleotide of coding EphA2 antigenic peptide can produce from the nucleic acid in suitable source. If can't obtain to contain the clone of nucleic acid of concrete peptide of encoding, but the sequence of EphA2 antigenic peptide is known, the nucleic acid of encoded peptide can chemical synthesis or is obtained (for example cDNA library or produce from the cDNA library of any tissue of expressing EphA2 or cell or the nucleic acid that therefrom separates, preferably poly-A+RNA) from suitable source so. Method comprises: can carry out pcr amplification with the synthetic primer that 3 ' and 5 ' end of this sequence is hybridized by using, or by using the specific gene sequence to determining, for example from the cDNA clone of the cDNA library of expressing described peptide, there is specific oligonucleotide probe to clone. Can use subsequently the nucleic acid clone of the amplification that any method well known in the art will produce by PCR to advance in the reproducible cloning vector.

In addition, can operate with the method for operation nucleotide sequence well known in the art nucleic acid for this method, such as recombinant DNA technology, rite-directed mutagenesis, PCR etc. is (referring to such as at Sambrook etc. 1990, Molecular Cloning, A Laboratory Manual, the 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, the volume such as NY and Ausubel, 1998, Current Protocols in Molecular Biology, John Wiley ﹠ Sons, the technology of describing among the NY, these documents at this by intactly with reference to quoting), thereby produce the EphA2 antigenic peptide that amino acid sequence is different from amino acid sequence shown in the SEQ ID NO:2, for example produce 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, deletion and/or insertion.

5.3.EphA2 the analysis of antigenic peptide

The invention provides based on listerial EphA2 vaccine, this vaccine comprises the Listeria that is built into expression EphA2 antigenic peptide. Be used for determining peptide whether known in the art any analytical method of T cellular antigens determinant or B cell antigen determinant can be used for testing the well-formedness that the EphA2 peptide is used for method and composition of the present invention.

For example, in order to determine whether T cellular antigens determinant of peptide, can carry out with the method that ELISPOT analyzes and intracellular cytokine dyes the CD4 of antigentic specificity⁺And CD8⁺The cell count of T and sign. Lalvani etc., (1997) J.Exp.Med.186:859-865; Waldrop etc., (1997) J.Clin Invest.99:1739-1750.

Can determine the EphA2 antigenic peptide by screening and the synthetic peptide of the partial response of EphA2. Can be according to their sequence or the structure of the prediction antigenic peptide of identifying the candidate. Many algorithms can be used for this purpose.

Hereinafter provided the exemplary scheme of this detection.

5.3.1. show the immunogenic peptide of EphA2

Can analyze the EphA2 peptide and in mammal, cause the ability that the EphA2 specific antibody is replied, for example by each EphA2 peptide that is used in emulsification in Freund ' the s adjuvant animal (for example mouse, cavy or rabbit) be carried out immunity and analyze.

After 3 injections (per injection 5 to 100 μ g peptides), the ELISA by peptide specific and for the Western blotting test I gG antibody response of EphA2.

It is reported to produce the antigenicity that sero-fast EphA2 peptide shows EphA2, this antiserum and EphA2 peptide specific react and are identified by the EphA2 albumen of total length in Western blotting.

5.3.2.CD4 ⁺The T analysis of cell proliferation

For example, this analysis comprises external cell culture assays, wherein from the patient's that suffers from the disease relevant with the EphA2 overexpression new blood, obtain PMBC (" PBMC "), and basically such as Kruse and Sebald, described in 1992, the EMBO J 11:3237-3244, by using FICOLL-PLAQUE PLUS (Pharmacia, Upsalla, Sweden) carry out centrifugal purification. PMBC and candidate EphA2 antigenic peptide were cultivated 7-10 days. In order to process and to present antigen, optionally in culture, added antigen presenting cell in can be before analysis 24-48 hour. Subsequently by centrifugal cell harvesting, and in the middle washing of RPMI 1640 culture mediums (GibcoBRL, Gaithersburg, MD). In 96 orifice plates with 5 * 10⁴The T cells/well of individual activation places RPMI 1640 culture mediums that contain 10% hyclone, 10mM HEPES, pH7.5,2mM Glu, 100 units/ml benzyl penicillin and 100 μ g/ml streptomycin sulphates to put 72 hours at 37 ℃, and with every hole 1 μ Ci³H thymidine (DuPont NEN, Boston, MA) stimulated 6 hours, and results are also measured radioactivity in TOPCPOUNT scintillation counter (Packard Instrument Col., Meriden, CT).

5.3.3. cell within a cell factor dyeing (ICS)

Can be basically such as Waldrop etc., 1997, J.Clin.Invest.99:1739-1750; Openshaw etc., 1995, J.Exp.Med.182:1357-1367; Or Estcourt etc., 1997, Clin. Immunol.Immunopathol.83:60-67 is described, measures the antigentic specificity cell within a cell factor of T cell and replys. With the PBMC of purifying from the patient who suffers from the disease relevant with the EphA2 overexpression, with every pipe 1 * 10⁶The concentration of individual cell places in 12 * 75 millimeters polystyrene tissue culture pipes (Becton Dickinson, Lincoln Park, N.J.). The streptomysin, 2 mMs of L glutamine (Gibco BRL), various EphA2 antigenicity candidate's peptides of different amounts and the anti-CD28mAb (Becton-Dickinson of 1 unit that add every milliliter of penicillin, 100 unit containing every milliliter of 0.5 milliliter of HL-1 serum free medium, 100 units in each pipe, Lincoln Park, N.J.) solution. To resist CD3 mAb to join in two parts of normal PBMC cultures as positive control. Culture tube was cultivated 1 hour. Brefeldin A is joined in each pipe with 1 milligram every milliliter concentration, will manage and cultivate again 17 hours.

With the PBMC that twice results of solution washing cell stimulate as mentioned above, this solution comprises the Brefeldin A of Dulbecco ' s phosphate-buffered salt (dPBS) and 10 units. Fixed these washed cells in 10 minutes by in the solution that contains 0.5 milliliter of 4% paraformaldehyde and dPBS, cultivating. With the solution washing cell that comprises dPBS and 2% hyclone (FCS). Then as (Waldrop etc., 1997, J.Clin.Invest.99:1739-1750) described, this cell be used for is used for the dyeing of intracellular cytokine and surface markers at once, or freezingly in freezing culture medium is no more than 3 days.

Cell preparation is melted rapidly and with the dPBS washing once. With cell, no matter be fresh or freezing, be resuspended in 0.5 milliliter the penetrating solution (Becton Dickinson Immunocytometry systems, San Jose, Calif.), and at room temperature lucifuge was cultivated 10 minutes. Cell after penetrating with the dPBS washed twice and with the mAbs of direct coupling at room temperature lucifuge cultivated 20 minutes. The optimal concentration of antibody is determined in advance according to standard method. After dyeing, washed cell also retightens by cultivating in the solution that contains dPBS and 1% paraformaldehyde, and stores to be used for Flow Cytometry Analysis 4 ℃ of lucifuges.

5.3.4.ELISPOT analyze

The Th1-cell factor specificity that the ELISPOT analysis to measure occurs in the mouse boosting cell after the vaccine inoculation of Listeria is induced. Carry out ELISPOT and analyze frequency to determine that the endogenous antigenic peptide of T lymphocyte responses stimulates, this analyzes such as Geginat etc., described in 2001, the J.Immunol.166:1877-1884. With the listeria monocytogenes of expressing candidate EphA2 antigenic peptide or the inoculation of HBSS in contrast Balb/c mouse (3 every group). In rear 5 days of inoculation results and compile whole mice spleen. In the situation that various antigens exist, divide dish to be placed in 37 ℃ of incubators the single-cell suspension liquid of mouse boosting cell and spend the night.

In the celluloid substrate 96 hole droplet price fixings that have been coated with rat anti-mouse INF-γ mAb, test. In order to test candidate EphA2 antigenic peptide, preparation 1 * 10^-5The M peptide solution. In every hole of round bottom 96 hole droplet price fixings, 135 μ l culture mediums (10%FCS, 100U/ml penicillin, 100 μ g/ml streptomysins, 1 * 10 have been replenished^-5Eagle ' the s improvement culture medium (Life Technologies, Eggenstein, Germany) of M 2-ME and 2mM glutamine) 6 * 10 in⁵1 * 10 of individual not separating Morr. cell and 15 μ l^-5The M peptide solution is mixed, to produce 1 * 10^-6The final peptide concentration of M. 37 ℃ cultivate 6 hours after, firmly pressure-vaccum makes cell resuspended, and the cell suspending liquid of 100 μ l or 10 μ l (is respectively 4 * 10⁵/ hole or 4 * 10⁴/ hole) be transferred in the coated ELISPOT dish of Ab, and 37 ℃ of overnight incubation. In the ELISPOT dish, final volume is adjusted to 150 μ l distributes to guarantee the cell homogeneous.

CD4 with improvement analytical method test purifying as described below⁺Or CD8⁺T cell: with the prediluted peptide (1 * 10 of 15 μ l^-5M) directly join in the coated ELISPOT dish of Ab, and with from nonimmune animal as 4 * 10 of APC⁵Individual splenocyte is mixed, produces the final volume of 100 μ l. At 37 ℃ of preculture APC after 4 hours, will from the listeria monocytogenes immune mouse, be purified into 1 * 10⁵Individual CD4⁺Or CD8⁺Cell joins each hole with the volume of 50 μ l, and 37 ℃ of incubated overnight. With 1 * 10^-6Peptide carry out external MHC restriction analysis based on ELISPOT after 2 hours in 37 ℃ of clones that are added to expression specificity MHC I quasi-molecule. Subsequently, the unconjugated peptide of wash-out (four times) is to prevent peptide and to reply splenocyte and be combined. Each hole of ELISPOT dish is with 1 * 10⁵Individual APC and 4 * 10 with peptide⁵Or 4 * 10⁴The individual splenocyte of replying is blended into final volume 150 μ l. After 37 ℃ of incubated overnight, with the aminoethyl carbazole dye point of biotin labeled rat anti-mouse INF-γ mAb, HRP streptomysin conjugate and each splenocyte the ELISPOT dish is developed. Use has specificity and the sensitiveness of the control ELISPOT of the cd8 t cell system analysis of specific INF-γ secretion to contrast antigen.

5.4. preventing/treating method

The present invention has supplied treatment, prevention or the control illness relevant with the EphA2 overexpression and/or excessive proliferative cell illness, the method of cancer preferably comprises that one or more are of the present invention based on listerial EphA2 vaccine administration to the individual body and function that these needs are arranged.

The present invention includes the immunoreactive method that causes the antagonism EphA2 expression cell relevant with excessive proliferative cell illness, the method comprises to individuality expresses the immunoreactive effective dose of cell to cause antagonism EphA2, with of the present invention one or more based on listerial EphA2 vaccine administration.

In another particular, the described illness for the treatment of, prevention or control is to express relevant symptom precancer of cell with excessive EphA2. In more specific embodiment, precancer, symptom was high-level PIN (PIN), mammary gland fibroma, galactoncus disease or compound mole.

The invention provides treatment, prevention or the control illness relevant with the EphA2 overexpression and/or excessive proliferative cell illness, the method of cancer preferably, the method comprise to the individual body and function that these needs are arranged of the present invention one or more based on listerial EphA2 vaccine and one or more other treatment administration. The example of other treatment includes but not limited in the following 5.4.3. part listed. In one embodiment, of the present invention based on listerial EphA2 vaccine can be used for the treatment of, illness that prevention or control are relevant with the EphA2 overexpression and/or one or more other treatments (for example prevention or therapeutic agent) combination medicine-feeding of excessive proliferative cell illness such as cancer. In certain embodiments, one or more based on listerial EphA2 vaccine be used for the treatment of or one or more other treatments (for example therapeutic agent) of control cancer to individuality, preferably people's while administration. Term " simultaneously " is not limited to award treatment (for example prevention or therapeutic agent) in the accurate same time, and refer to of the present invention based on listerial EphA2 vaccine and another treatment in order within a time period to individual administration, thereby make this based on listerial EphA2 vaccine can with this other the treatment play a role, thereby than they otherwise administration better effect is provided. For example, each at one time administration for the treatment of (for example prevention or therapeutic agent), or at different time points with the in succession administration of any order; Yet if not at one time administration, they should be with enough approaching time administration to provide desirable treatment or preventive effect. Each treatment (for example prevention or therapeutic agent) can be respectively, with any suitable form and by any suitable administration. In some embodiment kind, of the present invention based on listerial EphA2 vaccine can be before operation, simultaneously or afterwards administration. Preferably, this operation has fully been removed local tumour or has been reduced the size of large tumour. Operation also can be used as preventive measure or carries out for palliating the agonizing sufferings.

In each embodiment, treating (for example preventing or therapeutic agent) can be to give less than 1 hour interval, about 1 hour interval, about 1 hour extremely about 2 hours interval, about 2 hours extremely about 3 hours intervals, about 3 hours extremely about 4 hours intervals, about 4 hours extremely about 5 hours intervals, about 5 hours extremely about 6 hours intervals, about 6 hours extremely about 7 hours intervals, about 7 hours extremely about 8 hours intervals, about 8 hours extremely about 9 hours intervals, about 9 hours extremely about 10 hours intervals, about 10 hours extremely about 11 hours intervals, about 11 hours extremely about 12 hours intervals, no more than 24 hours interval or no more than 48 hours intervals. In preferred embodiments, two or more treatments administration when accessing with a patient.

Dosage and frequency in this administration that provides are effectively effectively contained with prevention by the term treatment. This dosage and frequency usually also change according to each patient's specific factors, depend on approach and age, body weight, reaction and the patient medical history in the past of the order of severity of the concrete treatment that gives or preventative reagent, cancer and type, administration. Suitable scheme can be by those skilled in the art by considering these factors and abideing by, for example in the literature the report and Physician ' s Desk Reference (the 56th edition, 2002, the 57 editions, 2003 and the 58th editions, 2004) dosage of recommending in is selected.

5.4.1.1. patient group

The invention provides the particularly method of cancer for the treatment of, prevention and/or the control illness relevant with the EphA2 overexpression and/or excessive proliferative cell disease, the method comprise to individual body and function that these needs are arranged of the present invention one or more based on listerial EphA2 vaccine with treatment or the immunoreactive amount administration that prevents effective dose or resist the EphA2 expression cell relevant with excessive proliferative disorders with effective initiation. In another embodiment, other treatment of effective dose and one or more based on listerial EphA2 vaccine of the present invention (for example treatment or preventative reagent) combination medicine-feeding is with treatment, prevention and/or the control illness relevant with the EphA2 overexpression and/or excessive proliferative cell disease cancer particularly. Individuality is mammal preferably, such as non-Primates (such as ox, pig, horse, cat, dog, mouse etc.) and Primates (for example monkey, such as cercopithecus aethiops (cynomolgous monkey) and people). In preferred embodiments, this individuality is the people.

The object lesson of the cancer of the method treatment that available the present invention comprises includes but not limited to the cancer of overexpression EphA2. In one embodiment, cancer is the epithelium source. The example of these cancers is lung cancer, colon cancer, prostate cancer, breast cancer and cutaneum carcinoma. Other cancer comprises carcinoma of urinary bladder and cancer of pancreas and clear-cell carcinoma and melanoma. In other embodiments, cancer is solid tumor. In another embodiment, cancer is the T cell source. The example of these cancers is leukaemia and lymthoma. In following 5.4.1.1. part, enumerated as an example and without limitation other cancer. In specific embodiments, method of the present invention can be used for treating and/or preventing the transfer from primary tumor.

Method and composition of the present invention comprises may suffer from cancer to suffering from maybe, for example the cancer of particular type is had genetic predisposition, is exposed to carcinogenic substance or the individuality that takes a turn for the better from particular cancers/or patient, with of the present invention one or more based on listerial EphA2 vaccine administration. In this manual, " cancer " of use refers to primary or metastatic cancer. These patients may accept or not accept treatment of cancer before this. Method and composition of the present invention is used as the treatment of cancer of any line, for example a line, two wires or three line treatments of cancer. The present invention comprises that also the patient to accepting other treatment of cancer treats, and method and composition of the present invention can occur using before any side effect or the intolerance in these other treatments of cancer. The present invention also comprise with of the present invention one or more based on listerial EphA2 vaccine administration, with treatment or improve the method for the patient's be difficult to treat symptom. In certain embodiments, cancer is that at least some signal portions that refer to cancer cell that therapy is difficult to treat are not killed or their cell division is not stopped. Can be by known in the art for detection of any technology for the treatment of to the validity of cancer cell, in vivo or external definite cancer cell whether " do not reply/be difficult to treat ", in these contexts, use the implication of " being difficult to treat " that this area accepts. In various embodiments, cancer is difficult to treat, and cancer cell number wherein is not significant to be reduced or increase to some extent. The present invention also comprises the method that is easy to outbreak or the recurrence of cancer in the cancered patient body with one or more based on Listeria EphA2 vaccine administration with prevention.

In specific embodiments, with of the present invention based on listerial EphA2 vaccine administration with inverse cancer cell to the resistance of some hormone, radiation and chemotherapy agents or the sensitiveness of reduction, thus so that cancer cell is again responsive to one or more these reagent, can give subsequently (or continuing to give) these reagent with treatment or control cancer, comprise the prevention transfer. In specific embodiments, with patient's administration of increasing to cell factor IL-6 level based on listerial EphA2 vaccine of the present invention, this factor is relevant to the generation of the resistance of different therapeutic schemes (such as chemotherapy and hormone therapy) from cancer cell. In another embodiment, with of the present invention based on listerial EphA2 vaccine to patient's administration of suffering from breast cancer, these patients have the responsiveness of reduction to tamoxifen therapy or are difficult to treat. In another particular, with patient's administration of increasing to cell factor IL-6 level based on listerial EphA2 vaccine of the present invention, this factor is relevant to the generation of the resistance of different therapeutic schemes (such as chemotherapy and hormone therapy) from cancer cell.

In optional embodiment, the invention provides the method for the treatment of or control patient cancer, comprise to the patient with of the present invention one or more based on listerial EphA2 vaccine and any other therapeutic combination administration, or be that other therapies is difficult to treat and no longer carry out patient's administration of this therapy to confirming. In certain embodiments, by the patient of method of the present invention treatment be receive chemotherapy, the patient of radiotherapy, hormone therapy or biology treatment/immunization therapy. But these patients comprise the patient who is difficult to treat with existing treatment of cancer, and have accepted this existing treatment still suffer from the patient of cancer. In other embodiments, the patient has been treated and has not had disease activity, comes the recurrence of pre-anti-cancer based on listerial EphA2 vaccine administration with of the present invention one or more.

In preferred embodiments, above-mentioned existing treatment is chemotherapy. In specific embodiments, this existing treatment comprises and awards chemotherapy, includes but not limited to methotrexate (MTX), taxol, mercaptopurine, thioguanine, hydroxycarbamide, cytarabine, endoxan, ifosfamide, Nitrosourea, cis-platinum, carboplatin, mitomycin, Dacarbazine, procarbizine, etoposide, campathecins, bleomycin, adriamycin, idarubicin, daunomycin, dactinomycin D, plicamycin, mitoxantrone, asparaginase, vincaleukoblastinum, vincristine, vinorelbine, Paclitaxel, Docetaxel etc. These patients comprise the patient who accepted radiotherapy, hormone therapy and/or biology treatment/immunization therapy. These patients also are included as treatment or control the patient that cancer has undergone surgery.

Selectively, the present invention also comprises treatment or controls the method for accepting or accepting the patient of radiotherapy. These patients comprise accept or receive chemotherapy before this, the patient of hormone therapy and/or biology treatment/immunization therapy. These patients also comprise the patient who accepted operation in order to treat cancer.

In another embodiment, the present invention includes the method that the patient of hormone therapy and/or biology treatment/immunization therapy was being accepted or accepting in treatment. These patients comprise and accept or receive chemotherapy and/or the patient of the treatment of radiotherapy. These patients also comprise the patient who accepted operation in order to treat cancer.

In addition, when being proved, chemotherapy, radiotherapy, hormone therapy and/or biology treatment/immunization therapy may prove that maybe toxicity is excessive, namely the individuality of receiving treatment has been caused and to have accepted or during intolerable side effect, the present invention also provides the replacement method of these treatments for the treatment of or control cancer. Accept the individuality of method of the present invention treatment and can optionally accept other treatment of cancer, for example operation, chemotherapy, radiotherapy, hormone therapy or biology treatment, which kind for the treatment of this depends on is can't accept or intolerable.

In other embodiments, the invention provides with of the present invention one or more and treat cancer based on listerial EphA2 vaccine administration, and do not need other treatment of cancer, this cancer has confirmed it is that these therapies are difficult to treat. In specific embodiments, lacking in the situation for the treatment of of cancer, with one or more EphA2 vaccines to the patient's administration that is difficult to treat with other cancer therapy.

In other embodiments, can to suffer from excessive EphA2 express cell relevant precancer symptom the patient use vaccine administration of the present invention, to treat illness and to reduce the possibility that it develops into malignant cancer. In specific embodiments, precancer, symptom was high-level PIN (PIN), mammary gland fibroma, galactoncus disease or compound mole.

In other embodiments, the invention provides the method for the treatment of, prevention and/or the excessive proliferative cell illness of control except cancer, particularly the illness relevant with the EphA2 overexpression includes but not limited to asthma, chronic obstructive pulmonary disease (COPD), fibrillatable (such as lung, kidney, heart and liver fibrosis), ISR (smooth muscle and/or endothelium), psoriasis etc. These methods comprise to mentioned above and are used for the treatment of, prevent the method similar with the method for control cancer, for example by EphA2 vaccine administration of the present invention, combined therapy (referring to, 5.4.3. part hereinafter for example, for example other treatment and the combination of EphA2 vaccine are awarded individuality, treat, prevent or control excessive proliferative disorders except cancer), to patient's administration that is difficult to treat with particular treatment etc.

5.4.1.2. cancer

Can be by the cancer of method and composition treatment of the present invention, prevention or control and the cancer that associated conditions includes but not limited to epithelial cell source and/or endothelium source. The example of these cancers comprises: leukaemia, such as but not limited to acute leukemia, ALL, acute bone marrow cell leukaemia for example myeloblast, promyelocyte, myelomonocyte, monocyte, red blood cell leukaemia and Myelodysplastic syndromes; Chronic leukemia is such as but not limited to chronic bone marrow cell (granulocyte) leukaemia, chronic lymphocytic leukemia, hairy cell leukemia; Polycythemia vera; Lymthoma is such as but not limited to lymphogranulomatosis, Non-Hodgkin lymphoma; Huppert's disease is such as but not limited to smouldering Huppert's disease, non-secretion myeloma, hardening myeloma, plasma cell leukaemia, unicity plasmacytoma and EMP; Macroglobulinemia; The MG that meaning is uncertain; Benign monoclonal gammopathy; Heavy chain disease; Bone and connective tissue sarcoma are such as but not limited to osteoma, osteosarcoma, chondrosarcoma, Ewing ' s knurl, pernicious giant-cell tumor, the fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma, fibrosarcoma, Kaposi sarcoma, leiomyosarcoma, embryonal-cell lipoma, lymphangioendothelial sarcoma, neurinoma, rhabdomyosarcoma, synovial sarcoma; Brain tumor is such as but not limited to glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glioma, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pinealocytoma, pinealoblastoma, former Brain lymphoma; Breast cancer includes but not limited to duct carcinoma, gland cancer, leaflet (cellule) cancer, intraductal carcinoma, marrow sample breast cancer, mucus breast cancer, tubulose breast cancer, mamillary breast cancer, Paget ' s disease and inflammation breast cancer; Adrenal is such as but not limited to pheochromocytoma and adrenocortical carcinoma; Thyroid cancer is such as but not limited to mamillary or folliculus shape thyroid cancer, medullary thyroid carcinoma and undifferentiated thyroid carcinoma; Cancer of pancreas is such as but not limited to insulinoma, gastrinoma, glucagon knurl, vasoactive intestinal peptide tumor, somatostatin secretion knurl and carcinoid or islet cell tumor; The hypophysis cancer is such as but not limited to Cushing syndrome, galactin knurl, acromegalia and diabetes insipidus; Cancer eye, such as but not limited to ophthalmomelanoma, for example iris melanoma, choroidal melanoma and ciliary body melanoma and cancer eye; Carcinoma of vagina, for example flakey cell cancer, gland cancer and melanoma; Carcinoma of vulva, for example flakey cell cancer, melanoma, gland cancer, basal-cell carcinoma, sarcoma and Paget ' s are sick; Cervix cancer is such as but not limited to flakey cell cancer and gland cancer; The cancer of the uterus is such as but not limited to carcinoma of endometrium and sarcoma of uterus; Oophoroma is such as but not limited to epithelial ovarian cancer, borderline tumor, gonioma and stromal tumor; Cancer of the esophagus is such as but not limited to flakey cancer, gland cancer, adenoid cystic carcinoma, mucoepidermoid carcinoma, gland carcinoma squamosum, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, oat cell (cellule) cancer; Cancer of the stomach is such as but not limited to gland cancer, gill fungus umbrella (polyp), ulcer, diffusion into the surface, extensive diffusive, malignant lymphoma, embryonal-cell lipoma, fibrosarcoma and carcinosarcoma; Colon cancer; The carcinoma of the rectum; Liver cancer is such as but not limited to hepatocellular carcinoma and liver mother cell cancer; Carcinoma of gallbladder, for example gland cancer; Cholangiocarcinoma is such as but not limited to mamillary, knot shape and disperse shape cancer; Lung cancer, for example non-small cell lung cancer, squamous cell carcinoma (epidermis shape cancer), gland cancer, large cell carcinoma and ED-SCLC; Carcinoma of testis, such as but not limited to gonioma, seminoma, undifferentiated, classical (typically), sperm mother cell type, nonseminoma, embryonal carcinoma, teratoma cancer, choriocarcinoma (yolk sac tumor); Prostate cancer is such as but not limited to PIN, gland cancer, leiomyosarcoma and rhabdomyosarcoma; The penal cancer; Carcinoma of mouth is such as but not limited to the flakey cell cancer; The substrate cancer; Salivary-gland carcinoma is such as but not limited to gland cancer, mucoepidermoid carcinoma and adenoid cystic carcinoma; Laryngocarcinoma is such as but not limited to flakey cell cancer and verrucous carcinoma; Cutaneum carcinoma, such as but not limited to basal-cell carcinoma, flakey cell cancer and melanoma, surface expansion melanoma, joint junction type melanoma, freckle chromoma, acra black mole melanoma; Kidney is such as but not limited to clear-cell carcinoma, gland cancer, hypernephroma, fibrosarcoma, transitional cell carcinoma (renal plevis and/or uterer); Wilms ' tumour; Carcinoma of urinary bladder is such as but not limited to transitional cell carcinoma, flakey cell cancer, gland cancer, carcinosarcoma. In addition, cancer comprises that myxosarcoma, osteosarcoma, endotheliosarcoma, lymph vessels endotheliosarcoma (lymphangioendotheliosarcoma), celiothelioma, synovial membrane cancer, hemangioblastoma, epithelioma, cystadenoma, bronchiolar carcinoma, syringocarcinoma, fatty gland cancer, papillary carcinoma and papillary adenocarcinoma are (about the summary of these illnesss, referring to Fishman etc., 1985, Medicine, second edition, J.B.Lippincott company, Philadelphia and Murphy etc., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A. company, the U.S.).

Method and composition of the present invention also can be used for treatment or prevents various cancers or other abnormality proliferation disease, include but not limited to: cancer comprises carcinoma of urinary bladder, breast cancer, colon cancer, kidney, liver cancer, lung cancer, oophoroma, cancer of pancreas, cancer of the stomach, cervix cancer, thyroid cancer and cutaneum carcinoma; The flakey cell cancer; The hematopoiesis tumour of Lymphatic System comprises leukaemia, acute lymphoblastic leukaemia, acute one-tenth leukemic lymphoblastoid, B cell lymphoma, t cell lymphoma, Burkitt ' s lymthoma; The tumour of spinal cord system comprises acute and chronic myeloid leukemia and becomes myeloid leukemia; The tumour in mesenchymal cell source comprises fibrosarcoma and rhabdomyosarcoma; Other tumour comprises melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; The tumour of maincenter and peripheral neverous system comprises astrocytoma, neuroblastoma, glioma and epilemma knurl; The tumour in mesenchymal cell source comprises fibrosarcoma, rhabdomyosarcoma and osteosarcoma; And other tumour, comprise melanoma, angioderma pigmentosum, keratoactanthoma, seminoma, follicular carcinoma of thyroid and teratocarcinoma. The cancer that expection is caused by abnormal apoptosis also can be by method and composition treatment of the present invention. These cancers can include but not limited to folliculus shape lymthoma, with the cancer of p53 sudden change, the hormone-dependent neoplasm of mammary gland, prostate and ovary; Premalignant lesion, for example familial adenomatous polyposis patients and Myelodysplastic syndromes. In specific embodiments, pernicious or ill propagation variation (for example transforming and dysplasia) or the excessive proliferative disorders in skin, lung, colon, mammary gland, prostate, bladder, kidney, pancreas, ovary or uterus are treated or prevent. In other particular, sarcoma, melanoma or leukaemia are treated or prevent.

In certain embodiments, cancer is pernicious and overexpression EphA2. In other embodiments, the illness that is treated is to express relevant symptom precancer of cell with excessive EphA2. In specific embodiments, precancer, symptom was high-level PIN (PIN), mammary gland fibroma, galactoncus disease or compound mole.

In preferred embodiments, method and composition of the present invention is used to treat and/or prevent breast cancer, oophoroma, cancer of the esophagus, colon cancer, oophoroma, lung cancer and prostate cancer and melanoma and provides for example without limitation hereinafter.

In another preferred embodiment, method and composition of the present invention is used to treat and/or prevent the cancer of T cell source, includes but not limited to leukaemia and lymthoma.

5.4.1.3. the treatment of breast cancer

In specific embodiments, to the patient who suffers from breast cancer with effective dose of the present invention one or more based on listerial EphA2 vaccine administration. In another embodiment kind, peptide of the present invention can be used for the treatment of other agent combination administration of breast cancer with one or more of effective dose, this reagent includes but not limited to: adriamycin, Epi-ADM, the combination of adriamycin and cyclophosphamide (AC), the combination of endoxan, adriamycin and 5 FU 5 fluorouracil (CAF), the combination of endoxan, Epi-ADM and 5 FU 5 fluorouracil (CEF), her-2 antibody is Trastuzumab, tamosifen for example, the combination of tamosifen and cytotoxicity chemotherapy, taxol (for example Docetaxel and Paclitaxel). In further embodiment, peptide of the present invention can add the together auxiliary curing of administration, Locally advanced breast cancer positive as tubercle of standard adriamycin and cyclophosphamide with taxol.

In specific embodiments, with of the present invention based on listerial EphA2 vaccine to patient's administration of suffering from mammary gland fibroma before the cancer or galactoncus disease, with the treatment illness with reduce it and develop into the possibility of malignant breast carcinomas. In another particular, treat particularly hormone therapy, more specifically tamoxifen therapy based on listerial EphA2 vaccine to using with of the present invention, patient's administration of being difficult to treat is with the treatment cancer and/or make the patient become medicable or reply.

5.4.1.4. the treatment of colon cancer

In specific embodiments, to the patient who suffers from colon cancer with effective dose of the present invention one or more based on listerial EphA2 vaccine administration. In another embodiment, peptide of the present invention can be used for the treatment of other agent combination administration of colon cancer with one or more of effective dose, and this reagent includes but not limited to: AVASTIN^IM(bevacizumab), the combination of 5-FU and formyl tetrahydrofolic acid, the combination of 5-FU and levamisol, Irinotecan (CPT-11), or the combination of Irinotecan, 5-FU and formyl tetrahydrofolic acid (IFL).

5.4.1.5. the treatment of prostate cancer

In specific embodiments, to the patient who suffers from prostate cancer with effective dose of the present invention one or more based on listerial EphA2 vaccine administration. In another embodiment kind, peptide of the present invention can be used for the treatment of other agent combination administration of prostate cancer with one or more of effective dose, and this reagent includes but not limited to: shine radiotherapy outward, radio isotope (namely¹²⁵I, palladium, iridium) intraluminal grafting, Leuprorelin or other LHRH activator, on-steroidal antiandrogen agent (Flutamide, Nilutamide, Bicalutamide), steroids antiandrogen agent (cyproterone acetate), the combination of Leuprorelin and Flutamide, estrogen for example the estrogen U.S.P., DES-diphosphonic acid, radio isotope of DES, Chlorotrianisene, ethinyl estradiol, coupling for example strontium 89, outer combination, two wires hormone therapy such as Aminoglutethimide, hydrocortisone, Flutamide according to radiotherapy and strontium 89 are given up, the meeting of progesterone and ketoconazole, low dosage prednisone or other report makes symptom obtain subjective the improvement and chemotherapy regimen that the PSA level is reduced, comprise Docetaxel, Paclitaxel, Estramustine/Docetaxel, Estramustine/etoposide, Estramustine/vincaleukoblastinum and Estramustine/Paclitaxel.

In specific embodiments, to the patient who suffers from high-level PIN before the cancer with EphA2 vaccine administration of the present invention, with the treatment illness and reduce the possibility that it will develop into the malignant prostate cancer.

5.4.1.6. melanomatous treatment

In specific embodiments, to suffer from melanomatous patient with effective dose of the present invention one or more based on listerial EphA2 vaccine administration. In another embodiment kind, peptide of the present invention can be used for the treatment of melanomatous other agent combination administration with one or more of effective dose, this reagent includes but not limited to: Dacarbazine (DTIC), Nitrosourea is such as card chlorine mustard (BCNU) and sendoxan (CCNU), reagent with moderate single reagent activity comprises vinca alkaloids, platinum compounds and taxol, Dartmouth scheme (cis-platinum, BCNU and DTIC), interferon-' alpha ' (IFN-α) and interleukin-22 (IL-2). In specific embodiments, one or more EphA2 vaccines of the present invention of effective dose can pour into (ILP), melphalan (L-PAM) and be with or without tumor necrosis factor α (TNF-α) combination with isolation high temperature limbs and award the patient who suffers from multiple brain transfer, bone transfer and vertebra compressing, so that sx↓ also makes the tumour atrophy with radiotherapy to a certain extent.

In the particular kind, with of the present invention based on listerial EphA2 vaccine to patient's administration of suffering from compound mole before the cancer, with the treatment illness and reduce the possibility that it develops into chromoma.

5.4.1.7. the treatment of oophoroma

In specific embodiments, to the patient who suffers from oophoroma with effective dose of the present invention one or more based on listerial EphA2 vaccine administration. In another embodiment kind, peptide of the present invention can be used for the treatment of other agent combination administration of oophoroma with one or more of effective dose, and this reagent includes but not limited to: radiotherapy such as P in the abdominal cavity³²Treatment, full belly and pelvis radiotherapy, cis-platinum, the combination of Paclitaxel (taxol) or Docetaxel (docetaxel) and cis-platinum or carboplatin, the combination of endoxan and cis-platinum, the combination of endoxan and carboplatin, the combination of 5-FU and formyl tetrahydrofolic acid, etoposide, liposomal doxorubicin, Ji Xitating or TPT. Expection is made up the patient who suffers from the disease that platinum is difficult to treat with of the present invention one or more of effective dose based on listerial EphA2 vaccine and taxol. Treatment to the patient that suffers from the oophoroma that is difficult to treat comprises: use the ifosfamide administration to the patient who suffers from the disease that platinum is difficult to treat, after based on the failure of the assembled scheme of platinum with hexamethyl melamine (HMM) administration as the remedial chemotherapy, but and use the tamosifen administration to the patient who has the cytoplasmic oestrogen-receptor of detection level in its tumour.

5.4.1.8. the treatment of lung cancer

In specific embodiments, to the patient who suffers from little pneumonocyte cancer with effective dose of the present invention one or more based on listerial EphA2 vaccine administration. In another embodiment, peptide of the present invention can be used for other agent combination administrations of lung cancer therapy with one or more of effective dose, this reagent includes but not limited to: chest radiotherapy alone or in combination, cis-platinum, vincristine, adriamycin and etoposide, part alleviation, bronchus inner support and/or the brachytherapy of the combination of endoxan, adriamycin, vincristine/etoposide and cis-platinum (CAV/EP), the treatment of bronchus inner laser.

In other embodiments, to the patient who suffers from non-little pneumonocyte cancer one or more one or more other agent combination administrations for lung cancer therapy based on listerial EphA2 vaccine and effective dose of the present invention with effective dose, this reagent includes but not limited to: the property alleviated radiotherapy, the combination of cis-platinum, vincaleukoblastinum and mitomycin, the combination of cis-platinum and vinorelbine, Paclitaxel, Docetaxel or Ji Xitating, the combination of carboplatin and Paclitaxel is to interstital brachytherapy,IBT or the operation of three-dimensional located irradiation that damages in the bronchus.

5.4.1.9.T the treatment of cell malignancies

In specific embodiments, to suffering from the T cell malignancies, as the patient of leukaemia and lymthoma (referring to for example 5.8.1.1. part) with effective dose of the present invention one or more based on listerial EphA2 vaccine administration. In another embodiment kind, peptide of the present invention can be used for prevention with one or more of effective dose, treat or improve other agent combination administration of cancer (particularly T cell malignancies or its one or more symptoms), described combined therapy comprise to the individuality of needs prevent or treat effective dose of the present invention one or more based on listerial EphA2 vaccine, with one or more treatments of cancer of prevention or treatment effective dose, this treatment of cancer comprises chemotherapy, hormone therapy, biology treatment, immunization therapy or radiotherapy.

In another embodiment, with effective dose of the present invention one or more based on listerial EphA2 vaccine and one or more cancer chemotherapy reagent to suffering from the pernicious patient's combination medicine-feeding of T cell, this reagent includes but not limited to: adriamycin, Epi-ADM, endoxan, 5 FU 5 fluorouracil, taxol such as Docetaxel and Paclitaxel, formyl tetrahydrofolic acid, levamisol, Irinotecan, Estramustine, etoposide, vincaleukoblastinum, Dacarbazine, Nitrosourea are such as card chlorine mustard and sendoxan, vinca alkaloids, platinum compounds, cis-platinum, mitomycin, vinorelbine, Ji Xitating, carboplatin, hexamethyl melamine and/or TPT. These methods randomly further comprise and give other treatment of cancer, such as but not limited to radiotherapy, biology treatment, hormone therapy and/or operation.

In another embodiment, one or more radiotherapies based on listerial EphA2 vaccine and one or more types of the present invention with effective dose, to patient's combination medicine-feeding of suffering from the T cell malignancies, radiotherapy is for example outer transplants (I-125, platinum, iridium), radio isotope such as strontium-89, chest radiotherapy, abdominal cavity P-32 radiotherapy and/or full belly and pelvis radiotherapy according to radiotherapy, a matter. These methods randomly further comprise and give other treatment of cancer, such as but not limited to chemotherapy, biology treatment/immunization therapy, hormone therapy and/or operation.

In another embodiment, of the present invention one or more with effective dose are used for biology treatment/immunization therapy or hormone therapy based on listerial EphA2 vaccine and one or more, to suffering from the pernicious patient's combination medicine-feeding of T cell, described treatment is tamosifen for example, and Leuprorelin or other LHRH activator, non-steroid antiandrogen (Flutamide, Nilutamide, Bicalutamide), steroid antiandrogen (cyproterone acetate), estrogen (DES), Chlorotrianisene, ethinyl estradiol, the estrogen U.S.P. of coupling, DES-diphosphonic acid, Aminoglutethimide, hydrocortisone, Flutamide are given up, progesterone, ketoconazole, prednisone, interferon-' alpha ', interleukin-22, tumor necrosis factor α and/or melphalan. The biology treatment also can comprise cell factor, such as but not limited to the tnf ligand family member, for example the anticancer activator of apoptosis-induced TRAIL, be also referred to as the TRAIL acceptor 1 of DR4 and DR5 (death domain comprises acceptor 4 and 5) and TRAIL antibody and DR4 and the DR5 of 2 combinations. TRAIL and TRAIL antibody, part and acceptor are known in the art, and at U.S. Patent number 6,342, explanation are arranged in 363,6,284,236,6,072,047 and 5,763,223. These methods randomly further comprise and give other treatment of cancer, such as but not limited to radiotherapy, chemotherapy and/or operation.

In another particular, with one or more standard and experimental therapies based on listerial EphA2 vaccine and T cell malignancies of the present invention of effective dose, to patient's combination medicine-feeding of suffering from the T cell malignancies. Can be used for the cell-mediated treatment of offensive combination chemotherapy, purine analogue, HSCT and T (the CD8+T cell that for example has the anti-leukocythemia liveness of antagonism target antigen includes but not limited to leukaemia specific proteins (for example bcr/abl, PML/RARa, EMV/AML-1), leukemia associated protein white (for example protease 3, WT-1, h-TERT, hdm-2)) that the standard of T cell malignancies of method and composition of the present invention and experimental therapy include but not limited to Antybody therapy (for example Campath , anti-Tac, HuM291 (humanization mouse IgG 2 monoclonal antibodies of anti-CD3), antibody drug conjugates (for example WAY-CMA 676), radiolabeled monoclonal antibody (for example Bexxar, select baby's spirit, Lym-1)), cytokine therapy, use or do not use cytotoxic reagent. (see Riddell etc., 2002, Cancer Control, 9 (2): 114-122; Dearden etc., 2002, Medical Oncology, 19, Suppl.S27-32; Waldmann etc., 2000, Hematology (Am Soc Hematol Educ Program): 394-408).

5.4.2. treatment or the prevention of the illness relevant with the abnormal vascular generation

EphA2 is the mark of the blood vessel that occurs of blood vessel, and blood vessel occur or neovascularization in play a significant role and (see such as Ogawa etc., 2000, Oncogene.19 (52): 6043-52; Hess etc., 2001, Cancer Res.61 (8): 3250-5). What blood vessel occured is characterized as smooth muscle and endothelial cell invasion, moves and breeds. Growth or the blood vessel of new blood vessel cause pathological state, such as DRP (Adonis etc., 1994, Amer.J.Ophthal., 118:445), rheumatic arthritis (Peacock etc., 1992, J.Exp.Med., 175:1135) and osteoarthritis (Ondrick etc., 1992, Clin.-Podiatr.-Med.-Surg.9:185).

Therefore of the present invention can have this individuality that needs with abnormal vascular relevant illness or its one or more symptoms to occur with prevention, control, treatment or improvement based on listerial composition. Occur relevant with abnormal vascular or take it as feature, and can include but not limited to tumor disease (non-limitative example is transfer and the leukaemia of tumour) with the illness of preventing, treat, controlling or improve based on listerial composition of the present invention; The disease of ocular angiogenesis (non-limitative example is AMD, DRP and retinopathy of prematurity, reangiostenosis); Skin disease (non-limitative example be infantile hemangioma, verruca vulgaris, psoriasis, basal cell and flakey cell cancer, cutaneous melanoma, Kaposi sarcoma, neural line fibroma, recessive atrophic type bubble epithelium dissociate disease); Arthritis (non-limitative example is rheumatic arthritis, stiff spondylitis, systemic lupus, chronic eczema arthropathy, Lay Te Shi syndrome and house Ge Lunshi syndrome); Gynaecological disease (non-limitative example is disease before mullerianosis, the pregnancy period eclampsia, oophoroma, carcinoma of endometrium and cervix cancer); And angiocardiopathy (non-limitative example is formation, arteriosclerosis and the coronary artery disease of atherosclerotic plaque).

In specific embodiments, occur relevant with abnormal vascular or take it as feature, and can comprise capillary proliferation and cancer in chronic rheumatic arthritis, psoriasis, DRP, neovascular glaucoma, macular degeneration, the atherosclerotic plaque with the illness of preventing, treat, controlling or improve based on listerial composition of the present invention, wherein EphA2 expresses in vascular system. These cancer illnesss for example comprise tunica fibrosa hyperplasia behind solid tumor, metastases, angiofibroma, the crystalline lens, hemangioma, Kaposi sarcoma.

In certain embodiments, will be used for comprising based on listerial composition the combined therapy scheme of other treatment. The non-limitative example of these treatments comprises anodyne, angiogenesis inhibitor, anticancer therapy and anti-inflammatory reagent, particularly anodyne and angiogenesis inhibitor.

5.4.2.1. patient group

The present invention includes illness relevant with the abnormal vascular generation in treatment, control and/or the prevention individuality or the method for its symptom, the method comprises with one or more based on listerial EphA2 vaccine administration. Method of the present invention comprise to suffer from maybe may with suffer from relevant disease occurs (for example having the patient of genetic predisposition or the patient who suffered from before this) and abnormal vascular patient with one or more based on listerial EphA2 vaccine administration. Such patient may accept before this treatment or accept the treatment of this illness at present. According to the present invention, can be used as the treatment of any line based on listerial EphA2 vaccine, include but not limited to the treatment of a line, two wires, three lines and four lines. In addition, according to the present invention, can before occuring based on any side effect of listerial EphA2 vaccine or non-tolerance, this use based on listerial EphA2 vaccine. The present invention includes with of the present invention one or more and with abnormal vascular the outbreak of relevant illness or the method for recurrence occur with prevention based on listerial EphA2 vaccine administration.

In one embodiment, the present invention also provides treatment or control and abnormal vascular that the alternative of the existing treatment of relevant illness occurs. In specific embodiments, existing treatment has confirmed maybe may confirm to patient's toxicity excessive (for example causing unacceptable or intolerable side effect). In another embodiment, the patient has confirmed that existing therapy is difficult to treat. In this embodiment, the invention provides with of the present invention one or more and treat based on listerial EphA2 vaccine administration or relevant illness occurs for control and abnormal vascular, and do not need other treatment. In certain embodiments, of the present invention one or more based on listerial EphA2 vaccine can replace other treatment to patient's administrations of needs with treatment or the control illness relevant with the abnormal vascular generation.

The present invention also comprises with of the present invention one or more and treats based on listerial EphA2 vaccine administration or improve the method that the symptom of relevant disease occurs for patient and blood vessel, and these patients are or have become the patient who is difficult to treat based on listerial EphA2 vaccine therapy with non-. Determine symptom whether be difficult to treatment can be in vivo or external use any technology known in the art finish, these technology can be analyzed treatment for the validity of the influenced cell among illness or the patient, this illness and abnormal vascular occur relevant, and this patient is or has become and non-ly be difficult to treat based on listerial EphA2 vaccine therapy.

5.4.3. other treatment

In some embodiments, will be with one or more based on the treatment of listerial EphA2 vaccine administration and one or more therapeutic combination, this treatment includes but not limited to chemotherapy, radiotherapy, hormone therapy and/or biology treatment. Preventing/treating reagent includes but not limited to protein molecule, includes but not limited to peptide, polypeptide, and protein includes but not limited to albumen, peptide after the posttranslational modification etc.; Or little molecule (less than 1000 dalton), inorganic or organic compound; Or nucleic acid molecules includes but not limited to two strands or single stranded DNA or two strands or single stranded RNA and triple helical nucleic acid molecules. Preventing/treating reagent can be derived from any known biology (including but not limited to animal, plant, bacterium, fungi and protist or virus) or derived from the library of synthetic molecules.

In specific embodiments, method of the present invention comprises the administration combination medicine-feeding based on listerial EphA2 vaccine and one or more preventing/treating reagent of the present invention, this reagent comprises antibody, it is kinase whose inhibitor, kinases is such as but not limited to ABL, ACK, AFK, AKT (AKT-1 for example, AKT-2 and AKT-3), ALK, AMP-PK, ATM, Aurora1, Aurora2, bARK1, bArk2, BLK, BMX, BTK, CAK, the CaM kinases, CDC2, CDK, CK, COT, CTD, DNA-PK, EGF-R, ErbB-1, ErbB-2, ErbB-3, ErbB-4, ERK (ERK1 for example, ERK2, ERK3, ERK4, ERK5, ERK6, ERK7), ERT-PK, FAK, FGR (FGF1R for example, FGF2R), FLT (FLT-1 for example, FLT-2, FLT-3, FLT-4), FRK, FYN, GSK (GSK1 for example, GSK2, GSK3-α, GSK3-β, GSK4, GSK5), G-G-protein linked receptor kinases (GRK), HCK, HER2, HKII, JAK (JAK1 for example, JAK2, JAK3, JAK4), JNK (JNK1 for example, JNK2, JNK3), KDR, KIT, the IGF-1 acceptor, IKK-1, IKK-2, INSR (insulin receptor), IRAK1, IRAK2, IRK, ITK, LCK, LOK, LYN, MAPK, MAPKAPK-1, MAPKAPK-2, MEK, MET, MFPK, MHCK, MLCK, MLK3, NEU, NIK, pdgf receptor α, pdgf receptor β, PHK, the PI-3 kinases, PKA, PKB, PKC, PKG, PYK1, PYK2, the p38 kinases, p135tyk2, p34cdc2, p42cdc2, p42mapk, p44mpk, RAF, RET, RIP, RIP-2, RK, RON, the RS kinases, SRC, SYK, S6K, TAK1-TEC, TIE1, TIE2, TRKA, TXK, TYK2, UL13, VEGF, VEGFR1, VEGFR2, YES, YRK, ZAP-70, (see for example Hardie and Hanks (1995) The Protein Kinase Facts Book I and II with these kinase whose all hypotypes, Academic publishing house, San Diego, Calif.). In preferred embodiments, with the administration combination medicine-feeding based on listerial EphA2 vaccine and one or more reagent of the present invention, this reagent is the inhibitor of Eph receptor kinase (for example EphA2, EphA4). In a more preferred embodiment, with the administration combination medicine-feeding of EphA2 vaccine of the present invention and one or more preventing/treating reagent, this reagent is the inhibitor of EphA2.

In specific embodiments, method of the present invention comprise with usefulness EphA2 vaccine administration of the present invention with make up with one or more therapeutic antibodies administrations. The example that can be used to the therapeutic antibodies of method of the present invention includes but not limited to AVASTIN , and it is VEGF antibody; Antibody (being the EphA2 agonistic antibody) with EphA2 immunologic opsonin zygotic induction signal transduction; The antibody of being combined with the EphrinA1 immunologic opsonin; HERCEPTIN  (Herceptin) (Genentech, CA), it is the Humanized anti-HER 2 monoclonal antibody that treatment suffers from the patient of metastatic breast cancer; REOPRO  (Abciximab) (Centocor), it is to prevent anti-glycoprotein receptor on the blood platelet that grumeleuse forms; ZENAPAX  (daclizumab) (Roche Pharmaceuticals, Switzerland), it is the inhibitive ability of immunity humanized recombined CD25 monoclonal antibody that the prophylaxis of acute kidney allograft repels; PANOREX^TM, it is the anti-17-IA cell surface antigen of mouse IgG2a antibody (Glaxo Wellcome/Centocor); BEC2, it is mouse antiidiotype (GD3 antigenic determinant) IgG antibody (ImClone System); IMC-C225, it is inosculating antibody EGFR IgG antibody (ImClone System); VITAXIN^TM, it is humanized anti-alpha_vβ ₃Integrin antibody (Applied Molecular Evolution/MedImmune); Campath 1H/LDP-03, it is humanized anti-CD52 IgG1 antibody (Leukosite); Smart M195, it is humanized anti-CD 33 IgG antibody (Protein Design Lab/Kanebo); RITUXAN^TM, it is inosculating antibody CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE^TM, it is humanized anti-CD22 IgG antibody (Immunomedics); LYMPHOCIDE^TMY-90 (Immunomedics); Lymphoscan (the Tc-99m-mark, radiophotography; Immunomedics); Nuvion (anti-CD3; Protein Design Labs); CM3, it is the anti-ICAM3 antibody of humanization (ICOS Pharm); IDEC-114, it is spirit lengthization Anti-CD80 McAb (IDEC Pharm/Mitsubishi); ZEVALIN^TM, it is radiolabeled mouse anti-CD 20 antibodies (IDEC/Schering AG); IDEC-131, it is humanization anti-CD40L antibodies (IDEC/Eisai); IDEC-151, it is spirit lengthization anti-CD 4 antibodies (IDEC); IDEC-152, it is the spirit anti-CD23 antibody of lengthization (IDEC/Seikagaku); The anti-CD3 of SMART, it is Humanized CD 3-resisting IgG (Protein Design Lab); 5G1.1 it is the humanization anticomplement factor 5 (C5) antibody (Alexion Pharm); D2E7, it is humanization anti-TNF alpha antibodies (CAT/BASF); CDP870, it is humanization TNF alpha antibody Fab fragment (Celltech); IDEC-151, it is spirit lengthization anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4, it is the anti-CD4 IgG of people antibody (Medarex/Eisai/Genmab); CD20-streptavidin (+biotin-Y90; NeoRx); CDP571, it is humanization TNF alpha antibody IgG4 antibody (Celltech); LDP-02, it is humanized anti-alpha_vβ ₇Antibody (LeikoSite/Genentech); OrthoClone OKT4A, it is the anti-CD4 IgG of humanization antibody (Ortho Biotech); ANTOVA^TM, it is humanization anti-CD 40 L IgG antibody (Biogen); ANTEGREN^TM, it is the anti-VLA-4 IgG of humanization antibody (Elan); And CAT-152, it is people's anti-TGF-beta 2 antibody (Cambridge Ab Tech).

In another particular, method of the present invention comprise with usefulness of the present invention based on listerial EphA2 vaccine administration with make up with one or more preventing/treating reagent administrations, this reagent is angiogenesis inhibitor, such as but not limited to angiostatin (plasminogen fragment); Anti-angiogenic generation Antithrombin III; Angiozyme; ABT-627; Bay 12-9566; Benfluralin; Bevacizumab (AVASTIN^TM); BMS-275291; The cartilage inhibitor (CDI) of deriving; CAI; CD59 complement fragment; CEP-7055; Col 3; Windmill presses down alkali A-4; Endostatin (collagen XVIII fragment); The fibronectin segment; Gro-β; Halofuginone; Heparinase; Heparin six bglii fragments; HMV833; Human chorionic promoting sexual gland hormone (hCG); IM-862; Interferon-' alpha '/β/γ; Interferon inducible protein (IP-10); IL-12; Kringle 5 (plasminogen fragment); Horse grain Ma Sita; Metal protease inhibitors (TIMP); Methoxyestradiol; MMI 270 (CGS 27023A); MoAb IMC-1C11; The spirit of shark cancer; NM-3; Panzem; PI-88; Placental ribonuclease inhibitor; The inhibitor of plasminogen activator; PF4 (PF4); Puli department he (Prinomastat); Prolactin 16kD fragment; Proliferin GAP-associated protein GAP (PRP); PTK 787/ZK 222594; Biostearin; Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SU11248; Tetrahydro cortisone S; Four sulfydryl molybdates; The Sa Li polyamines; Thrombospondin-1 (TSP-1); TNP-470; Transforming growth factor-beta; Angiostatin; Blood vessel inhibiting factor (calprotectin fragment); ZD6126; ZD6474; Farnesyl transferase inhibitor (FTI); And Diphosphonate.

In another embodiment, method of the present invention comprise with usefulness EphA2 administration of the present invention with one or more antitumor and anticancer agent administrations combination, this reagent is such as but not limited to the A Xi reform, Aclarubicin, the hydrochloric acid acodzole, acronine, Adozelesin, Aldesleukin, hexamethyl melamine, ambomycin, the acetic acid Ametantrone, Aminoglutethimide, amsacrine, the bent azoles of arna, Anthramycin, asparaginase, the woods aspergillin, azacitidine, Azetepa, azotomycin, Batimastat, Benzodepa, Bicalutamide, bisantrene hydrochloride, two methanesulfonic acid bisnafides, Bizelesin, bleomycin sulfate, brequinar sodium salt, Bropirimine, busulfan, act-C, Calusterone, Caracemide, Carbetimer, carboplatin, card chlorine mustard, carubicin hydrochloride, the card folding comes star, ground, west sweet smell cuts down, Chlorambucil, Cirolemycin, cis-platinum, Cladribine, methanesulfonic acid crisnatol, endoxan, cytarabine, Dacarbazine, dactinomycin D, the hydrochloric acid daunomycin, Dacarbazine, Decitabine, Dexormaplatin, Dezaguanine, the methanesulfonic acid Dezaguanine, diaziquone, Docetaxel, adriamycin, ADMh, Droloxifene, droloxifene citrate, dromostanolone propionate, diazomycin, Edatrexate, DFMO, Elsamitrucin, Enloplatin, the general amine ester of grace, solvable EphrinA1, the EphrinA1-Fc polypeptide, the EphA2-Fc polypeptide, the EphA2 antisense, the EphrinA1 antisense, Epipropidine, Farmorubine Hydrochloride, Erbulozole, esorubicin hydrochloride, Estramustine, estramustine phosphate sodium, etanidazole, etoposide, the phosphoric acid etoposide, etoprine, CGS-16949A, fludarabine, Suwei A amine, azauridine, fludarabine phosphate, fluorouracil, flurocitabine, Fosquidone, the Fostriecin sodium salt, Ji Xitating, his spit of fland of gemcitabine hydrochloride, hydroxycarbamide, idarubicin hydrochloride, ifosfamide, Yi Mofuxin, interleukin-22 (comprising recombinant interleukin 2 or rIL2), Intederon Alpha-2a, Interferon Alpha-2b, interferon alfa-n1, Alferon N, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, Letrozole, leuprorelin acetate, liarozole hydrochloride, the Lometrexol sodium salt, sendoxan, losoxantrone hydrochloride, Masoprocol, maytansine, mustine hydrochlcride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate (MTX), the methotrexate (MTX) sodium salt, metoprine, Meturedepa, mitindomide, mitocarcin, mitocromin, Mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, Mycophenolic Acid, Nitrosourea, nocodazole, nogalamycin, Ormaplatin, Oxisuran, Paclitaxel, Pegaspargase, Peliomycin, neptamustine, Peplomycin sulfate, Perfosfamide, pipobroman, piposulfan, the hydrochloric acid Piroxantrone, plicamycin, Plomestane, Porfimer Sodium, porphyromycin, prednimustine, PRO, Puromycin, puromycin hydrochloride, pyrazofurin, riboprine, Rogletimide, Safingol, the hydrochloric acid Safingol, Semustine, simtrazene, the sparfosate sodium salt, sparsomycin, spirogermanium hydrochloride, spiromustine, Spiroplatin, broneomycin, streptozotocin, Sulofenur, Talisomycin, the tecogalan sodium salt, Tegafur, teloxandrone hydrochloride, Temoporfin, Teniposide, teroxirone, Testolactone, thiamiprine, thioguanine, phosphinothioylidynetrisaziridine, Tiazofurine, Tirapazamine, FC-1157a, Herceptin (HERCEPTIN^TM), Triamcinolone prop Long, phosphoric Qu West Li Bin, Acamprosate, trimetrexate glucuronate ester, triptorelin, hydrochloric proper cloth chlorazol, Ula Secretary Ting Mo, Ury Tepa, Vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, Vindesine, vindesine sulfate, Changchun horses scheduled sulfates, monoglyceride sulfates, Changchun, Changchun Luo New sulfate, vinorelbine tartrate, sulfate Luoding Changchun, Changchun Lee sulfate, furosemide chlorine Azole, folding Nepal platinum, net cilastatin, Zuo doxorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20 - Table -1,25 - Dihydroxy vitamin D3, 5 - acetylene uracil, abiraterone, aclarubicin, acyl fulvene, adecypenol, Addo to new, A to interleukin, ALL-TK antagonists, Rokko melamine, ammonia Secretary Mo Ting, amidox, amifostine, aminolevulinic acid, ammonia, doxorubicin, amsacrine, anagrelide, A Anastrozole, andrographolide, angiogenesis inhibitors, antagonists D, antagonist G, antarelix, Anti back morphogenetic protein-1, anti-androgen agents, anti-estrogen agents, anti-tumor substances, glycine Aphid intestinal vancomycin, apoptotic gene regulators, apoptosis regulators, off purine nucleic acids, A-CDP-DL-PTBA, Arginine deaminase, asulacrine, A he exemestane, Amos Ting, axinastatin 1, axinastatin 2, axinastatin 3, Azasetron, diazo toxin (azatoxin), diazo tyrosine, Baccatin III derivatives, balanol, batimastat, BCR / ABL antagonist, benzo chlorin (Benzochlorins), benzoylstaurosprine, β-lactam derivatives, β-alethine, betaclamycin B, birch acid ester, bFGF inhibitors, bicalutamide than cohort, bisaziridinylspemine, double Nai Fade, bistratene A, than off to new, breflate, bromine horses stand Ming, cloth degree titanium, butyl thionine imines, calcipotriol, calphostin C, camptothecin derivatives, gold Wire bird pox IL-2, capecitabine, formamide - amino - triazole, formamide triazole, CaRest M3, CARN 700, cartilage derived inhibitor Kazhe to new, casein kinase inhibitor (ICOS), millet spermine, cecropin B, cetrorelix, chlorine quinoxaline sulfa drugs, Sika prostaglandin, cis - porphyrins, cladribine, Clomiphene analogues, clotrimazole, collismycin A, collismycin B, wind suppression alkali car A4, Wind base analogues inhibit the car, conagenin, crambescidin 816, crisnatol, Nostoc cyclic peptide (Cryptophycin) 8, Nostoc cyclic peptide A derivatives, curacin A, ring five anthraquinone (Cyclopentanthraquinones), cycloplatam, cypemycin, cytosine arabinoside, cell Soluble factor, cytostatin, up to infliximab, decitabine, dehydrogenation film ecteinascidins B, Deslorelin, Dexamethasone, the right ifosfamide, dexrazoxane, Right verapamil, acridine quinones, film ecteinascidins (Didemnin) B, didox, diethylnorspermine, dihydro-5 - aza-cytidine, dihydro- Paclitaxel, dioxamycin, biphenyl Lo Secretary Ting Mo, docetaxel, Docosanol, dolasetron, Doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, according to test Secretary Ting Mo, Yiddish Fuxin, according to decisions Los mAb, eflornithine, Elemene, ethyl acetate for fluorine, epirubicin, according to Li finasteride, estramustine analogues, estrogen antagonist, estrogen antagonist, according to his metronidazole, phosphorus Acid etoposide, exemestane, fadrozole, Fazha La Marina, Fenway A amine, filgrastim, finasteride Finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorunicin Hydrochloric acid, phenol Meike blessing, Formestane, Fotemustine, Germany porphyrin gadolinium gallium nitrate, Gallo gemcitabine, plus Nirui Ke, gelatinase inhibitor, gemcitabine, glutathione inhibitors, hepsulfam, heregulin, Hexamethylene bisacetamide, hypericin, ibandronate, idarubicin, Addo raloxifene, Iraq decided Meng Ketones, Yimo Fu Xin, Yi Luoma Division he, imidazolyl acridinone, imiquimod, immune-stimulating peptides, class Insulin-like growth factor-1 receptor inhibitors, interferon agonists, interferons, interleukins, MIBG, Iodine doxorubicin, sweet alcohol, Elohim Pula, Irsogladine, isobengazole, isohomohalicondrin B, Iraq he granisetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, to filgrastim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibitory Factor, leukocyte interferon α, leuprolide + estrogen + progesterone, leuprolide, levamisole, Liarozole linear polyamine analogues lipophilic two glycopeptides, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lombricine, Lome song Faso, chlorine Nida Ming, loxoprofen anthraquinone, lovastatin, Loxoprofen Li Philippines, Leto irinotecan, Germany porphyrin lutetium, lysofylline, lytic peptides, America tansin, mannostatin A, His horse flew Division, Ma Suoluo phenol, maspin, matrix cracking inhibitors, matrix metalloproteinase inhibition Agents, Miele Li Er, Maier Barron, meterelin, methionine enzymes, metoclopramide, MIF inhibitors, Mifepristone, miltefosine, m vertical Division kiosks, mismatched double-stranded RNA, mitoxantrone guanidine hydrazone, dibromo-dulcitol, Mitomycin analogues, mitoxantrone naphthylamine, mitomycin (mitotoxin) fibroblast growth factor - soap Grass plain, mitoxantrone, Mofaluoting, Mora Secretary kiosks, human chorionic gonadotropin, monophosphoryl lipid A + Binding bacterial cell wall sk, MO piperazine of alcohol, multidrug resistance gene inhibitor, based on multiple tumor suppression Formulation 1 treatment, mustard anticancer agent, Indian sponge (mycaperoxide) B, tuberculosis fine Cell wall extract, myriaporone, N-acetyldinaline, N-substituted benzamide, nafarelin, nagrestip, naloxone + pentazocine, napavin, naphterpin, it asked the Secretary kiosks, nedaplatin, Nanaimo doxorubicin, Chennai stand acid, neutral endopeptidase enzyme Nilutamide, nisamycin, an oxidation Nitrogen modifiers, nitroxide antioxidant, nitrullyn, O6-benzyl guanine, octreotide, okicenone, Oligonucleotides, Iona mifepristone, ondansetron, oracin, oral cytokine inducer, Omar platinum, Osage Trondheim, oxaliplatin, oxaunomycin, paclitaxel, paclitaxel analogues, Paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronate, ginseng alkyne three Alcohol, Pano citrate, Vice bacteria ferritic (parabactin), Pa fold leptin, pegaspargase, peldesine, Wood sodium polysulfide, pentostatin, pentrozole, perfluorinated alkyl bromide, training phosphorus amide, erucic alcohol, phenazinomycin, phenyl acetate, phospholipase inhibitors, picibanil preparation, pilocarpine hydrochloride, Pirarubicin, topiramate song Corzine, placetin A, placetin B, plasminogen activator inhibitor, platinum Complex, a platinum compound, a platinum - triamine complex, porfimer sodium, seaweed adriamycin, prednisone, propyl Biacridine ketone, prostaglandin J2, proteasome inhibitors, protein A-based immune modulator, Protein kinase C inhibitor, protein kinase C inhibitors, microalgal, protein tyrosine phospholipase inhibitors, Purine nucleoside inhibitors, purpurin, topiramate Junior acridine (pyrazoloacridine), pyridoxylated blood Myoglobin polyoxyethylene conjugate, raf antagonist, ralititrexd, ramosetron, ras farnesyl protein Transferase inhibitors, ras inhibitors, ras-GAP inhibitor demethylation retelliptine, etidronate Phosphonate rhenium Re 186, Rhizopus factors, nucleic acid enzymes, RII-dimensional amine resin, Luo Gu imine, rohitukine, Romo peptides, Luo quinoline Meike, rubiginone B1, Shafen Ge, saintopin, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetics, semustine, aging derived inhibitor 1, sensor signal transduction Inhibitors, signal transduction regulator, single-chain antigen-binding protein, Xizuo furans Sobuzoxane, boron card Sodium, sodium phenylacetate, solverol, promoting growth factor binding protein, Suo Naming, Spa Fox acid, spicamycin D, Lo Secretary Ting Mo, splenopentin, sponge hormone (spongistatin) 1, dogfish amines, Stem cell inhibitors, stem cell division inhibitors, stipiamide, matrix cracking inhibitors, ultra live Of vasoactive intestinal peptide antagonist, suradista, suramin, Swainsonine, synthetic glycosaminoglycan, he Secretary Ting Mo, tamoxifen methiodide, taurocholic Secretary Ting Mo, paclitaxel, tazarotene, tecogalan Sodium, tegafur, tellurapyrylium, telomerase inhibitors, MO parked for fun, temozolomide, for Teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide, thiocoraline, thioguanine, thrombopoietin, thrombopoietin mimetic, thymus AFP, Epo receptor agonists promote thymus, thymus Tonin, thyroid stimulating hormone, tin ethyl early purpurin, Tirapazamine, titanium dichloride ene, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, Accutane, triacetyl uridine, Qu Li Bin West, Acamprosate, triptorelin, Qu Xi horses, properly Rochon urea, tyrosine kinase inhibitors, tyrosine phosphorylation inhibitor, UBC inhibitors, bestatin Secretary urogenital Dou Yansheng growth inhibitory factor, urokinase receptor antagonist, Vapreotide, variolin B, Vector system, red blood cells, gene therapy, Wei Lalei Suo, veramine, verdins, vinorelbine, vinxaltine, vinxaltine, vitaxin, volt chlorazol, Provenzano Trondheim, folding Nigeria platinum, vitamin C and benzylidene Net Division he Martins esters. Other preferred anti-cancer drugs are 5 - fluorouracil and folinic acid. ...

In more specific embodiment, the present invention also comprise with usefulness of the present invention one or more based on listerial EphA2 vaccine administration with one or more treatment administrations combinations, this treatment is preferably used for treating breast cancer mentioned above, oophoroma, melanoma, prostate cancer, colon cancer and lung cancer such as but not limited to for example disclosed antitumor and anticancer agent in following table 5.

Table 5

Therapeutic agent	Administration	Dosage	The interval
				ADMh (Doxorubicin RDF  and Doxorubicin PFS )	Intravenous	The 1st day 60-75 mg/m 2	21 days intervals
Farmorubine Hydrochloride	Intravenous	In first of each circulation	3-4 week circulation

(Ellence TM)		It 100-120mg/m2Or mean allocation gave at 1 to 8 day that circulates
				Fluorouracil	Intravenous	How administration: 5ml and 10ml bottle (comprise respectively 250 and 500mg fluorouracil)
Docetaxel (docetaxel )	Intravenous	60-100 mg/m in 1 hour2	Per 3 weeks 1 time
				Paclitaxel (PTX )	Intravenous	175 mg/m in 3 hours2	Per 3 weeks 1 time, carry out 4 times (administration after containing the combination chemotherapy of adriamycin)
Citric acid tamosifen (Nolvadex/Nolvadex-D )	Oral (tablet)	20-40mg should be with the dosed administration (morning and evening) that separates more than the dosage of 20 mg	Every day
				The injection Calciumlevofolinate	Intravenous or intramuscular injection	How administration: 350mg bottle	Dosage in the textbook is unclear. PDR3610
Acetic acid luprolide (Leuprorelin )	Single subcutaneous injection	1mg (0.2ml or 20 unit markings)	Every day 1 time
				Flutamide (Eulexin )	Oral (capsule)	250mg (capsule that respectively comprises the 125mg Flutamide)	With 8 hours interval every day 3 times (total every daily dose 750mg)
Nilutamide (Nilandron )	Oral (tablet)	300mg or 150mg (tablet that respectively comprises 50 or 150 mg Nilutamides)	Every day 300mg, totally 30 days, afterwards every day 150mg
				Bicalutamide (Casodex )	Oral (tablet)	50mg (tablet that respectively comprises 50 mg Bicalutamides)	Every day 1 time
Progesterone	Injection	USP in the sesame oil, 50mg/ml
				Ketoconazole (Nizoral )	Creme	According to symptom every day using 2% cremes 1 time or 2 times
Prednisone	Oral (tablet)	According to the disease specific entity of accepting, initial dose can be at 5mg to 60mg every day

		Between change
				Estramustine phosphate sodium (Emcyt )	Oral (capsule)	14mg/kg body weight (being 1 140mg capsule of every 10kg or 22lb body weight)	Every day the dosed administration to separate for 3 times or 4 times
Etoposide or VP-16	Intravenous	The 20mg/ml solution (100mg) of 5ml
				Dacarbazine (DTIC-Dome )	Intravenous	2-4.5mg/knowing	Every day 1 time, totally 10 days. Can repeat with the interval in 4 weeks.
Polifeprosan 20 and BCNU implant (BCNU) (nitroso ureas) (Gliadel )	Place the thin slice of resection cavity	If the size and dimension of resection cavity allows, 8 thin slices, each comprises the BCNU of 7.7mg, total amount 61.6mg
				Cis-platinum	Injection	How administration: the solution of 1 mg/ml in the multiple dose vials of 50ml and 100ml
Mitomycin	Injection	With 5mg and 20mg bottle (comprising 5mg and 20mg mitomycin) supply
				Gemcitabine hydrochloride (Gemzar )	Intravenous	For NSCLC-2, after deliberation plan but also determine one 30 minutes intravenous administration 1000mg/m of optimal planning 4 week plan2-30 minutes intravenous administration 1250mg/m of 3 weeks plan2Gemzar	4 weeks were planned the 1st, 8 and 15 day of circulation in-per 28 days. The 1st day after the Gemzar transfusion with 100 mg/m2Intravenous carries out the 1st and 8 day of cis-platinum administration 3 week plan circulation in-per 21 days. After Gemzar administration in the 1st day with 100mg/m2The dosage intravenous carry out the cis-platinum administration
Carboplatin (Paraplatin )	Intravenous	Single reagent treatment: at the 1st day 360	Per 4 weeks

		mg/m 2Other Rapid Dose Calculation of intravenous administration (continuing 15 minutes and transfusion for more time): regulate suggestion, formula dosage etc. with combined therapy, the dosage of endoxan
				Ifosfamide (Ifex )	Intravenous	Every day 12g/m2	Repeated in continuous 5 days per 3 weeks or after from hematotoxicity, recovering
Topotecan hydrochloride (Hycamtin )	Intravenous	30 minutes every days 15 mg/m2Venous transfusion	Continuous 5 days, since the 1st day of 21 day course for the treatment of

The present invention also comprises and comes destruction of cancer cells with of the present invention based on listerial EphA2 vaccine and radiotherapy combination, and radiotherapy comprises X ray, gamma-rays and other radioactive source. In preferred embodiments, carry out according to radiation or remote radiation beyond the radiotherapy, wherein radioactive ray are from radioactive source at a distance. In other preferred embodiment, radiotherapy gives with internal therapentics or short distance treatment, wherein the radioactivity source is placed in the body place near cancer cell or tumour material.

In specific embodiments, method of the present invention comprise with usefulness EphA2 vaccine administration of the present invention with make up with one or more anti-inflammatory reagent administrations. Any anti-inflammatory reagent comprises the reagent well known to those skilled in the art that is used for the treatment of inflammatory conditions, all can be used for the compositions and methods of the invention. The non-limitative example of anti-inflammatory reagent comprises nonsteroid anti-inflammatory drugs (NASID), SAID, anticholinergic agent (for example atropine sulfate, atropine methyl nitrate and Ipratropium Bromide (ATROVENT^TM)), β2agonists (salbutamol (VENTOLIN for example^TMAnd PROVENTIL^TM), bitolterol (TORNALATE^TM)、levalbuterol(XOPONEX ^TM), orciprenaline (ALUPENT^TM), pirbuterol (MAXAIR^TM)terbutlaine (BRETHAIRE ^TMAnd BRETHINE^TM), salbutamol (PROVENTIL^TM、 REPETABS ^TMAnd VOLMAX^TM), Formoterol (FORADIL AEROLIZER^TM) and salmeterol (SEREVENT^TMWith SEREVENT DISKUS^TM)) and methyl xanthine (theophylline (UNIPHYL for example^TM、THEO-DUR ^TM、SLO-BID ^TMAnd TEHO-42^TM)). The example of NSAID includes but not limited to aspirin, brufen, Celecoxib (CELEBREX^TM), Diclofenac (VOLTAREN^TM), Etodolac (LODINE^TM), fenoprofen (NALFON^TM), Indomethacin (INDOCIN^TM)、ketoralac(TORADOL ^TM), olsapozine (DAYPRO^TM)、nabumentone(RELAFEN ^TM), sulindac (CLINORIL^TM), tolmetin (TOLECTIN^TM), rofecoxib (VIOXX^TM), naproxen (ALEVE^TM、NAPROSYN ^TM), Ketoprofen (ACTRON^TM) and Nabumetone (RELAFEN^TM). These NSAID are by suppressing cyclooxygenase (for example COX-1 and/or COX-2) performance function. The example of SAID includes but not limited to glucocorticoid, dexamethasone (DECADRON^TM), corticosteroid (methylprednisolone (MEDROL for example^TM)), cortisone, hydrocortisone, prednisone (PREDNISONE^TMAnd DELTASONE^TM), prednisolone (pRELONE^TMAnd PEDIAPRED^TM), fluoxyprednisolone, willow nitrogen sulphur be than the inhibitor (for example prostaglandin, thromboxan and leukotrienes (seeing following table 6 about the non-limitative example of leukotriene and the exemplary dosage of these reagent)) of pyridine and eicosanoid.

In certain embodiments, anti-inflammatory reagent is for the reagent that prevents, controls, treats, improves asthma or its one or more symptoms. The non-limitative example of these reagent comprises adrenotrophin (catecholamine (adrenaline for example for example, isoprel and dilabron), Resorcino (orciprenaline for example, Terbutaline and fenoterol), and salicoside (for example salbutamol)), AC, blucocorticoids, corticosteroid (beclomethadonse for example, budesonide, flunisolide, fluticasone, fluoxyprednisolone, methylprednisolone, prednisolone and prednisone), other steroids, β 2 antagonists (albtuerol for example, bitolterol, fenoterol, dilabron, orciprenaline, pirbuterol, salbutamol, Terbutaline, Formoterol, salmeterol and albutamol Terbutaline), anticholinergic agent (for example Ipratropium Bromide and oxitropium bromide), IL-4 antagonist (comprising antibody), IL-5 antagonist (comprising antibody), IL-13 antagonist (comprising antibody), the PDE4-inhibitor, NF-κ-beta inhibitor, the VLA-4 inhibitor, CpG, anti--CD23, select plain antagonist (TBC 1269), mast cell protease 1 inhibitor (trypsinlike enzyme inhibitors of kinases (GW-45 for example for example, GW-58 and genisteine), phosphatidylinositols-3 ' (PI3) inhibitors of kinases (such as calphostin C) and other inhibitors of kinases (such as staurosporine) (are seen Temkin etc., 2002 J Immunol 169 (5): 2662-2669; Vosseller etc., 1997 Mol.Biol.Cell 8 (5): 909-922; With Nagai etc., 1995 Biochem Biophys Res Commun 208 (2): 576-581)), C3 receptor antagonist (comprising antibody), immunodepressant (for example methotrexate (MTX) and golden salt), loose molecular regulation agent (nasmil (INTAL for example^TM) and Nedocromil Na (TILADE^TM) and mucolytic agent (for example acetyl cysteine)). In specific embodiments, anti-inflammatory reagent is leukotriene inhibitors (montelukast (SINGULAIR for example^TM), zafirlukast (ACCOLATE^TM), Pranlukast (ONON^TM) or Zileuton (ZYFLOTM) (seeing Table 6)).

Table 6. is used for the treatment of the leukotriene inhibitors of asthma

The leukotriene dressing agent	Daily dosage
		Montelukast (SINGULAIRTM)	2-5 year 4mg 6-15 year 5mg 15 years old and above 10mg
Zafirlukast (ACCOLATETM)	5 to 12 years old 10mg b.i.d., 12 years old every day 2 times or above 20mg b.i.d., every day 2 times
		Pranlukast (ONONTM)	Only can use in the Asia
Zyleuton(ZYFLO TM)	12 years old and above 600mg, every day 4 times

Table 7.H1 antihistaminic

Chemical classes and represent medicine	Daily dosage
		The monoethanolamine diphenhydramine	Every 4-6 hour 25-50mg

Clemastine	Per 12 hours 0.34-2.68mg
		The ethylenediamine Tripelennamine	Every 4-6 hour 25-50mg
Alkylamine Brompheniramine chlorphenamine triprolidine (1.25mg/5ml)	Every 4-6 hour 4mg; Or the every 8-12 hour every 4-6 of 8-12mg SR form hour 4mg; Or the every 8-12 hour every 4-6 of 8-12mg SR form hour 2.5mg
		The phenothiazine fenazil	25mg before the sleep
The piperazine hydroxyzine	Every 6-8 hour 25mg
		Piperidines astemizole (non-sedating) azatadine cetirizine Cyproheptadine fexofenadine (non-sedating) loratadine (non-sedating)	Per 24 hours 10mg of per 12 hours 60mg of 10mg/ days per 12 hours 1-2mg 10mg/ days every 6-8 hour 4mg

The treatment for the treatment of of cancer and the excessive proliferative cell illness except cancer and the usage of their dosage, method of administration and recommendation are well known by persons skilled in the art, and such as Physician ' s Desk Reference (the 56th edition, 2002, the 57th edition, 2003, with the 58th edition, 2004) in the document explanation is arranged.

5.5. BA

Toxicity and the effect that prevents and/or treats scheme of the present invention can be determined in animal used as test by the standard pharmaceutical step, for example determines LD₅₀(lethal dose of population 50%) and ED₅₀(the treatment effective dose of population 50%). Dose ratio between toxic effect and the result for the treatment of is the treatment coefficient, and it can be expressed as ratio LD₅₀/ED ₅₀ The preventing and/or treating property reagent that shows large treatment coefficient is preferred. Although can use the preventing and/or treating property reagent that shows toxic and side effect, but should carefully design delivery system, so that these reagent are navigated to affected tissue site, minimized by the latent lesion of unaffected cell thereby make, thereby reduce side effect.

The data that obtain from zooscopy can be used for formulating the dosage range for the preventing/treating reagent of human body. The dosage of these reagent preferably drops on and comprises ED₅₀And have very low or do not have in the virose circulation composition scope. This dosage can change in this scope according to the formulation of using and used method of administration. For reagent of the present invention, can at first from cell culture assays, estimate the treatment effective dose for any. Can formulate the dosage in animal model, to obtain the circulating plasma concentration range, this scope is included in the IC that determines in the zooscopy₅₀(namely realizing the maximum vaccine that suppresses of half of symptom or the concentration of test compounds). The useful dosage of such Information Availability in determining more accurately human body. Blood plasma level can be measured, for example by the high-efficient liquid phase color spectrometry.

The active anticancer of the treatment that the present invention uses also can be determined with various experimental animal models for cancer research, immunocompetent mouse model for example, such as Balb/c or C57/B1/6 or transgenic mice, its small mouse EphA2 employment EphA2 replaces, be built into the mouse model of the mouse tumor cell system of expressing people EphA2 to it, below the animal model that illustrates in the 6th part or any known in the art and Relevance of Tumor Models for Anticancer Drug Development (1999, Fiebig and Burger compile); Contributions to Oncology (1999, Karger); The Nude Mouse in Oncology Research (1991, Boven and Winograd compile), and Anticancer Drug Development Guide (1997, Teicher compiles) in the animal model (comprising hamster, rabbit etc.) of explanation, these documents at this by intactly with reference to quoting.

Before the human test, can in other suitable animal model, test the compound that is used for the treatment of, this animal model includes but not limited to rat, mouse, chicken, ox, monkey, rabbit, hamster etc., for example above-described animal model. Compound can be used in the suitable clinical testing subsequently.

In addition, any analytical method well known by persons skilled in the art all can be used to estimate combined therapy disclosed in this invention is used for the treatment of or pre-anti-cancer prevent and/or treat effect.

5.6. vaccine combination

Composition of the present invention comprises for the preparation of the bulk drug composition of non-Pharmaceutical composition (for example impure or non-sterile composition) with for the preparation of the Pharmaceutical composition of unit dosage forms (namely being fit to the composition to individuality or patient's administration). These compositions comprise prevention or the preventing and/or treating property reagent disclosed in this invention for the treatment of effective dose or the combination of these reagent and pharmaceutically acceptable carrier. Preferably, composition of the present invention comprise the prevention or the treatment effective dose of the present invention one or more based on listerial EphA2 vaccine. Listeria and the pharmaceutically acceptable carrier that comprises one or more expression EphA2 antigenic peptide based on listerial EphA2 vaccine of the present invention.

In specific embodiments, composition of the present invention comprises based on listerial EphA2 vaccine and other prevention or therapeutic agent, for example antitumor and anticancer agent. According to this embodiment, said composition can further comprise pharmaceutically acceptable carrier.

In specific embodiments, term " pharmaceutically acceptable " refers to pass through federation or state government's approved by management or list in American Pharmacopeia or other universally recognized pharmacopeia, can be used for animal, particularly people. Term " carrier " refer to diluent, adjuvant (for example Freund ' s adjuvant (completely with incomplete) or preferred can be from Chiron, the MF59C.1 adjuvant that Emeryville, CA obtain), excipient or with the excipient of therapeutic agent administration. These pharmaceutical carriers can be sterile liquids, and for example water or oil comprise oil, animal, plant or synthetic oil of originating, for example peanut oil, soya-bean oil, mineral oil, sesame wet goods. When pharmaceutical composition was intravenous administration, water was preferred carrier. Salting liquid and aqueous dextrose and glycerite also can be used as liquid-carrier, are used for especially Injectable solution. Suitable materia medica excipient comprises starch, glucose, lactose, sucrose, gel, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, odium stearate, glycerol monostearate, mica, sodium chloride, drying defatted milk, glycerine, propylene, ethylene glycol, water, ethanol etc. If necessary, said composition can also comprise a small amount of wetting agent or emulsifying agent or pH buffer. These compositions can be the forms such as solution, suspending agent, emulsion, tablet, pill, capsule, pulvis, sustained release agent.

Usually, the composition of composition of the present invention with unit dosage forms individually or mix and provide, for example at airtight container, such as mark in the ampulla or sachette of amount of active agent, provide as dry freeze-dried powder or water-free concentrate. If said composition will by the transfusion administration, be dispersed in it in infusion bottle that contains aseptic pharmaceutical grade water or salting liquid. If said composition is by drug administration by injection, provide the ampulla that contains aseptic injection water or salting liquid so that described composition can mix before injection.

Composition of the present invention can be mixed with the form of neutrality or salt. Pharmaceutically acceptable salt comprises the salt with anion, such as the salt derived from hydrochloric acid, phosphoric acid, acetic acid, creeping oxalis, tartaric acid etc.; And with cationic salt, such as the salt derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine etc.

Various delivery systems are known, and can be used to give of the present invention based on listerial EphA2 vaccine, or EphA2 vaccine of the present invention be used for prevention or the prevention for the treatment of cancer or the combination of therapeutic agent, this delivery system (is seen for example Wu and Wu such as the inclusion enclave, microparticle, microcapsule, the recombinant cell that can express the EphA2 antigenic peptide, the receptor mediated endocytosis that are wrapped in the liposome, 1987, Biol.Chem.262:4429-4432), as structure of the nucleic acid of the part of retrovirus or other carrier etc. Give based on listerial EphA2 vaccine, or the method for the combination based on listerial EphA2 vaccine and prevention or therapeutic agent of the present invention includes but not limited to: parenteral (for example in intracutaneous, the muscle, in the abdominal cavity, intravenous and subcutaneous), epidural administration and mucosa delivery (for example nose is interior, suction and oral cavity route). In specific embodiments, of the present invention based on listerial EphA2 vaccine, or the combination of EphA2 vaccine of the present invention and prevention or therapeutic agent by in the muscle, intravenous or subcutaneous administration. Of the present invention based on listerial EphA2 vaccine, or combination based on listerial EphA2 vaccine and prevention or therapeutic agent of the present invention can be by any classical pathway administration, for example by inculcating or fast injection, absorb (such as oral mucosa, rectum and mucous membrane of small intestine etc.) administration by epithelium or mucocutaneous layer, and can be with other BA reagent administration. Administration can be system or local.

In specific embodiments, give partly of the present inventionly based on listerial EphA2 vaccine on the zone of needs treatments, or combination based on listerial EphA2 vaccine and prevention of the present invention or therapeutic agent of the present invention may be desirable. This can pass through, such as but not limited to, the part is inculcated, is injected or transplant and carries out. Described transplanting is to transplant porous, atresia or gel rubber material, comprises film such as sialastic film, or fiber.

In another embodiment, of the present invention based on listerial EphA2 vaccine, or combination based on listerial EphA2 vaccine and prevention or therapeutic agent of the present invention can be passed through controlled release or slow-released system administration. In one embodiment, can with pump realize controlled or delay to discharge (see Langer, the same; Sefton, 1987, CRC Crit.Ref Biomed.Eng.14:20; Buchwald etc., 1980, Surgery 88:507; Saudek etc., 1989, N.Engl.J.Med.321:574). In another embodiment, can with polymeric material realize the listerial controlled or court of a feudal ruler slowly-releasing of expression of the present invention EphA2 antigenic peptide put (referring to, Medical Applications of Controlled Release for example, Langer and Wise compile, CRC publishing house, Boca Raton, FL (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball compile, Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol.Sci.Rev. Macromol.Chem.23:61; Also referring to Levy, 1985, Science 228:190; During etc., 1989, Ann.Neurol.25:351; Howard etc., 1989, J.Neurosurg.71:105); U.S. Patent number 5,679,377,5,916,597,5,912,015,5,989,463,5,128,326; International publication number WO 99/15154 and WO 99/20253. The example that is used for the polymer of sustained release preparation includes but not limited to: poly-(2-HEMA), PMMA, polyacrylic acid, poly-(ethene-altogether-vinyl acetate), polymethylacrylic acid, poly-Glycolic acid (PLG), poly-acid anhydrides, poly-(NVP), poly-(vinyl alcohol), polyacrylamide, polyethylene glycol, polyactide (PLA), poly-(lactide-altogether-Glycolic acid) are (PLGA) and poe. In preferred embodiments, the polymer that is used for sustained release preparation is inertia, that can not filter out impurities, stable storing, aseptic and biodegradable. In another embodiment, controlled or delay delivery system can be positioned at the prevention or the treatment target spot near, therefore only need the part of systemic dosage (referring to for example Goodson, see among the Medical Applications of Controlled Release, the same, the 2nd volume, 115-138 page or leaf (1984)).

Controlled release system has discussion in the summary (1990, Science 249:1527-1533) of Langer. Any technology well known by persons skilled in the art all can be used for preparing the sustained release preparation that comprises one or more therapeutic agent of the present invention. Referring to, for example U.S. Patent number 4,526, and 938; International publication number WO 91/05548 and WO 96/20698; Ning etc., 1996, Radiotherapy ﹠ Oncology 39:179-189; Song etc., 1995, PDA Journal of Pharmaceutical Science ﹠ Technology 50:372-397; Cleek etc., 1997, Pro.Int ' l.Symp.Control.Rel.Bioact.Mater.24:853-854; With Lam etc., 1997, Proc.Int ' l.Symp.Control Rel.Bioact.Mater.24:759-760, each document are intactly drawn at this is reference.

5.6.1. preparation

The pharmaceutical composition that the present invention uses can use conventional method to prepare with one or more physiology acceptable carriers or excipient.

Therefore, the Listeria of expression of the present invention EphA2 antigenic peptide and their physiology acceptable salt and solvate can be prepared administration, and this administration is by sucking or be blown into (by mouth or nose) or oral, parenteral or mucous membrane (for example oral cavity, vagina, rectum, hypogloeeis) administration. In preferred embodiments, use topical or systemic parenteral.

For oral administration, can use pharmaceutically acceptable excipient to be prepared into by conventional method based on listerial EphA2 vaccine, the form of tablet or capsule for example, this excipient such as adhesive (for example cornstarch of in advance gel, polyvinylpyrrolidone or hydroxypropyl methylcellulose); Filler (for example lactose, microcrystalline cellulose or calcium monohydrogen phosphate); Lubricant (for example dolomol, mica or silica); Disintegrant (for example potato starch or Sodium Carboxymethyl Starch); Or wetting agent (for example lauryl sodium sulfate). This tablet can use technology well known in the art coated. The liquid preparation that is used for oral administration can be, the form of solution, syrup or suspending agent for example, or they can be dry products, mixed with water or other suitable carrier before use. These liquid preparations can prepare with pharmaceutically acceptable additive by conventional method, this additive such as suspension reagent (for example sorbitol syrups, cellulose derivative or hydrogenation edible fat); Emulsifying agent (for example lecithin or gum arabic); Non-aqueous carrier (for example vegetable oil of apricot kernel oil, grease, ethanol or fractionation); And anticorrisive agent (for example methyl or propyl group-p-hydroxybenzoate or sorbic acid). If suitable, said preparation can also comprise suitable buffer salt, flavor enhancement, colouring agent or sweetener.

The preparation of oral administration can be mixed with suitably and make the reactive compound controlled release.

For oral administration, composition can be mixed with usual manner the form of tablet or lozenge.

For inhalation, prevention or therapeutic agent that the present invention uses can be sent easily by using suitable propellant form with gas atomization from pressurized package or sprayer, and this propellant is suitable other of dicholorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane, carbon dioxide or other for example. In the example of pressurised aerosol, can determine that dosage unit is to send predetermined amount by valve. The capsule and the pencil that are used for inhalant or insufflation can be mixed with such as the capsule of gel and pencil and to contain compound and suitable powdery matrix, for example powder mixture of lactose or starch.

Can be mixed with by injection based on listerial EphA2 vaccine and to carry out parenteral, for example fast injection or inculcate continuously. The preparation that is used for injection can exist with the form of UD, for example in ampulla or in multi-dose container, and is added with anticorrisive agent. Said composition can be the form such as the emulsion in suspending agent, solution or oiliness or the aqueous carrier, and can contain reagent preparation, such as suspending agent, stabilizing agent and/or dispersant. Selectively, described active component can be powder form, and before use with suitable carrier, for example aseptic apirogen water is mixed.

Vaccine of the present invention also can be mixed with rectal compositions, for example suppository or enema,retention, and for example said composition contains conventional suppository bases, such as cocoa butter or other glyceride.

Except above-mentioned preparation, described prevention or therapeutic agent can also be mixed with durative action preparation. Such durative action preparation can be by implanting (for example subcutaneous or muscle is implanted into) or intramuscular injection administration. Therefore, for example, this prevention or therapeutic agent can be prepared together with suitable polymer or hydrophobic material (for example, the emulsion in acceptable oil) or ion exchange resin, or as the slightly soluble derivative, for example slightly soluble salt is prepared.

The present invention also provides and has been packaged in airtight container with of the present invention based on listerial EphA2 vaccine, for example indicates in the ampulla or sachette of quantity. In one embodiment, vaccine provides as the dry aseptic freeze-dried powder in the airtight container or without aqueous concentrate, and can again be mixed into suitable concn to individual administration with for example water or salting liquid.

In a preferred embodiment of the invention, preparation and the administration of various chemotherapy, biology treatment/immunization therapy and the hormone therapy reagent that is used in combination with vaccine of the present invention are known in the art, and usually in Physician ' s Desk Reference (the 56th edition, 2002), explanation is arranged. For example, in some particular of the present invention, this reagent can and provide such as the preparation of the method that provides in the table 3.

In other embodiments of the present invention, radiotherapy reagent, for example radio isotope can be used as the liquid in the capsule or gives as beverage is oral. Radio isotope can also be mixed with for intravenous injection. Experienced cancer expert can determine most preferred dosage form and the approach of administration.

In certain embodiments, the Listeria of expression of the present invention EphA2 antigenic peptide is mixed with 1mg/ml, 5mg/ml, 10mg/ml and 25mg/ml is used for intravenous injection, and 5mg/ml, 10mg/ml and 80mg/ml are used for repeatedly subcutaneous administration and intramuscular injection. In other embodiments, the scope of the amount of the Listeria preparation of expression EphA2 antigenic peptide of the present invention is about 1 * 10²CFU/ml is to about 1 * 10¹²CFU/ml, for example 1 * 10²CFU/ml、5×10 ²CFU/ml、1×10 ³ CFU/ml、5×10 ³CFU/ml、1×10 ⁴CFU/ml、5×10 ⁴CFU/ml、1×10 ⁵CFU/ml、5×10 ⁵ CFU/ml、1×10 ⁶CFU/ml、5×10 ⁶CFU/ml、1×10 ⁷CFU/ml、5×10 ⁷CFU/ml、1×10 ⁸ CFU/ml、5×10 ⁸CFU/ml、1×10 ⁹CFU/ml、5×10 ⁹CFU/ml、1×10 ¹⁰CFU/ml、 5×10 ¹⁰CFU/ml、1×10 ¹¹CFU/ml、5×10 ¹¹CFU/ml or 1 * 10¹²CFU/ml。

If necessary, above-mentioned composition may reside in packing or the packaging device, and this device can comprise one or more unit dosage forms that contain described active component. This packing can for example comprise metal or plastic sheeting, for example blister plastic packaging. Packing or packaging device can be enclosed the administration specification.

5.6.2. dosage

The amount of effective composition of the present invention can be determined by the research on standard technology in treatment, prevention or the control of cancer. For example, in the treatment of cancer, prevention or control effectively the dosage based on listerial EphA2 vaccine of the present invention can by with said composition to animal model, for example animal model administration disclosed in this invention or well known by persons skilled in the art is determined. In addition, randomly can carry out analyzed in vitro to help to determine the optimal dose scope.

Preferred effective dose can be determined (for example by clinical testing) based on some factors known to persons of ordinary skill in the art by those of ordinary skill. These factors comprise: the other factors of the accuracy of the disease that treat or prevent, the body weight about symptom, patient, patient's immune state and the pharmaceutical composition that the known reflection of those of ordinary skill gives.

The exact dose that uses in the described preparation also depends on the seriousness of method of administration and cancer, and should decide according to the judgement of medical practitioner and each patient's situation. Effective dose can obtain from the dose-response curve extrapolation from external or animal model test macro.

Based on the listerial dosage in the listerial EphA2 vaccine, this dosage is based on the amount of colony forming unit (c.f.u.) about of the present invention. Usually, in each embodiment, this dosage range is that about 1.0c.f.u./kg is to about 1 * 10¹⁰C.f.u./kg; Approximately 1.0c.f.u./kg is to about 1 * 10⁸C.f.u./kg; About 1 * 10²C.f.u./kg is to about 1 * 10⁸C.f.u./kg; About 1 * 10⁴C.f.u./kg is to about 1 * 10⁸C.f.u./kg. Effective dose can always be extrapolated from the dose-response curve of animal model test macro and be obtained. In some typical embodiments, this dosage range is mouse LD₅₀0.001 times to 10,000 times, 0.01 times to 1,000 times, 0.1 times to 500 times, 0.5 times to 250 times, 1 times to 100 times, 5 times to 50 times. In certain embodiments, this dosage range is mouse LD₅₀0.001 times, 0.01 times, 0.1 times, 0.5 times, 1 times, 5 times, 10 times, 50 times, 100 times, 200 times, 500 times, 1,000 times, 5,000 times, 10,000 times.

For other treatment of cancer reagent to patient's administration, provide the exemplary dosage of various cancer therapeutic agents known in the art in the table 3. In the present invention, some preferred embodiment is included in the combined therapy scheme to be lower than the dosed administration of the dosage that the single reagent administration recommended.

The invention provides with known prevention or therapeutic agent, use than thinking before this to treatment, the control of cancer or improving any method of the low dosed administration of effective dosage. Preferably, the known antitumor and anticancer agent of low dosage and low dosage more of the present invention based on listerial EphA2 vaccine combination medicine-feeding more.

5.7. kit

The invention provides packing or kit, it has comprised one or more container based on listerial EphA2 vaccine or the component based on listerial EphA2 vaccine of the present invention of the present invention has been housed. In addition, one or more other prevention or therapeutic agent that can be used for treating cancer or other excessive proliferative disorders also can be included in this packing or the kit. Randomly, such container can be with the notice of the government department that manufacturing, use or the sale of adjusting medicine or biological product are described, this this department of notice reflection has ratified to be used for manufacturing, use or the sale of human body administration.

The invention provides the kit that can be used for method mentioned above. In one embodiment, this kit comprise of the present invention one or more based on listerial EphA2 vaccine. In another embodiment, this kit further comprises one or more other prevention or therapeutic agent that can be used for treating cancer or other excessive proliferative disorders in one or more container. In other embodiments, this prevention or therapeutic agent are biology or hormone therapy agent.

6, embodiment: treatment and prevention benefit that the cancer of antagonism expression EphA2 is provided based on listerial EphA2 vaccine

Receptor tyrosine kinase EphA2 overexpression optionally in various malignant cell types and tumour. In addition, recent research has identified the T lymphocyte that derives from the patient, and it identifies EphA2. Therefore, EphA2 provides in demand target spot for the active immne treatment. At this, we have disclosed at the facultative intracellular bacteria of Gram-positive, and namely the people EphA2 of ectopic expression can cause in the inoculation animal that the antitumor of antigentic specificity reply in the listeria monocytogenes (Listeria). Crucial antigen presenting cell is infected in the Listeria, and thereby the treatment of cancer effect is provided because it can induce antagonism be encoded effective and strong CD4+ and the CD8+T cell response of antigen. Use attenuation mutant listeria strain bacterial strain, this bacterial strain has been kept the antigen transmission capacity of wild mushroom, but pathogenic in mouse reduced nearly 10,000 times. In order to confirm the effect based on listerial EphA2, Listeria actA-strain construction is become to express born of the same parents outer (ECD) or interior (ICD) domain (actA-hEphA2-ECD or actA-hEphA2-ICD) of born of the same parents of people EphA2. By the Western engram analysis confirmed hEphA2-EX and-expression and secretion of CO from the Listeria. The protective immunity of actA-hEphA2EX has suppressed to express the subcutaneous growth (p=0.0037) of the CT26 cell of total length hEphA2 significantly. In contrast, the mouse that has inoculated parental generation actA bacterial strain generates tumour, and this tumour is suitable with the control mice of using vehicle treated. The protective immunity of carrying out with actA-hEphA2CO has increased the survival rate of the mouse that is excited by RenCA-hEphA2 significantly. Subsequently, use the result for the treatment of of experiment CT26-hEphA2 lung neoplasm model evaluation actA-hEphA2-ECD or actA-hEphA2-ICD. After the tumour cell intravenous was transplanted, the Balb/c mouse was immune with actA, actA-hEphA2EX or actA-hEphA2-ICD. Compare with the contrast (the time-to-live intermediate value of carrier or actA be respectively 19 with 20 days) of coupling, the immunity of carrying out with actA-hEphA2-ECD or actA-hEphA2-ICD prolonged significantly survival (survival intermediate value＞43 day, p=0.0035). Importantly, after the huEphA2 immune mouse of 80 % is survived and implanted to tumour 43 days. In a word, these data acknowledgements the Listeria mediated immunity take the EphA2 tumour antigen as target can provide the antagonism various malignant tumours prevention and result for the treatment of.

6.1. embodiment 1: listerial life cycle

The life cycle that has shown listeria monocytogenes among Figure 1A-1B comprises that endocytosis, phagolysosome cracking and cell are to the diffusing step of cell.

6.2. embodiment 2: the structure of expressing EphA2 and contrast Liszt bacterial strain

6.2.1. background

Carry out the mechanism that heterologous antigen is presented according to the Listeria by MHC I class path, the expression of heterologous gene and new synthetic albumen are secreted into efficient in the kytoplasm of infected (antigen presentation) cell and the startup of CD8+T cell and/or the intensity of activation from bacterium have direct relation. Because the level that the Ag specific T-cells starts and the effect of vaccine have direct relation, so the efficient of heterologous protein expression and secretion is directly related with the intensity of vaccine. Therefore, just start and/or activate with regard to the CD8+T cell response of the EphA2 albumen that is specific to coding, the efficient of optimizing the EphA2 expression and secretion is with will be based on the maximum effect of listerial vaccine.

6.2.2. the preparation of sudden change Liszt bacterial strain

Liszt's bacterial strain is derived from 10403S (Bishop etc., J.Immnol.139:2005 (1987)). Get everything ready by SOE-PCR and equipotential exchange system with existing method (Camilli etc., Mol.Microbiol.8:143 (1993)) and to specify Liszt's bacterial strain of deleting in the gene frame. At Glomski etc., mutant strain LLO L461T (DP-L4017) is described among the J.Cell. Biol.156:1029 (2002), the document is referenced at this. ActA sudden change (DP-L4029) is the DP-L3078 bacterial strain, and this bacterial strain is at Skoble etc., and J.of Cell Biology has description among the 150:527-537 (2000), and it has been become prophage by self-healing, and the document is quoted by complete reference at this. (prophage self-healing has explanation at (Lauer etc., J Bacteriol.184:4177 (2002)) in the U.S. Patent Publication No. 2003/0203472).

In some vaccine, use mutant listeria strain strain (Genbank registration number AL591975 (listeria monocytogenes bacterial strain EGD, complete genome, the fragment 3/12 of internalization element B defective; InlB gene region: nts.97008-98963), quoted by complete reference at this, and/or Genbank registration number NC_003210 (listeria monocytogenes bacterial strain EGD, the complete genome group, the inlB gene region: nts.457008-458963) listed sequence, at this by intactly with reference to quoting). One special actA-inlB-bacterial strain (DP-L4029inlB) is deposited in U.S. typical case culture center (ATCC) on October 3rd, 2003, and the preserving number of appointment is PTA-5562.

6.2.3. cloning vector

Selected heterologous antigen expression cassette molecule construction body is inserted into pPL2 (Lauer etc., J.Bacteriol. 2002) or pAM401 (Wirth etc., J.Bacteriol.165:831-836) in, be modified into polyclone sequence (the AatII small fragment that contains pPL2,171bp), be inserted between tetracycline resistance gene (pAM401-MCS) the interior flat terminal Xba I and Nru I recognition site. Usually, hly promoter and (selecting) signal peptide sequence are inserted between pPL2 or the distinctive KpnI of pAM401-MCS plasmid vector and the Bam HI site. Subsequently with selected EphA2 gene (the modified terminal and C terminal antigenic determinant label of N that comprises sometimes; See following explanation) clone between the distinctive Bam HI of these constructs and the SacI site. The method of using those skilled in the art to commonly use by electroporation, will be incorporated into based on the molecule construction body of pAM401-MCS plasmid vector in the selected also listeria monocytogenes through the lysozyme processing. From the clone that the BHI agar disks (10 μ g/ml) that contains chloramphenicol forms, isolate DNA, verify expection plasmid structure in the transformant of Listeria by restricted enzyme cutting analysis. As mentioned below, the recombinant listeria bacterium that will transform with various heterologous protein expression frame construction bodies based on pAM401-MCS is for detection of the expression and secretion of heterologous protein.

According to method mentioned above (Lauer etc., 2002, J.Bacteriol.184:4177-4186), will be incorporated into based on the heterologous protein expression frame construction body of pPL2 in the genomic tRNAArg gene of selected Liszt's bacterial strain. In brief, at first by electroporation or chemical method pPL2 heterologous protein expression frame construction physique grain is incorporated into (Simon etc., 1983, Bio/Technology 1:784-791) among the e. coli host bacteria strain SM10. Subsequently, will transfer to the selected Listeria from the SM10 that transforms based on the plasmid of pPL2 by engaging. After the aforesaid drug selectivity BHI agar disks that contains every milliliter of 7.5 μ g chloramphenicol and every milliliter of 200 μ g streptomysins is cultivated, go down to posterity by the dish in same composition and to come the selected group of purifying for 3 times. In order to confirm that the pPL2 carrier has been integrated into phage attachment site, select single group and by PCR screening, use the primer pair of forward primer NC16 (5 '-gtcaaaacatacgctcttatc-3 ') (SEQ ID NO:47) and reverse primer PL95 (5 '-acataatcagtccaaagtagatgc-3 ') (SEQ ID NO:48). Have the selected group based on the plasmid of pPL2 in the tRNAArg gene that is incorporated into selected Listeria pnca gene group and produce the diagnostic DNA amplicon of 499bps.

6.2.4. promoter

The heterologous protein expression frame comprises prfA-dependence hly promoter, and this promoters driven coding Liszt bacteriolysin O (LLO) transcribes, and is activated in the microenvironment of infection cell. Use primer shown below pair, by PCR amplification of nucleotide 205586-206000 (414bps) from listeria monocytogenes bacterial strain DP-L4056. The zone of amplification comprises front 28 amino acid of hly promoter and LLO, comprises secA1 signal peptide (the same) and PEST domain. This regional expected sequence of listeria monocytogenes bacterial strain EGD can find (registration number: gi/6802048/ref/NC_003210.1/[16802048]) in GenBank.

Primer pair

Forward (KapnI-LLO nts.1257-1276):

5’CTCT GGTACCTCCTTTGATTAGTATATTC(SEQ ID NO：49)

Oppositely (Bam HI-LLO nts.X-x):

5’CTCT GGATCCATCCGCGTGTTTCTTTTCG(SEQ ID NO：50)

(underscore is the restriction enzyme enzyme recognition site)

According to manufacturer's specification, 422bp pcr amplification clone is advanced among the plasmid vector TOPO (Invitrogen, Carlsbad, CA). Determine the nucleotide sequence of the Listeria specificity base among the pCR-XL-TOPO-hly promoter plasmid clone. Compare with the EGD bacterial strain, 8 nucleotide bases that listeria monocytogenes bacterial strain DP-L4056 is included in prfA frame both sides change. In figure below, shown the hly promoter comparison (being respectively SEQ ID NO:68 and 69) of listeria monocytogenes DP-L4056 and EGD bacterial strain.

Listeria hly DP-L4056 and EGD comparison

Query: Listeria EGD

Subject:DP-L4056 (wild type, Portnoy strain)

                                                                   prfA Box

Query：1   ggtacctcctttgattagtatattcctatcttaaagtgacttttatgttgaggcattaac 60

           ||||||||||||||||||||||||||||||||||||| |||||||||| |||||||||||

Sbjct：1   ggtacctcctttgattagtatattcctatcttaaagttacttttatgtggaggcattaac 6O

Query：61  atttgttaacgacgataaagggacagcaggactagaataaagctataaagcaagcatata 120

           ||||||||| ||||  ||| ||| |||| |||||||||||||||||||||||||||||||

Sbjct：61  atttgttaatgacgtcaaaaggatagcaagactagaataaagctataaagcaagcatata 120

Query：121 atattgcgtttcatctttagaagcgaatttcgccaatattataattatcaaaagagaggg 180

           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct：121 atattgcgtttcatctttagaagcgaatttcgccaatattataattatcaaaagagaggg 180

                                                 Shine-Delgarno     LLO start

Query：181 gtggcaaacggtatttggcattattaggttaaaaaatgtagaaggagagtgaaacccatg 240

           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct：181 gtggcaaacggtatttggcattattaggttaaaaaatgtagaaggagagtgaaacccatg 240

By digesting with Kpn I and Bam HI, can from pCR-XL-TOPO-hly promoter plasmid clone, discharge the 422bp DNA corresponding to hly promoter and secA1 LLO signal peptide, and according to conventional method well known to those skilled in the art it is cloned into pPL2 plasmid vector (Lauer etc., 2002, J.Bact). This plasmid is known as pPL2-hlyP (natural).

6.2.5. EphA2 is cloned and is inserted in the pPL2 carrier to express in selected restructuring listeria monocytogenes bacterial strain

Clone respectively external structure territory (EX2) and kytoplasm (CO) domain of the EphA2 of EphA2 transbilayer helix both sides, be used for being inserted into various pPL2-signal peptide expression construct. Use the gene corresponding with natural mammal sequence or codon, this sequence or codon are optimized to express EphA2 EX2 and CO domain in listeria monocytogenes. In the Listeria, the optimal codon of each in 20 seed amino acids all is used for codon optimized EphA2 EX2 and EphA2 CO. Use the general technology of those skilled in the art, synthesize codon optimized EphA2 EX2 and CO domain by extending overlapping oligonucleotides. Verify the expected sequence of the EphA2 construct that all are synthetic by nucleotide sequencing.

SEQ ID NO:23,21 and 22 has represented respectively primary amino acid sequence and natural and the codon optimized nucleotide sequence of the EX2 domain of EphA2.

SEQ ID NO:34,32 and 33 has represented respectively primary amino acid sequence and natural and the codon optimized nucleotide sequence of the CO domain of EphA2.

In addition, FLAG (Stratagene, La Jolla, CA) and myc antigenic determinant label be inserted into synthetic EphA2 EX2 and amino and the carboxyl terminal of CO gene with frame respectively, thereby can detect with the specific antibody of FLAG and albumen the EphA2 of expression and secretion by the Western engram analysis. Therefore, the protein of expression has following sequential element: NH2-signal peptide-FLAG-EphA2-myc-CO2. Below be FLAG and the amino acid of myc antigenic determinant label and codon optimized nucleotide sequence.

FLAG

5’-GATTATAAAGATGATGATGATAAA(SEQ ID NO：51)

NH2-DYKDDDDK-CO2(SEQ ID NO：52)

Myc

5’-GAACAAAAATTAATTAGTGAAGAAGATTTA(SEQ ID NO：53)

NH2-EQKLISEEDL-CO2(SEQ ID NO：54)

6.2.6. detect synthetic and heterologous protein secretion by the Western engram analysis

Inoculum by Western engram analysis trichloroacetic acid (TCA) precipitation detects the synthetic of EphA2 albumen and from the secretion of various selected recombinant listeria bacterium EphA2 bacterial strains. In brief, be collected in the listerial exponential phase of growing in the BHI culture medium mid-term culture in 50mL taper centrifuge tube, precipitum adds ice-cold TCA so that the bacterial cultures supernatant reaches ultimate density [6%], and cultivates at least 90 minutes on ice or spend the night. By collecting the TCA protein precipitations at 2400 * g in centrifugal 20 minutes at 4 ℃. Subsequently sediment is resuspended in volume and is the comprising among the red TE of 15 μ g/ml phenol peptides of pH8.0 of 300-600 μ l. Dissolving by the facilitated sample. If necessary add NH₄OH regulates sample pH value, until the color pinkiness. By adding the SDS sample loading buffer and preparing all samples in 10 minutes to be used for electrophoresis 90 ℃ of placements. Subsequently in micro-centrifuge tube with 14,000 rpm with centrifugal 5 minutes of sample, collect supernatant and also be stored in-20 ℃. For the Western engram analysis, according to those skilled in the art's common method, the part that 20 μ l are made (is equivalent to 1～4 * 10⁹The nutrient solution of individual bacterium) application of sample is to the SDS-PAGE glue of 4-12%, electrophoresis and with protein delivery to the PDDF film. By vibration under the room temperature in the PBS that contains 5% milk powder 2 hours, preparation was with the transfer membrane of antibody incubation. Antibody carries out following dilution in PBST buffer solution (0.1%Tween 20 among the PBS): the anti-Myc polyclonal antibody of (1) rabbit (ICL laboratories, Newberg, Oregon), and with 1: 10,000 dilution; (2) the anti-FLAG monoclonal antibody of mouse (Stratagene, the same), with 1: 2,000; And the anti-EphA2 of (3) rabbit (carboxyl terminal is specific) polyclonal antibody (sc-924, Santa Cruz Biotechnology company, Santa Cruz, CA). By carrying out second incubation with goat-anti rabbit or anti-mouse antibody with the coupling of horseradish peroxidase, and detect and be exposed to the specific binding that film is estimated antibody and protein target with ECL chemiluminescence analysis kit.

6.2.7. the recombinant listeria bacterium secretion EphA2 by coding various forms EphA2

6.2.7.1. Listeria: [DP-L4029 (actA) or DP-L4017 (LLO L461T) bacterial strain]

Expression cassette construct: LLOss-PEST-CO-EphA2 (SEQ ID NO:35)

Native sequences and the natural secA1 LLO sequence of EphA2 CO domain are carried out gene fusion, and as indicated above, will be inserted at the heterologous antigen expression cassette under the control of Listeria hly promoter between the Knp I and Sac I of pPL2 plasmid. As indicated above, by engaging pPL2-EphA2 plasmid construction body is imported among Listeria bacterial strain DP-L4029 (actA) and the DP-L4017 (L461T LLO). Fig. 2 has shown the Western engram analysis result of the inoculum of the 4029-EphA2 CO of TCA precipitation and 4017-EphA2 CO. This analytical proof make up comprise by the Listeria of the heterologous protein expression frame that forms with secA1 and the corresponding native sequences of EphA2 CO fusion and express a plurality of EphA2 specific fragments, these fragments are less than the expection molecular weight of 52kDa, and this has proved needs to modify expression cassette.

6.2.7.2. Listeria: [DP-L4029 (actA)]

The expression cassette construct:

(the SEQ ID NO:26) that natural LLOss-PEST-FLAG-EX2_EphA2-myc-is codon optimized

(codon optimized) LLOss-PEST-(codon optimized) FLAG-EX2_EphA2-myc (SEQ ID NO:28)

Natural secA1 LLO signal peptide sequence or codon be optimized to be optimized with the secA1 LLO signal peptide sequence of expressing in the Listeria and codon carry out gene fusion with the EphA2 EX2 domain sequence of in the Listeria, expressing, and as indicated above, the heterologous antigen expression cassette under the control of Listeria hly promoter is inserted between the Kpn I and Sac I site of pPL2 plasmid. As mentioned above, by engaging pPL2-EphA2 plasmid construction body is imported among the Listeria bacterial strain DP-L4029 (actA). Fig. 3 has shown the Western engram analysis result of inoculum of the Listeria actA of TCA precipitation, the natural or codon optimized secA1 LLO signal peptide that this Listeria actA coding and codon optimized EphA2 EX2 domain merge. This analytical proof be used in combination the signal peptide sequence of preference codon of codon optimized one-tenth listeria monocytogenes and the total length EphA2 EX2 domain protein that the heterologous protein sequence has given expression to expectation. Only use codon optimized total length EphA2 coded sequence, the expression of total length EphA2 EX2 domain protein is undesirable. When the son that accesses to your password was optimized with the listeria monocytogenes LLO secA1 signal peptide of expressing in listeria monocytogenes, the level of heterologous protein expression (fragment or total length) was the highest.

6.2.7.3. Listeria: [DP-L4029 (actA)]

The expression cassette construct:

Natural LLOss-PEST-(codon optimized) FLAG-EphA2_CO-myc (SEQ ID NO:37)

Codon optimized LLOss-PEST-(codon optimized) FLAG-EphA2_CO-myc (SEQ ID NO:39)

Codon optimized PhoD-(codon optimized) FLAG-EphA2_CO-myc (SEQ ID NO:41)

Natural secA1 LLO signal peptide sequence or codon are optimized with the secA1 LLO signal peptide sequence of expressing in the Listeria, or selectively, codon is optimized and carries out gene fusion with being optimized from the Tat signal peptide of bacillus subtilis phoD gene and codon of expressing in the Listeria with the EphA2 domain sequence of showing in the Listeria, and will be inserted at the heterologous antigen expression cassette under the control of Listeria hly promoter as mentioned above, between the Knp I and Sac I site of pAM401-MCS plasmid. As mentioned above, by electroporation pAM401-EphA2 plasmid construction body is incorporated among the bacterial strain DP-L4029 of Listeria. Fig. 4 has shown the Western engram analysis result of inoculum of the Listeria actA of TCA precipitation, the natural or codon optimized secA1 LLO signal peptide that this Listeria actA coding and codon optimized EphA2 CO domain merge or codon optimized bacillus subtilis phoD Tat signal peptide. This analysis has confirmed to be used in combination the signal peptide sequence of preference codon of codon optimized one-tenth listeria monocytogenes and the total length EphA2 CO domain protein that the heterologous protein sequence has given expression to expectation again. In addition, give expression to the total length EphA2 CO domain protein of expectation from the recombinant listeria bacterium expression and secretion, the codon optimized bacillus subtilis phoD Tat signal peptide that this recombinant listeria bacterium coding and codon optimized EphA2 CO domain merge. This result has confirmed novel and beyond thought discovery, and the signal peptide that namely belongs to from different bacterium can be used for heterologous protein secretion from recombinant listeria bacterium. Only the EphA2 sequence is carried out codon optimizedly, the expression of total length EphA2 CO domain protein is undesirable. When the son that accesses to your password was optimized with the signal peptide of expressing in listeria monocytogenes, the expression of heterologous protein was the highest.

6.2.8. express the structure of the Listeria bacterial strain of AH1/OVA or AH1-A5/OVA

Preparation mutant listeria strain strain, this mutant strain express the clipped form of model antigen ovalbumin (OVA), from the advantage immune epitope (SPSYVYHWF) that is called AH1 (SEQ ID NO:55) and the antigenic determinant AH1-A5 (SPSYAYHQF (SEQ ID NO:56) through changing of mouse colorectal cancer (CT26); Slansky etc., 2000, Immunity, 13:529-538). Use pPL2 integration vector (Lauer etc., J.Bacteriol.184:4177 (2002); U.S. Patent Publication No. 2003/0203472) derive OVA and AH1-A5/OVA recombinant listeria bacterium bacterial strain, this bacterial strain comprise the single copy that is integrated into this genomic nontoxic site, Listeria.

6.2.9. express the listerial structure (DP-L4056) of OVA

At first prepare the antigen presentation frame, this expression cassette by the deletion of merging with the OVA of brachymemma the LLO of hemolysin form, and be included in the pPL2 integration vector (pPL2/LLO-OVA). By PSA (from the bacteriophage of ScottA) the attachment site tRNAArg-attBB ' that pPL2/LLO-OVA is imported to curing listeria monocytogenes bacterial strain DP-L4056, thereby derive Liszt-OVA vaccine strains.

Use pcr amplification to delete the LLO of hemolysin, use with lower bolster and primer:

Source: DP-L4056 genomic DNA

Primer:

Forward (Knp I-LLO nts 1257-1276):

5’-CTCT GGTACCTCCTTTGATTAGTATATTC(SEQ ID NO：57)

(T _m: LLO-spec:52 ℃ is overall: 80 ℃)

Oppositely (Bam HI-XhoI-LLO nts 2811-2792)

5’-CAAT GGATCCCTCGAGATCATAATTTACTTCATCCC

(SEQ ID NO：58)

(T _m: LLO-spec:52 ℃ is overall: 102 ℃)

PCR also is used for using following template and the OVA of primer amplification brachymemma:

Source: from the colibacillary pDP3616 DNA of DP-E3616 (Higgins etc., Mol. Molbiol.31:1631-1641 (1999))

Primer:

Forward (XhoI-NcoI cDNA nts.174-186):

5’-ATTT CTCGAGT CCATGGGGGGTTCTCATCATC

(SEQ ID NO：59)

(T _m: LLO-spec:60 ℃ is overall: 88 ℃)

Oppositely (XhoI-NotI-HindIII):

5’-GGTG CTCGAGT GCGGCCGCAAGCTT

(SEQ ID NO：60)

(T _m: overall: 82 ℃)

A scheme finishing building process comprises at first shears the LLO amplicon with KpnI and BamHI, and the KpnI/BamHI carrier is inserted in the pPL2 carrier (pPL2-LLO). Then shear the OVA amplicon with XhoI and NotI, and be inserted among the pPL2-LLO that sheared with XhoI/NotI. (note: the pPL2 carrier does not comprise any XhoI site; PPD-3616 comprises an XhoI site, and it is present in the design of OVA reverse primer. ) by restricted enzyme cutting analysis (KpnI-LLO-XhoI-OVA-NotI) and sequence verification pPL2/LLO-OVA. By transforming plasmid pPL2/LLO-OVA imported to Escherichia coli, to import and be integrated into by joint subsequently in the Listeria (DP-L4056), fully such as the description (or being integrated in listerial other desirable strain) of Lauer etc.

6.2.10. express the structure of the Listeria bacterial strain of AH1/OVA or AH1-A5/OVA

In order to prepare the Listeria of expressing AH1/OVA or AH1-A5/OVA antigen sequence, at first from the insert of oligonucleotides preparation with antigen, and connect subsequently in carrier pPL2-LLO-OVA (as mentioned above preparation).

Following oligonucleotides is for the preparation of AH1 or AH1-A5 insert:

AH1 antigenic determinant insert (the compatible end of ClaI-PatI):

Top chain oligonucleotides (AH1 Top):

5’-CGATTCCCCTAGTTATGTTTTACCACCAATTTGCTGCA

(SEQ ID NO：61)

End chain oligonucleotides (AH1 Bottom):

5’-GCAAATTGGTGGTAAACATAACTAGGGGAAT

(SEQ ID NO：62)

AH1-A5 antigenic determinant insert (the compatible end of ClaI-AvaII):

The sequence of AH1-A5 antigenic determinant is SPSYAYHQF (SEQ IDNO:56) (5 '-AGT CCA AGT Tat GCA Tat CAT CAA TTT-3 ') (SEQ ID NO:63).

Top: 5 '-CGATAGTCCAAGTTATGCATATCATCAATTTGC (SEQ ID NO:64)

The end: 5 '-GTCGCAAATTGATGATATGCATAACTTGGACTAT (SEQ ID NO:65)

The oligonucleotides of given antigen is mixed in together with equimolar ratio, 95 ℃ of heating 5 minutes. Then allow the oligonucleotide mixture Slow cooling. Subsequently with the oligonucleotides of annealing to being connected with plasmid pPL2-LLO/OVA with the relevant limit enzymic digestion with 200: 1 mol ratio. Can verify the identity of novel constructs by restricted enzyme cutting analysis and/or order-checking.

Plasmid can be imported in the Escherichia coli by transforming subsequently, to import and be integrated into by joint afterwards in the Listeria (DP-L4056), fully such as the description of Lauer etc., or to be integrated in listerial other desirable strain, for example actA^-Mutant strain (DP-L0429), LLO L461T strain (DP-L4017) or actA^-/inlB ^-Strain (DP-L4029inlB).

6.3. embodiment 3: express the generation of the mouse tumor cell system of people EphA2

6.3.1. background

Setting up mouse immune treatment model is used for testing of the present invention based on listerial vaccine. Set up three kinds of mouse tumor cell systems, CT26 mouse colonic cell system, B16F10 mouse black-in tumor cell system and RenCa mouse kidney cell cancerous cell line come high level expression huEphA2 albumen. Carry out the FACS cell classification and analyze to identify the CT26 of high level expression huEphA2, B16F10 and RenCa tumour cell, assemble these cells and also analyze with the Western engram analysis. Further assemble the clone to produce the subclone of high level expression huEphA2 by the FACS cell classification.

6.3.2. the selection of the CT26 mouse colonic cell of high level expression huEphA2

6.3.2.1.Lipofectamine ^TMThe transfection analysis

The rotaring dyeing technology of Application standard and commercial retrievable Lipofectamine^TM, illustrate with the construct transfection CT26 cell that comprises huEphA2 according to the manufacturer.

6.3.2.2. flow cytometry (FACS) is analyzed

Carry out unicellular FACS classification analysis with the CT26 mouse tumor cell of identification high level expression people EphA2 with standard technique.

Fig. 5 has shown representative experiment, has shown the EphA2-3 clone of high level expression people EphA2 albumen.

6.3.2.3. the Western trace of the gathering group of high level expression huEphA2

To drop into capable FACS classification with the gathering groups of the cell of various constructs (comprising the huEphA2 gene) transfection after, go back the Application standard technology and carry out the Western trace, to measure people EphA2 protein expression level in the CT26 cell. Fig. 6 has illustrated the result of representative experiment. Compare with the clone of various tests, the people EphA2 albumen of huEphA2-3 clonal expression highest level is selected for effect research in the body hereinafter described. Assemble further cell and express the subclone of huEphA2 with the production highest level.

6.3.3. the selection of the B16F10 mouse black-in tumor cell of high level expression huEphA2

6.3.3.1. retrovirus transduction

By the retrovirus transduction method people EphA2 is imported in the B16F10 melanoma cells, to set up the clone of this albumen of high level expression.

6.3.3.2. flow cytometry (FACS) is analyzed

With the same to the CT26 cell, by standard technique the B16F10 cell of expressing huEphA2 is carried out unicellular FACS classification analysis, to produce the clone of high level expression huEphA2. Assemble the clone that highest level is expressed huEphA2, and by the Western engram analysis it is done further to detect. Shown representational FACS experiment among Fig. 7, it has shown B16F10 subclone high level expression huEphA2.

6.3.3.3. the Western trace of the gathering group of high level expression huEphA2

After the gathering groups of the cell that comprises the huEphA2 gene that imports by retrovirus transduction is dropped into capable FACS classification, also carry out as mentioned above the Western trace, to measure huEphA2 protein expression level in the B16F10 cell. Further assemble cell and express the subclone of huEphA2 to produce highest level.

6.3.4. the selection of the RenCa mouse kidney cell cancer cell of high level expression huEphA2

6.3.4.1.Lipofectamine ^TMThe transfection analysis

The rotaring dyeing technology of Application standard and commercial retrievable Lipofectamine^TMIllustrate with the construct transfection RenCa cell that comprises huEphA2 according to the manufacturer.

6.3.4.2. flow cytometry (FACS) is analyzed

Carry out unicellular FACS classification analysis with the RenCa clear-cell carcinoma tumour cell of identification high level expression people EphA2 with standard technique.

6.3.4.3. the Western trace of the gathering group of high level expression huEphA2

To after dropping into capable FACS classification with the gathering groups of the cell of the construct transfection of the various huEphA2 of comprising genes, go back the Application standard technology and carry out the Western trace, to measure the level of people EphA2 protein expression in the RenCa cell. Assemble further cell and express the subclone of huEphA2 to produce highest level.

6.3.5. pCDNA4 plasmid transfection 293 cells with coding total length EphA2

The expression cassette construct:

pCDNA4-EphA2

Natural total length EphA2 Gene cloning is advanced among the expression plasmid pCDNA4 (Invitrogen, Carlsbad, CA) based on protokaryon CMV promoter. Fig. 8 has shown with the Western engram analysis result of the cell pyrolysis liquid of 293 cells of pCDNA4-EphA2 plasmid transfection, has confirmed total length EphA2 albumen great expression in mammalian cell.

6.4. embodiment 4: the evaluation of antigen-specific immune response after the vaccine inoculation

Can estimate vaccine of the present invention with method in the various external and bodies. Some analyses comprise to be analyzed the T cells with antigenic specificity of the spleen of vaccinated mouse. For example, by intravenous injection 0.1 LD₅₀The Listeria bacterial strain of expression OVA (or other suitable antigen) come C57B1/6 or Balb/c are inoculated. After inoculation 7 days, spleen is put into ice-cold RPMI 1640 culture mediums and from its preparation single-cell suspension liquid, collected mouse boosting cell (typically, every group of 3 mouse). Selectively, can similarly collect the lymph node of mouse, be prepared into the splenocyte in the hereinafter described analysis of single-cell suspension liquid and replacement. Usually, splenocyte is used for the intravenous of vaccine or the evaluation of intraperitoneal administration, and splenocyte and LNC are interior, subcutaneous for the muscle of vaccine or the evaluation of intradermal administration.

Unless otherwise indicated, all antibody that are used for these embodiment can be from Pharmingen, San Diego, and CA obtains.

6.4.1.ELISPOT analyze:

The Listeria bacterial strain that use has OVA antigen uses the ELISPOT assay to produce the quantity frequency of T cells with antigenic specificity by immunity in mouse model as an example. The T cells with antigenic specificity of estimating is OVA specific C D8+ or LLO specific C D8+ or CD4+T cell. This OVA antigen model evaluation is to the immune response of the allos tumour antigen that is inserted into vaccine, and available any other interested antigen replaces. LLO antigen is the Listeria specific antigen. Estimate specific T-cells by the release that detects cell factor (for example IFN-γ) behind the identification specific antigen. With anti-mouse IFN-γ monoclonal antibody (mAb R4; 5 μ g/ml) in 4 ℃ of coated 96 orifice plates (BD Biosciences, San Jose, CA) based on PVDF that spend the night. Wash plate is also sealed plate 2 hours in room temperature with the complete RPMI of 200 μ l. With every hole 2 * 10⁵Individual cell adds the splenocyte from Mice Inoculated (or nonvaccinated control mice), and cultivates 20 to 22 hours at 37 ℃ under the various peptide concentrations of 0.01 to 10 μ M. The peptide that is used for OVA and LLO is SL8, the MHC I class antigenic determinant of OVA; LLO190, (NEKYAQAYPNVS, Invitrogen), the MHC II class antigenic determinant (Listeria antigen) of Liszt's bacteriolysin O; LLO₂₉₆(VAYGRQVYL), the MHC I class antigenic determinant of Liszt's bacteriolysin O; Or LLO₉₁(GYKDGNEYI), the MHC I class antigenic determinant of Liszt's bacteriolysin O. LLO₁₉₀And LLO₂₉₆Be used for the C57B1/6 model, and LLO₉₁Be used for the Balb/c model. After washing, plate and the secondary biotinylated antibody of IFN-γ specificity (XMG1.2) that is diluted to 0.5 μ g/ml in PBS are cultivated. In incubated at room temperature after 2 hours, wash plate and 37 ℃ with the 1nm gold goat-anti biotin conjugates (GAB-1 that is diluted among the PBS that contains 1%BSA; With dilution in 1: 200; Ted Pella, Redding, CA) cultivated 1 hour. After thoroughly washing, will coil at room temperature and substrate (Silver Contrast agent box; The 30ml/ hole; Ted Pella) cultivates 2 to 10 minutes to form spot. Subsequently with the distilled water wash plate to stop substrate reactions. After with plate air drying, use automation ELISPOT to read plate instrument (CTL, Cleveland, OH) and calculate spot in each hole. For OVA specific T-cells or Listeria specific T-cells, cytokine response is expressed as per 2 * 10⁵The quantity of IFN-γ spot formation cell in the individual splenocyte.

6.4.2. cell within a cell factor staining analysis (ICS):

Be associated with the result of acquisition in the ELISPOT analysis for the quantity of further evaluation antigentic specificity CD8+ or CD4+T cell and with the result, carry out ICS and estimate cell by Flow Cytometry Analysis. The splenocyte that will obtain from inoculation group and control group mice and SL8 (stimulating OVA specific C D8+ cell) or LLO190 (stimulating LLO specific C D4+ cell) cultivated 5 hours in the presence of Brefeldin A (Pharmingen). Brefeldin A suppresses owing to the cytokine secretion that stimulates the T cell to occur. The splenocyte that the MHC I class peptide of nothing to do with is cultivated is with comparing. 20ng/ml PMA (phorbol-12-myristic acid-13-acetic acid, Sigma) and the splenocyte that stimulates of 2 μ g/ml ionomycin (Sigma) as the positive control of IFN-γ and the dyeing of TNF-α intracellular cytokine. In order to detect the expression of intracellular cytokine, cell with FITC-anti--CD4 mAb (RM4-5) and PerCP-be anti--CD8 mAb (53-6.7) dyeing, and fixing and penetrating with Cytofix/CytoPerm solution (Pharmingen), dyeed on ice 30 minutes with the TNF alpha antibody mab (MP6-XT22) of PE coupling and the anti-IFN-γ mab (XMG 1.2) of APC coupling. By flow cytometry (FACScalibur, Becton Dickinson, Mountain View, CA) also analyze the percentage that data are determined the cell of the interior INF-γ of expression born of the same parents and/or TNF-α with CELLQuest software (Becton Dickinson immunocyte number system). Because fluorescence labeling on various antibody all can be identified by FACScalibur, therefore by selecting paintedly have anti-INF-γ or TNF alpha antibody dyeing or both CD8+ and CD4+ to identify suitable cell.

6.4.3. the cytokine-expressing of the splenocyte of irriate:

Can also estimate the cytokine secretion level of the mouse boosting cell of contrast and inoculation C57B1/6 mouse. With SL8 or LLO₁₉₀Stimulated splenocyte 24 hours. With irrelevant peptide HSV-gB²(Invitrogen, SSIEFARL) stimulates with comparing. Collect the supernatant of the cell that stimulates and use elisa assay (eBiosciences, CO) or cytometer beads array kit (Pharmingen) to determine the level of T auxiliary-1 and auxiliary-2 cell factors of T.

6.4.4. the evaluation of cytotoxic t cell activity:

Can be further by external or directly estimate OVA specific C D8+T cell at its cellular cytoxicity activity of Mice Body inner evaluation. The CD8+T cell is identified the also target cell of their correspondences of cracking in the mode of antigentic specificity. Discharge analysis with chromium and measure vitro cytotoxicity. In order in the splenocyte group, to expand the OVA specific T-cells, with postradiation EG7.OVA cell (through the EL-4 of transfection expression OVA tumor cell line, ATCC, Manassas, VA) or 100nM SL8 with the splenocyte of 10: 1 proportional stimulation mouse natural and Listeria-OVA (inner) inoculation. Cultivate after 7 days, 4 hours of standard⁵¹Determine effector cell's cytotoxic activity during Cr-discharge to analyze, analyze use EL-4 cell (ATCC, Manassas, VA) that EG7.OVA or SL8 stimulate as target cell and EL-4 cell itself as negative control. For the activity of distinguishing the NK cell and the activity of T cell, YAC-1 clone (ATCC, Manassas, VA) is determined the activity of NK cell as target spot. The percentage of specificity toxicity is calculated does 100 * (experiment release-spontaneous release)/(maximum release-spontaneous release). There is not effector cell's target cell to determine spontaneous release by cultivation. By determining maximum release with 0.1%Triton X-100 cell lysis. If 20% of spontaneous release＜maximum release, experiment is considered to effective for analysis.

In order to estimate the cells in vivo cytotoxic activity of OVA specific C D8+T cell, be divided into average two parts from the splenocyte of natural C57B1/6 mouse. Use specific peptide for every group, target (SL8) or contrast (HSV-gB²) stimulated 90 minutes at 37 ℃ with 0.5 μ g/ml. Washed cell 3 times in culture medium subsequently is with PBS+0.1%BSA washing 2 times. With every milliliter 1 * 10⁷Individual cell is resuspended in cell among the warm PBS+0.1%BSA (10ml or still less), is used for mark Fluoresceincarboxylic acid diacetic acid amber imines fat (CFSE, Molecular Probes, Eugene, OR). The 5mM CFSE liquid storage of 1.25 μ l is joined in the target cell suspension liquid also by the mixed sample of vibration. In order to control cell suspending liquid, 10 times of dilutions that add the CFSE liquid storage doubly and by vibration mix sample. 37 ℃ of cultured cells 10 minutes. (＞40ml) ice-cold PBS stops dyeing by adding large volume. At room temperature use twice of PBS washed cell and subsequently with its suspension and counting. Each cell suspending liquid is diluted to 50 * 10⁶Every milliliter, tail 100 μ l of each group are mixed and by natural mouse or Mice Inoculated carries out intravascular injection. After 12-24 hour, gather in the crops spleen and amount to 5 * 10 by Flow Cytometry Analysis⁶Individual cell. High (target) and low (contrast) fluorescence peak are counted, and the ratio of two quantity is used for setting up the percentage of target cell cracking. The in vivo cytotoxicity analysis can be estimated the lytic activity of T cells with antigenic specificity, and does not need external again stimulation. In addition, the T cell function in the natural surroundings has been estimated in this analysis.

6.5. embodiment 5: EphA2 efficacy study in the body

6.5.1. background

In order to characterize the GVT of huEphA2, in the mouse of the CT26 tumour cell of having inoculated the ectodomain (ED) of expressing people EphA2, carry out efficacy study. The terminal point of measuring is gross tumor volume and the survival percentage of mouse behind the tumor inoculation. The approach of inoculation is subcutaneous (s.c.) and intravenous (i.v.). HBSS and Listeria administration are in contrast.

6.5.2. contrast inoculation with the Listeria of expressing AH1-A5

With 1 * 10⁵Behind the individual CT26 cell intravenous inoculation 3 days, use 0.1 LD₅₀The Listeria Balb/c mouse (n=5) is carried out immunity. Fig. 9 A has confirmed to treat the survival that immunity has improved the animal of inoculation with the Listeria of expressing AH1-A5. Fig. 9 B has shown an independence but the result of the experiment that is equal to, wherein at postvaccinal the 19th day results of cell and the fixing lung of experiment mice. Carried out the cross assessment of lung nodules yet, confirmed to compare with the control-animal of accepting Listeria contrast, accepting does not have tumour in the lung of test animal of Listeria AH1-A5.

6.5.3. preventative EphA2 inoculation

6.5.3.1. carry out immunity to the effect of CT26-hEphA2 tumor growth and survival with the Listeria of the ECD that expresses huEphA2

Use the CT26 cell mass of the expression huEphA2 that unicellular facs analysis mentioned above produces to carry out preventative research. With transfection the group of every group of 10 Balb/c mouse of the CT26 colon cancer cell of people EphA2 (" CT26-hEphA2 ") subcutaneous vaccination, and the group of every group of 5 mouse of intravenous inoculation. Use 0.1 LD₅₀The Listeria of the ECD of Listeria contrast or expression hEphA2 is carried out immunity with the heavy dose of 200 μ l to mouse. In order to study the subcutaneous vaccination of CD26, use the AH1/A5 Listeria as positive control. Before exciting, the CT26-hEphA2 tumour carried out immunity in 14 days and 4 days. During studying, carry out weekly twice gross tumor volume and measure to determine the antitumous effect of inoculation.

Figure 10 A shown with negative control and compared (^*P=0.0012), the antitumous effect of the subcutaneous vaccination of the CT26 cell of the Listeria of the ECD of expression hEphA2 and expression huEphA2. Its data are summarized as follows table 8:

Inoculation group

Gross tumor volume (mm3±s.e.m.)

P with respect to HBSS

P with respect to the Listeria contrast

	(the 42nd day)
				HBSS	1202.9(±321)	-	0.5528
The Listeria contrast	945.5(±338)	0.5528	-
				Listeria AH1/A5	392.5(±225)	0.0471	0.1895
Listeria-hEphA2-ECD	0.0(±0.0)	0.0012	0.0118

Table 8

Figure 10 B confirmed to compare with negative control (^*P=0.0017), the antitumous effect of the intravenous inoculation of the CT26 cell of the Listeria of the ECD of expression hEphA2 and expression huEphA2. Its data are summarized as follows table 9:

Inoculation group	The survival intermediate value (my god)	P with respect to HBSS	# survivor (the 65th day)
				HBSS	18	-	0
The Listeria contrast	18	0.754	0
				Listeria AH1/A5	＞65	0.0017	5
Listeria-hEphA2-ECD	＞65	0.0017	5

Table 9

Carry out preventative research according to plan hereinafter described. The CT26 cell mass of the expression huEphA2 that is produced by above-mentioned unicellular facs analysis is used in these researchs.

Group: 8 groups, every group of 10 mouse. As shown in the following Table 10, with transfection CT26 colon cancer cell subcutaneous vaccination group 1-4 and the intravenous inoculation group 5-8 of people EphA2:

Treatment group	Every group mouse quantity
		1, contrast-HBSS	10
2, L4029-contrast listeria monocytogenes	10
		3, L4029-EphA2 exFlag-expresses the listeria monocytogenes of the ectodomain of people EphA2	10
4, L4029-AH1 listeria monocytogenes	10
		5, contrast-HBSS	10
6, L4029-contrast listeria monocytogenes	10
		7, L4029-EphA2 exFlag-expresses the listeria monocytogenes of the ectodomain of people EphA2	10
8, L4029-AH1 listeria monocytogenes	10

Table 10

Plan: animal is at the intravenous administration of the reagent as listed above of the 0th day and the 10th day acceptance 200 μ l heavy doses. At the 14th day, under the animal skins or intravenous (experiment lung metastasis model) inoculation by CT26 colon cancer cell (L4029EphA2-exFLAG), the Listeria contrast (L4029) of people EphA2 transfection or comprise Listeria positive control (L4029-AH1) (per 100 μ l volumes 5 * 10 of AH1 albumen⁵Individual cell). Per two weeks measure gross tumor volume (only to subcutaneous vaccination) and measure weekly the weight of animals. Tumour is greater than 2,000mm³Or any animal that has presented ill sign (bow-backed attitude, hypopnea, mobility reduce, lose weight greater than 20% etc.) is sentenced euthanasia humanly. Its data are summarized as follows table 11:

Group	The cell route of inoculation is (among the 100 μ l 5 * 105Individual cell) (the 14th day)	Primary vaccination (the 0th day)	Booster shot (the 10th day)
				1, contrast	Subcutaneous	HBSS	HBSS
2、L4029	Subcutaneous	2×10 7CFU	2×10 7 CFU
				3、L4029 EphA2-   exFlag	Subcutaneous	2×10 7CFU	2×10 7CFU
4、L4029-AH1	Subcutaneous	2×10 7CFU	2×10 7 CFU
				5, contrast	Intravenous	HBSS	HBSS
6、L4029	Intravenous	2×10 7CFU	2×10 7 CFU
				7、L4029 EphA2-   exFlag	Intravenous	2×10 7CFU	2×10 7CFU
8、L4029-AH1	Intravenous	2×10 7CFU	2×10 7CFU

Table 11

In this research, with the Listeria-the huEphA2 inoculation has proved in subcutaneous and experiment lung metastasis model (intravenous) significant GVT is arranged all. In subcutaneous model, make the remarkable reduction of tumor growth and 3 mouse not have tumour. Than contrast Listeria and vehicle treated animal, this is replied also is specific. In experiment lung metastasis model, than vehicle treated group, also confirm to have effect with Listeria huEphA2-exFlag inoculation.

Figure 11 A-11D has shown the result of preventive experiment. Figure 11 A has shown with carrier (HBSS), Listeria (L4029) and Listeria positive (L4029-AH1) compares, and begins from postvaccinal the 21st day significantly to reduce and lasted till the 32nd day with the gross tumor volume of the mouse of the CT26 inoculation of the ECD that expresses huEphA2. Figure 11 B has also shown the result of preventive experiment, shown again with Listeria (L4029) and compared, significantly reduced from postvaccinal the 21st day and lasted till the 32nd day with the gross tumor volume of the mouse of CT26 cell (L4029-EphA2 exFlag) inoculation of the ECD that expresses huEphA2. Figure 11 C has shown the preventative result of study in subcutaneous model, has measured the survival percentage of the mouse behind the CT26 tumor cell inoculation. Compare with all control groups, L4029-EphA2 exFlag group has the most significant survival rate (being represented by triangle). Figure 11 D has shown the Prevention Research in the lung metastasis model, has measured the survival percentage of the mouse behind tumor cell inoculation. Compare with all control groups, L4029-EphA2 exFlag group has the most significant survival rate.

Aforementioned data confirmed with the Listeria of the ECD that expresses hEphA2 carry out preventative immunosupress the CT26-hEphA2 tumour growth and prolonged survival.

6.5.3.2. carry out immunity to the effect of the survival of the mouse that inoculated RenCa-hEphA2 with the Listeria of the ICD that expresses huEphA2

The RenCa cell mass of the expression huEphA2 that use produces and screens by said method (U.S. typical case's culture center, Manassas, VA) carries out preventative research. Group with every group of 10 Balb/c mouse of RenCa clear-cell carcinoma cell (" RenCa-hEphA2 cell ") inoculation of expressing people EphA2. Use 0.1 LD₅₀The Listeria of the ICD of Listeria contrast or expression hEphA2 is carried out immunity with the heavy dose of 200ml to mouse. Before exciting, the RenCa-hEphA2 cell tumour carried out immunity in 18 days and 4 days. During studying, carry out weekly twice gross tumor volume and measure to determine the antitumous effect of inoculation.

Figure 12 has confirmed to compare expression with negative control, the antitumous effect of the subcutaneous vaccination of the RenCa cell of the Listeria of the ICD of hEphA2 and expression huEphA2. Compare with the animal with Listeria inoculation only, in the animal with the Listeria inoculation of the ICD that expresses hEphA2, observe significantly antitumor replying (^*P=0.0079), this can survive to estimate by the prolongation that Kaplan-Meier analyzes.

6.5.4. therapeutic EphA2 inoculation

Utilization is treated research by the CT26 cell mass of the expression huEphA2 that above-mentioned unicellular facs analysis produces.

Followingly carry out representational treatment research:

Group: 6 groups, every group of 10 mouse. As shown in table 12 below, use CT26 mouse colonic cell subcutaneous vaccination group 1-3 and intravenous inoculation group 4-6:

Treatment group	Every group mouse quantity
		1, contrast-HBSS	10
2, L4029-contrast listeria monocytogenes	10
		3, L4029-EphA2 exFlag-expresses the listeria monocytogenes of the ectodomain of people EphA2	10
4, contrast-HBSS	10
		5, L4029-contrast listeria monocytogenes	10
6, L4029-EphA2 exFlag-expresses the listeria monocytogenes of the ectodomain of people EphA2	10

Table 12

Plan: use CT26 colon cancer cell (L4029EphA2 exFLAG), Listeria contrast (L4029 contrast) or carrier (HBSS) (every 100ml volume 5 * 10 by people EphA2 transfection⁵Individual cell) carry out subcutaneous to animal or intravenous (experiment lung metastasis model) inoculation. At cell postvaccinal the 3rd day, the intravenous administration of the above-mentioned listed reagent of animals received 200ml heavy dose, after two weeks, animals received booster shot. Per two weeks measure gross tumor volume (only to subcutaneous vaccination) and measure weekly the weight of animals. Tumour is greater than 2,000mm³Or any animal that has presented ill sign (bow-backed attitude, hypopnea, mobility reduce, lose weight greater than 20% etc.) is sentenced euthanasia humanly. Program overview such as following table 13.

Group	The cell route of inoculation is (among the 100 μ l 5 * 105Individual cell)	Primary vaccination (the 3rd day)	Booster shot (the 17th day)
				1, contrast	Subcutaneous	HBSS	HBSS
2、L4029	Subcutaneous		6×10 6To 2 * 107 CFU					6×10 6To 2 * 107 CFU
				3、L4029   EphA2-exFlag	Subcutaneous		6×10 6To 2 * 107 CFU		6×10 6To 2 * 107CFU
4, contrast	Intravenous	HBSS	HBSS
				5、L4029	Intravenous	6×10 6To 2 * 107 CFU	6×10 6To 2 * 107 CFU
6、L4029   EphA2-exFlag	Intravenous	6×10 6To 2 * 107 CFU	6×10 6To 2 * 107CFU

Table 13

Figure 13 A-13C has shown the result of typical curative test. In Figure 13 A, after inoculation with 7 days interval measurement gross tumor volume. Compare with carrier (HBSS) and Listeria, had the gross tumor volume that significantly reduces at the 14th day and lasted till the 28th day with the mouse of the CT26 inoculation of the ECD that expresses huEphA2. Figure 13 B has shown the mean tumour volume with the mouse of the CT26 cell inoculation that comprises Listeria contrast or huEphA2. Compared with the control, the mouse with the CT26 cell inoculation of expressing huEphA2 has the mean tumour volume that reduces. Figure 13 C has shown the result of the treatment research of using the lung metastasis model, has measured mouse with HBSS or Listeria contrast or express the survival percentage of listerial CT26 cell postvaccinal mouse of the ECD of huEphA2. Compared with the control, the animal that inoculates with the listerial CT26 cell (being represented by triangle) of the ECD that expresses huEphA2 has shown the survival rate of higher percentage.

Another research in, with transfection the CT26 colon cancer cell of people EphA2 (" CT26-hEphA2 ") group of every group of 10 Balb/c mouse is carried out subcutaneous or intravenous inoculation. Use 0.1 LD₅₀The Listeria of the ICD of the contrast of actA Listeria or expression hEphA2 is carried out immunity with the heavy dose of 200 μ l to mouse. In a scheme, after the subcutaneous vaccination of CT26-hEphA2 tumour, carried out immunity in 6 days and 14 days. In another program, behind CT26-hEphA2 tumour intravenous inoculation, carried out immunity in 3 days and 14 days. Determine antitumous effect by semiweekly measurement of tumor value and survival rate.

In the animal with Listeria-hEphA2 inoculation, observe significant antitumous effect (p=0.0035).

Figure 14 A has illustrated the measurement of tumor value of the animal after the immunity. These data are summarized as follows table 14:

Inoculation group	Gross tumor volume (mm3± s.e.m.) (the 21st day)	P with respect to HBSS	P with respect to the Listeria contrast
				HBSS	1827(±518)	-	0.961
The Listeria contrast	1799(±267)	0.961	-
				Listeria AH1/A5	0	0.0005	0.000003
Listeria-hEphA2-ICD-1	694(±232)	0.0054	0.006
				Listeria-hEphA2-ICD-2	731(±176)	0.052	0.004

Table 14

Figure 14 B has illustrated the time-to-live of the animal after the immunity. These data are summarized as follows table 15:

Inoculation group	Survive whole intermediate value (my god)	P with respect to HBSS	# survivor (the 65th day)
				HBSS	19	-	0
The Listeria contrast	20	Ns	0
				Listeria-hEphA2-ICD-1	＞65	0.0035	3
Listeria-hEphA2-ICD-2	＞65	0.0035	4
				Listeria-hEphA2-ICD-3	＞65	0.0035	4

Table 15

Recombinant listeria bacterium with coding OVA.AH1 (MMTV gp70 advantage immune epitope) or OVA.AH1-A5 (MMTV gp70 advantage immune epitope has the irregular variation that strengthens the φt cell receptor combination) carries out the result (Figure 14 C) that immunity has produced long-term surviving to the Balb/c mouse that suffers from CT26.24 (huEphA2+) lung neoplasm.

EphA2 CO domain is immunogenic by force, and when immune with the recombinant listeria bacterium of coding password EphA2 CO domain sequence that optimize or natural, in the Balb/c mouse that suffers from CT26.24 (huEphA2+) lung neoplasm, observe significant long-term surviving (Figure 14 D).

EphA2 EX2 domain is weak immunity, if and only if during the recombinant listeria bacterium immunity of the codon optimized secA1 signal peptide that merges with coding and codon optimized EphA2 EX2 domain sequence, in the Balb/c mouse that suffers from CT26.24 (huEphA2+) lung neoplasm, observe the survival of prolongation. When the recombinant listeria bacterium immunity of the natural secA1 signal peptide that merges with coding and codon optimized EphA2 EX2 domain sequence, in mouse, do not observe result for the treatment of (Figure 14 E). SecA1 signal peptide and EphA2 EX2 domain sequence that the son that accesses to your password is optimized are desirable, and this has obtained the statistically support of significant therapeutic anti-tumour effect, and is as shown in the table.

Compare by the survival curve that shows among the logarithm level resolution chart 14E, such as the general introduction in the following table 16:

Experimental group	The survival intermediate value (my god)	Conspicuousness (p value) with respect to HBSS group	The secA1/EphA2 EX2 group's natural with respect to actA-conspicuousness (p value)
				HBSS	19	-	-
ActA	20	NS	NS
				The secA1-EphA2 EX2 (natural) that actA-is natural	19	NS	-
The secA1-EphA2 EX2 (codon optimized) that actA-is natural	24	0.0035	NS
				The secA1-EphA2 EX2 (codon optimized) that actA-is codon optimized	37	0.0035	0.0162
The secA1-EphA2 CO (codon optimized) that actA-is natural	＞99	0.0035	0.0015

Table 16

Significantly, although 293 cells of pCDNA4-EphA2 plasmid transfection have produced very high-caliber protein expression, with the pCDNA4-EphA2 plasmid Balb/C mouse that suffers from CT26.24 (huEphA2+) lung neoplasm is carried out immunity and do not observe any therapeutic anti-tumour effect (Figure 14 F).

In order to carry out therapeutic in-vivo tumour research, transplant in female Balb/C mouse with 5 * 105 CT26 cell IV of stably express EphA2. After 3 days, with the mouse randomization and with the recombinant listeria bacterium bacterial strain IV Mice Inoculated of the various EphA2 that encode. (indicate in the drawings) in some cases, mouse is inoculated at front shin flesh with pCDNA4 plasmid or the pCDNA4-EphA2 plasmid of 100 μ g. As positive control, with the recombinant listeria bacterium bacterial strain of coding OVA.AH1 or OVA.AH1-A5 albumen block polymer mouse is carried out the IV inoculation. Mouse was inoculated in the 3rd day and 14 days after tumour cell is implanted. Injected the listerial mouse of Hanks balanced salt solution (HBSS) buffer solution or unmodified as negative control. All experimental group comprise 5 mouse. For survival research, when mouse begins to show any anxiety or dyspneic sign, mouse is put to death.

Above-mentioned data declaration carry out therapeutic immunization with the Listeria of expressing hEphA2 and suppressed the growth of ripe CT26-hEphA2 tumour and prolonged survival.

6.6. embodiment 6: the long term inhibition that excites again rear CT26-hEphA2 tumor growth

After carrying out preventative immunity with the Listeria of the ICD of the hEphA2 that expresses antagonism CT26-hEphA2 tumour or ECD, the Balb/c mouse of failing to form tumour is excited (subcutaneous) again, excited again after first tumour excites 56 days and front primary immune response after carried out at opposite side with CT26 mother cell system and CT26-hEphA2 cell in 60 days. The mouse of age-matched in this experiment with comparing.

Excite again the tumor growth difference (data do not show) that between group, does not show statistical significance with female CT26 cell. But, as shown in Figure 15, with the group of the Listeria inoculation of the ICD that expresses hEphA2 or ECD shown remarkable inhibition to the tumor growth that excites again (^*p＜0.041)。

6.7. embodiment 7: carry out immunity with the Listeria of expressing hEphA2 and cause EphA2 specific C D8+T cell response

Be separated by with the Listeria L461T of born of the same parents' intracellular domain (hEphA2-ICD) of expressing hEphA2 or the Δ act that expresses the ectodomain (hEphA2-ECD) of codon optimized hEphA2 and two weeks ABalb/c mouse (n=3) carried out immunity. Immunity after 6 days is sentenced mouse euthanasia and results and is assembled spleen the last time. Analyze in order to carry out ELISPOT, cell is stimulated external with the P815 cell of expression total length hEphA2 or the cell pyrolysis liquid for preparing from these cells again. P815 mother cell or cell pyrolysis liquid are as negative control. Same hEphA2 Fc fusion irritation cell with restructuring. Use 96 hole spot reading apparatus to measure INF-γ positive spots and form group (SFC).

As shown in Figure 16, observing INF-γ SFC in the splenocyte from Listeria-hEphA2 Mice Inoculated increases. Express the cell of hEphA2 or the stimulation of cell pyrolysis liquid and cause IFN-γ SFC to increase, this has shown EphA2 specific C D8+ and CD4+T cell response. The splenocyte that contrasts in the mouse that inoculates with the Listeria mother cell does not show IFN-γ SFC increase.

6.8. embodiment 8:CD4+ and CD8+T cell response all are that the maximization of hEphA2 antitumous effect is necessary

With 2 * 10⁵Individual CT26-hEphA2 carried out intravenous inoculation at the 0th day to Balb/c mouse (n=10). By removing CD4+ cell and CD8+ cell at the 1st day and the 3rd day injection anti-CD4 of 200 μ g (ATCC hybridoma GK1.5) or anti-CD8 (ATCC hybridoma 2.4-3), this verifies (data do not show) by facs analysis. Use subsequently 0.1 LD₅₀The Listeria L461T that expresses hEphA2 ICD carried out the intravenous immunity and monitored its survival mouse at the 4th day.

As shown in figure 17, CD4+ and CD8+ removal group all do not show at non-T cell and remove antitumor the replying of observing in the animal. Data are summarized as follows table 17:

Inoculation group	The survival intermediate value (my god)	P with respect to HBSS	# survivor (the 67th day)
				HBSS	17	-	0
Listeria-hEphA2-ICD	＞67	＜0.0001	7
				The anti-CD4 of Listeria-hEphA2-ICD+	19	0.03	2
The anti-CD8 of Listeria-hEphA2-ICD+	24	0.0002	0

Table 17

Above-mentioned data declaration CD4+ and CD8+T cell in the optimal inhibition of tumor growth, be essential.

6.9. embodiment 9: carrying out the therapeutic inoculation with the Listeria of expressing people EphA2 ICD, to strengthen CD45+ tumor-infiltrated

After the subcutaneous vaccination of CT26-hEphA2 tumour 6 days, use 0.1 LD₅₀Immunity is carried out to Balb/c mouse (n=3) in the ECD of the contrast of actA-Listeria or expression hEphA2 or the Listeria of ICD. After vaccine inoculation 9 days, the results tumour, and fixing in 10% neutral buffered formalin, be embedded in the paraffin, cut into slices with 4 μ m. Prepare microscopic section from tumor biopsy. Tissue in the section is taken off paraffin and following heavy water to be closed: change 4 times each 5 minutes with dimethylbenzene; Change 2 times each 5 minutes with absolute alcohol; Change 1 time with 95% alcohol, carried out 5 minutes; Change 1 time with 70% alcohol, carried out 5 minutes; Change twice with distilled water.

Use target antigen to reclaim (TAR) solution (DakoCytomation, Carpinteria, CA) and carry out the recovery of steam antigen with the improvement project of manufacturer's explanation at Black and Decker Rice steam engine. Section placed be preheated to the TAR solution that only is lower than boiling temperature and placed 20 minutes. To cut into slices subsequently from TAR solution and to take out and it was at room temperature cooled off 20 minutes, in the PBS analysis buffer, wash twice.

With biotinylated antibody section is dyeed, step is as follows:

Section immersed in the methanol solution of 3% hydrogen peroxide 10 minutes with endogenous peroxidase sealing, carry out subsequently 2 distilled waters and change each 5 minutes. 1 * Tris buffer salt solution (containing 0.01% Tween 20) that 5% bovine serum albumin(BSA) is immersed in section (TBST) at least 30 minutes.

On will cutting into slices after the too much BSA solution removal, make " pond " to be organized as the center, lie in section in the moist case and be provided in the 1% BSA/TBST solution biotinylation rat anti-mouse CD45/B220 (Pharmingen) with dilution in 1: 100. To spend the night under the room temperature in the case that lies in carefully humidity of cutting into slices, dry to prevent histotomy.

Morning next day, use TBST washing slice 2 times, continue 10 minutes for the second time. HRP or the AP of use and the coupling of strepto-biotin at room temperature placed 30 minutes. With TBST washing slice 2 times, with suitable substrate chromogen (to Strep-HRP, using DAB) colour developing. With after the distilled water washing, by in the immersion dyestuff of will cut into slices 2 minutes, with the Mayers haematine section is redyed. Subsequently with the running water flushing section of flowing until water becomes clear, will cut into slices and immerse in the bleaching agent (Scotts replaces running water) 30 seconds, and in running water, again wash. Section merges transparent in gradient alcohol by dimethylbenzene (or dimethylbenzene substituent) heavy water, washing step is as follows: 95% alcohol 1 minute, and 3 replacing absolute alcohols, each 1 minute, and change dimethylbenzene, each 1 minute 4 times.

Mounting glue is added in (for dimethylbenzene, use DPX mounting glue) and the dried overnight of will cutting into slices on the cover glass before observation.

Observe section and obtain picture (Figure 18 A) with the Nikon digital camera at Nikon Eclipse E400. With data based gross tumor volume standardization (Figure 18 B).

The result confirms that the infiltrating lymphocytes of Tumor-assaciated after the therapeutic inoculation has increased.

7. equivalent

By using the normal experiment method, those skilled in the art will recognize that the equivalent that maybe can understand many particular of the present invention described herein. These equivalents tend to be included in the following claim.

All publication, patents and patent applications of mentioning in this manual this with same degree with reference to being referenced in this specification, independently open, patent or patent application are referenced specially and independently as each.

Sequence table

Sequence table

＜110〉Medimmune Inc.

Seles Co., Ltd

＜120〉based on listerial EphA2 vaccine

<130>10271-146

<150>US 60/511,919

<151>2003-10-15

<150>US 60/511,719

<151>2003-10-15

<150>US 60/532,666

<151>2003-12-24

<150>US 60/556,631

<151>2004-03-26

<150>60/615,470

<151>2004-10-01

<150>60/617,544

<151>2004-10-07

<160>72

<170>PatentIn version 3.2

<210>1

<211>3963

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(138)..(3068)

<400>1

attaaggact cggggcagga ggggcagaag ttgcgcgcag gccggcgggc gggagcggac    60

accgaggccg gcgtgcaggc gtgcgggtgt gcgggagccg ggctcggggg gatcggaccg    120

agagcgagaa gcgcggc atg gag ctc cag gca gcc cgc gcc tgc ttc gcc       170

                   Met Glu Leu Gln Ala Ala Arg Ala Cys Phe Ala

                   1               5                   10

ctg ctg tgg ggc tgt gcg ctg gcc gcg gcc gcg gcg gcg cag ggc aag      218

Leu Leu Trp Gly Cys Ala Leu Ala Ala Ala Ala Ala Ala Gln Gly Lys

            15                  20                  25

gaa gtg gta ctg ctg gac ttt gct gca gct gga ggg gag ctc ggc tgg      266

Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp

        30                  35                  40

ctc aca cac ccg tat ggc aaa ggg tgg gac ctg atg cag aac atc atg      314

Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met

    45                  50                  55

aat gac atg ccg atc tac atg tac tcc gtg tgc aac gtg atg tct ggc    362

Asn Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly

60                  65                  70                  75

gac cag gac aac tgg ctc cgc acc aac tgg gtg tac cga gga gag gct    410

Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala

                80                  85                  90

gag cgt atc ttc att gag ctc aag ttt act gta cgt gac tgc aac agc    458

Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser

            95                  100                 105

ttc cct ggt ggc gcc agc tcc tgc aag gag act ttc aac ctc tac tat    506

Phe pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr

        110                 115                 120

gcc gag tcg gac ctg gac tac ggc acc aac ttc cag aag cgc ctg ttc    554

Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe

    125                 130                 135

acc aag att gac acc att gcg ccc gat gag atc acc gtc agc agc gac    602

Thr Lys Ile Asp Thr Ile Ala pro Asp Glu Ile Thr Val Ser Ser Asp

140                 145                 150                 155

ttc gag gca cgc cac gtg aag ctg aac gtg gag gag cgc tcc gtg ggg    650

Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly

                160                 165                 170

ccg ctc acc cgc aaa ggc ttc tac ctg gcc ttc cag gat atc ggt gcc    698

Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala

            175                 180                 185

tgt gtg gcg ctg ctc tcc gtc cgt gtc tac tac aag aag tgc ccc gag    746

Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys pro Glu

        190                 195                 200

ctg ctg cag ggc ctg gcc cac ttc cct gag acc atc gcc ggc tct gat    794

Leu Leu Gln Gly Leu Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp

    205                 210                 215

gca cct tcc ctg gcc act gtg gcc ggc acc tgt gtg gac cat gcc gtg    842

Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val Asp His Ala Val

220                 225                 230                 235

gtg cca ccg ggg ggt gaa gag ccc cgt atg cac tgt gca gtg gat ggc    890

Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys Ala Val Asp Gly

                240                 245                 250

gag tgg ctg gtg ccc att ggg cag tgc ctg tgc cag gca ggc tac gag    938

Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu

            255                 260                 265

aag gtg gag gat gcc tgc cag gcc tgc tcg cct gga ttt ttt aag ttt    986

Lys Val Glu Asp Ala Cys Gln Ala Cys Ser Pro Gly Phe Phe Lys Phe

        270                 275                 280

gag gca tct gag agc ccc tgc ttg gag tgc cct gag cac acg ctg cca    1034

Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro

    285                 290                 295

tcc cct gag ggt gcc acc tcc tgc gag tgt gag gaa ggc ttc ttc cgg    1082

Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg

300                 305                 310                 315

gca cct cag gac cca gcg tcg atg cct tgc aca cga ccc ccc tcc gcc    1130

Ala Pro Gln Asp Pro Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala

                320                 325                 330

cca cac tac ctc aca gcc gtg ggc atg ggt gcc aag gtg gag ctg cgc    1178

pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg

            335                 340                 345

tgg acg ccc cct cag gac agc ggg ggc cgc gag gac att gtc tac agc    1226

Trp Thr Pro Pro Gln Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser

        350                 355                 360

gtc acc tgc gaa cag tgc tgg ccc gag tct ggg gaa tgc ggg ccg tgt    1274

Val Thr Cys Glu Gln Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys

    365                 370                 375

gag gcc agt gtg cgc tac tcg gag cct cct cac gga ctg acc cgc acc    1322

Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr

380                 385                 390                 395

agt gtg aca gtg agc gac ctg gag ccc cac atg aac tac acc ttc acc    1370

Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr

                400                 405                 410

gtg gag gcc cgc aat ggc gtc tca ggc ctg gta acc agc cgc agc ttc    1418

Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe

            415                 420                 425

cgt act gcc agt gtc agc atc aac cag aca gag ccc ccc aag gtg agg    1466

Arg Thr Ala Ser Val Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg

        430                 435                 440

ctg gag ggc cgc agc acc acc tcg ctt agc gtc tcc tgg agc atc ccc    1514

Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser Trp Ser Ile Pro

    445                 450                 455

ccg ccg cag cag agc cga gtg tgg aag tac gag gtc act tac cgc aag    1562

pro Pro Gln Gln Ser Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys

460                 465                 470                 475

aag gga gac tcc aac agc tac aat gtg cgc cgc acc gag ggt ttc tcc    1610

Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser

                480                 485                 490

gtg acc ctg gac gac ctg gcc cca gac acc acc tac ctg gtc cag gtg    1658

Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val

            495                 500                 505

cag gca ctg acg cag gag ggc cag ggg gcc ggc agc aag gtg cac gaa    1706

Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu

        510                 515                 520

ttc cag acg ctg tcc ccg gag gga tct ggc aac ttg gcg gtg att ggc    1754

Phe Gln Thr Leu Ser pro Glu Gly Ser Gly Asn Leu Ala Val Ile Gly

    525                 530                 535

ggc gtg gct gtc ggt gtg gtc ctg ctt ctg gtg ctg gca gga gtt ggc    1802

Gly Val Ala Val Gly Val Val Leu Leu Leu Val Leu Ala Gly Val Gly

540                 545                 550                 555

ttc ttt atc cac cgc agg agg aag aac cag cgt gcc cgc cag tcc ccg    1850

Phe Phe Ile His Arg Arg Arg Lys Asn Gln Arg Ala Arg Gln Ser Pro

                560                 565                 570

gag gac gtt tac ttc tcc aag tca gaa caa ctg aag ccc ctg aag aca    1898

Glu Asp Val Tyr Phe Ser Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr

            575                 580                 585

tac gtg gac ccc cac aca tat gag gac ccc aac cag gct gtg ttg aag    1946

Tyr Val Asp Pro His Thr Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys

        590                 595                 600

ttc act acc gag atc cat cca tcc tgt gtc act cgg cag aag gtg atc    1994

Phe Thr Thr Glu Ile His Pro Ser Cys Val Thr Arg Gln Lys Val Ile

    605                 610                 615

gga gca gga gag ttt ggg gag gtg tac aag ggc atg ctg aag aca tcc    2042

Gly Ala Gly Glu Phe Gly Glu Val Tyr Lys Gly Met Leu Lys Thr Ser

620                 625                 630                 635

tcg ggg aag aag gag gtg ccg gtg gcc atc aag acg ctg aaa gcc ggc    2090

Ser Gly Lys Lys Glu Val Pro Val Ala Ile Lys Thr Leu Lys Ala Gly

                640                 645                 650

tac aca gag aag cag cga gtg gac ttc ctc ggc gag gcc ggc atc atg    2138

Tyr Thr Glu Lys Gln Arg Val Asp Phe Leu Gly Glu Ala Gly Ile Met

            655                 660                 665

ggc cag ttc agc cac cac aac atc atc cgc cta gag ggc gtc atc tcc    2186

Gly Gln Phe Ser His His Asn Ile Ile Arg Leu Glu Gly Val Ile Ser

        670                 675                 680

aaa tac aag ccc atg atg atc atc act gag tac atg gag aat ggg gcc    2234

Lys Tyr Lys Pro Met Met Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala

    685                 690                 695

ctg gac aag ttc ctt cgg gag aag gat ggc gag ttc agc gtg ctg cag    2282

Leu Asp Lys Phe Leu Arg Glu Lys Asp Gly Glu Phe Ser Val Leu Gln

700                 705                 710                 715

ctg gtg ggc atg ctg cgg ggc atc gca gct ggc atg aag tac ctg gcc    2330

Leu Val Gly Met Leu Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ala

                720                 725                 730

aac atg aac tat gtg cac cgt gac ctg gct gcc cgc aac atc ctc gtc    2378

Asn Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val

            735                 740                 745

aac agc aac ctg gtc tgc aag gtg tct gac ttt ggc ctg tcc cgc gtg    2426

Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val

        750                 755                 760

ctg gag gac gac ccc gag gcc acc tac acc acc agt ggc ggc aag atc    2474

Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile

    765                 770                 775

ccc atc cgc tgg acc gcc ccg gag gcc att tcc tac cgg aag ttc acc    2522

Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr

780                 785                 790                 795

tct gcc agc gac gtg tgg agc ttt ggc att gtc atg tgg gag gtg atg      2570

Ser Ala Ser Asp Val Trp Ser Phe Gly Ile Val Met Trp Glu Val Met

                800                 805                 810

acc tat ggc gag cgg ccc tac tgg gag ttg tcc aac cac gag gtg atg      2618

Thr Tyr Gly Glu Arg Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met

            815                 820                 825

aaa gcc atc aat gat ggc ttc cgg ctc ccc aca ccc atg gac tgc ccc      2666

Lys Ala Ile Asn Asp Gly phe Arg Leu Pro Thr Pro Met Asp Cys Pro

        830                 835                 840

tcc gcc atc tac cag ctc atg atg cag tgc tgg cag cag gag cgt gcc      2714

Ser Ala Ile Tyr Gln Leu Met Met Gln Cys Trp Gln Gln Glu Arg Ala

    845                 850                 855

cgc cgc ccc aag ttc gct gac atc gtc agc atc ctg gac aag ctc att      2762

Arg Arg Pro Lys Phe Ala Asp Ile Val Ser Ile Leu Asp Lys Leu Ile

860                 865                 870                 875

cgt gcc cct gac tcc ctc aag acc ctg gct gac ttt gac ccc cgc gtg      2810

Arg Ala Pro Asp Ser Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val

                880                 885                 890

tct atc cgg ctc ccc agc acg agc ggc tcg gag ggg gtg ccc ttc cgc      2858

Ser Ile Arg Leu Pro Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg

            895                 900                 905

acg gtg tcc gag tgg ctg gag tcc atc aag atg cag cag tat acg gag      2906

Thr Val Ser Glu Trp Leu Glu Ser Ile Lys Met Gln Gln Tyr Thr Glu

        910                 915                 920

cac ttc atg gcg gcc ggc tac act gcc atc gag aag gtg gtg cag atg      2954

His Phe Met Ala Ala Gly Tyr Thr Ala Ile Glu Lys Val Val Gln Met

    925                 930                 935

acc aac gac gac atc aag agg att ggg gtg cgg ctg ccc ggc cac cag      3002

Thr Asn Asp Asp Ile Lys Arg Ile Gly Val Arg Leu Pro Gly His Gln

940                 945                 950                 955

aag cgc atc gcc tac agc ctg ctg gga ctc aag gac cag gtg aac act      3050

Lys Arg Ile Ala Tyr Ser Leu Leu Gly Leu Lys Asp Gln Val Asn Thr

                960                 965                 970

gtg ggg atc ccc atc tga gcctcgacag ggcctggagc cccatcggcc             3098

Val Gly Ile Pro Ile

            975

aagaatactt gaagaaacag agtggcctcc ctgctgtgcc atgctgggcc actggggact    3158

ttatttattt ctagttcttt cctccccctg caacttccgc tgaggggtct cggatgacac    3218

cctggcctga actgaggaga tgaccaggga tgctgggctg ggccctcttt ccctgcgaga    3278

cgcacacagc tgagcactta gcaggcaccg ccacgtccca gcatccctgg agcaggagcc    3338

ccgccacagc cttcggacag acatatagga tattcccaag ccgaccttcc ctccgccttc    3398

tcccacatga ggccatctca ggagatggag ggcttggccc agcgccaagt aaacagggta    3458

cctcaagccc catttcctca cactaagagg gcagactgtg aacttgactg ggtgagaccc    3518

aaagcggtcc ctgtccctct agtgccttct ttagaccctc gggccccatc ctcatccctg    3578

actggccaaa cccttgcttt cctgggcctt tgcaagatgc ttggttgtgt tgaggttttt    3638

aaatatatat tttgtacttt gtggagagaa tgtgtgtgtg tggcaggggg ccccgccagg    3698

gctggggaca gagggtgtca aacattcgtg agctggggac tcagggaccg gtgctgcagg    3758

agtgtcctgc ccatgcccca gtcggcccca tctctcatcc ttttggataa gtttctattc    3818

tgtcagtgtt aaagattttg ttttgttgga catttttttc gaatcttaat ttattatttt    3878

ttttatattt attgttagaa aatgacttat ttctgctctg gaataaagtt gcagatgatt    3938

caaaccgaaa aaaaaaaaaa aaaaa                                          3963

<210>2

<211>976

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met Glu Leu Gln Ala Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys

1               5                   10                  15

Ala Leu Ala Ala Ala Ala Ala Ala Gln Gly Lys Glu Val Val Leu Leu

            20                  25                  30

Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr

        35                  40                  45

Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met Asn Asp Met pro Ile

    50                  55                  60

Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp

65                  70                  75                  80

Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile

                85                  90                  95

Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala

            100                 105                 110

Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu

        115                 120                 125

Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe Thr Lys Ile Asp Thr

    130                 135                 140

Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp Phe Glu Ala Arg His

145                 150                 155                 160

Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys

                165                 170                 175

Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala Cys Val Ala Leu Leu

            180                 185                 190

Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gln Gly Leu

        195                 200                 205

Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala

    210                 215                 220

Thr Val Ala Gly Thr Cys Val Asp His Ala Val Val pro Pro Gly Gly

225                 230                 235                 240

Glu Glu Pro Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro

                245                 250                 255

Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu Lys Val Glu Asp Ala

            260                 265                 270

Cys Gln Ala Cys Ser Pro Gly phe phe Lys Phe Glu Ala Ser Glu Ser

        275                 280                 285

Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro Ser pro Glu Gly Ala

    290                 295                 300

Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gln Asp Pro

305                 310                 315                 320

Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr

                325                 330                 335

Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gln

            340                 345                 350

Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Gln

        355                 360                 365

Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg

    370                 375                 380

Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser

385                 390                 395                 400

Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn

                405                 410                 415

Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val

            420                 425                 430

Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser

        435                 440                 445

Thr Thr Ser Leu Ser Val Ser Trp Ser Ile Pro Pro Pro Gln Gln Ser

    450                 455                 460

Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn

465                 470                 475                 480

Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp

                485                 490                 495

Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val Gln Ala Leu Thr Gln

            500                 505                 510

Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu phe Gln Thr Leu Ser

        515                 520                 525

pro Glu Gly Ser Gly Asn Leu Ala Val Ile Gly Gly Val Ala Val Gly

    530                 535                 540

Val Val Leu Leu Leu Val Leu Ala Gly Val Gly Phe Phe Ile His Arg

 545                550                 555                 560

Arg Arg Lys Asn Gln Arg Ala Arg Gln Ser Pro Glu Asp Val Tyr Phe

                565                 570                 575

Ser Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His

            580                 585                 590

Thr Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys Phe Thr Thr Glu Ile

        595                 600                 605

His pro Ser Cys Val Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe

    610                 615                 620

Gly Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu

625                 630                 635                 640

Val Pro Val Ala Ile Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gln

                645                 650                 655

Arg Val Asp Phe Leu Gly Glu Ala Gly Ile Met Gly Gln Phe Ser His

            660                 665                 670

His Asn Ile Ile Arg Leu Glu Gly Val Ile Ser Lys Tyr Lys Pro Met

        675                 680                 685

Met Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu

    690                 695                 700

Arg Glu Lys Asp Gly Glu phe Ser Val Leu Gln Leu Val Gly Met Leu

705                 710                 715                 720

Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val

                725                 730                 735

His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val

            740                 745                 750

Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp pro

        755                 760                 765

Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp Thr

    770                 775                 780

Ala Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val

785                 790                 795                 800

Trp Ser Phe Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg

                805                 810                 815

Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala Ile Asn Asp

            820                 825                 830

Gly Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr Gln

        835                 840                 845

Leu Met Met Gln Cys Trp Gln Gln Glu Arg Ala Arg Arg Pro Lys Phe

    850                 855                 860

Ala Asp Ile Val Ser Ile Leu Asp Lys Leu Ile Arg Ala Pro Asp Ser

865                 870                 875                 880

Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser Ile Arg Leu Pro

                885                 890                 895

Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp

            900                 905                 910

Leu Glu Ser Ile Lys Met Gln Gln Tyr Thr Glu His Phe Met Ala Ala

        915                 920                 925

Gly Tyr Thr Ala Ile Glu Lys Val Val Gln Met Thr Asn Asp Asp Ile

    930                 935                 940

Lys Arg Ile Gly Val Arg Leu Pro Gly His Gln Lys Arg Ile Ala Tyr

945                 950                 955                 960

Ser Leu Leu Gly Leu Lys Asp Gln Val Asn Thr Val Gly Ile Pro Ile

                965                 970                 975

<210>3

<211>12

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>3

Thr Leu Ala Asp Phe Asp Pro Arg Val Pro Arg Thr

1               5                   10

<210>4

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>4

Val Leu Leu Leu Val Leu Ala Gly Val

1               5

<210>5

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>5

Val Leu Ala Gly Val Gly phe Phe Ile

1               5

<210>6

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>6

Ile Met Asn Asp Met Pro Ile Tyr Met

1               5

<210>7

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>7

Ser Leu Leu Gly Leu Lys Asp Gln Val

1               5

<210>8

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>8

Trp Leu Val pro Ile Gly Gln Cys Leu

1               5

<210>9

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>9

Leu Leu Trp Gly Cys Ala Leu Ala Ala

1               5

<210>10

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>10

Gly Leu Thr Arg Thr Ser Val Thr Val

1               5

<210>11

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>11

Asn Leu Tyr Tyr Ala Glu Ser Asp Leu

1               5

<210>12

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>12

Lys Leu Asn Val Glu Glu Arg Ser Val

1               5

<210>13

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>13

Ile Met Gly Gln Phe Ser His His Asn

1               5

<210>14

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>14

Tyr Ser Val Cys Asn Val Met Ser Gly

1               5

<210>15

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>15

Met Gln Asn Ile Met Asn Asp Met Pro

1               5

<210>16

<211>15

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>16

Glu Ala Gly Ile Met Gly Gln Phe Ser His His Asn Ile Ile Arg

1               5                   10                  15

<210>17

<211>13

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>17

Pro Ile Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly

1               5                   10

<210>18

<211>16

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>18

Asp Leu Met Gln Asn Ile Met Asn Asp Met Pro Ile Tyr Met Tyr Ser

1               5                   10                  15

<210>19

<211>3105

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: fusion constructs

<400>19

atgaaaaaaa taatgctagt ttttattaca cttatattag ttagtctacc aattgcgcaa    60

caaactgaag caaaggatgc atctgcattc aataaagaaa attcaatttc atccatggca    120

ccaccagcat ctccgcctgc aagtcctaag acgccaatcg aaaagaaaca cgcggatctc    180

gagctccagg cagcccgcgc ctgcttcgcc ctgctgtggg gctgtgcgct ggccgcggcc    240

gcggcggcgc agggcaagga agtggtactg ctggactttg ctgcagctgg aggggagctc    300

ggctggctca cacacccgta tggcaaaggg tgggacctga tgcagaacat catgaatgac    360

atgccgatct acatgtactc cgtgtgcaac gtgatgtctg gcgaccagga caactggctc    420

cgcaccaact gggtgtaccg aggagaggct gagcgtatct tcattgagct caagtttact    480

gtacgtgact gcaacagctt ccctggtggc gccagctcct gcaaggagac tttcaacctc    540

tactatgccg agtcggacct ggactacggc accaacttcc agaagcgcct gttcaccaag    600

attgacacca ttgcgcccga tgagatcacc gtcagcagcg acttcgaggc acgccacgtg    660

aagctgaacg tggaggagcg ctccgtgggg ccgctcaccc gcaaaggctt ctacctggcc    720

ttccaggata tcggtgcctg tgtggcgctg ctctccgtcc gtgtctacta caagaagtgc    780

cccgagctgc tgcagggcct ggcccacttc cctgagacca tcgccggctc tgatgcacct    840

tccctggcca ctgtggccgg cacctgtgtg gaccatgccg tggtgccacc ggggggtgaa    900

gagccccgta tgcactgtgc agtggatggc gagtggctgg tgcccattgg gcagtgcctg    960

tgccaggcag gctacgagaa ggtggaggat gcctgccagg cctgctcgcc tggatttttt    1020

aagtttgagg catctgagag cccctgcttg gagtgccctg agcacacgct gccatcccct    1080

gagggtgcca cctcctgcga gtgtgaggaa ggcttcttcc gggcacctca ggacccagcg    1140

tcgatgcctt gcacacgacc cccctccgcc ccacactacc tcacagccgt gggcatgggt    1200

gccaaggtgg agctgcgctg gacgccccct caggacagcg ggggccgcga ggacattgtc    1260

tacagcgtca cctgcgaaca gtgctggccc gagtctgggg aatgcgggcc gtgtgaggcc    1320

agtgtgcgct actcggagcc tcctcacgga ctgacccgca ccagtgtgac agtgagcgac    1380

ctggagcccc acatgaacta caccttcacc gtggaggccc gcaatggcgt ctcaggcctg    1440

gtaaccagcc gcagcttccg tactgccagt gtcagcatca accagacaga gccccccaag    1500

gtgaggctgg agggccgcag caccacctcg cttagcgtct cctggagcat ccccccgccg    1560

cagcagagcc gagtgtggaa gtacgaggtc acttaccgca agaagggaga ctccaacagc    1620

tacaatgtgc gccgcaccga gggtttctcc gtgaccctgg acgacctggc cccagacacc    1680

acctacctgg tccaggtgca ggcactgacg caggagggcc agggggccgg cagcagggtg    1740

cacgaattcc agacgctgtc cccggaggga tctggcaact tggcggtgat tggcggcgtg    1800

gctgtcggtg tggtcctgct tctggtgctg gcaggagttg gcttctttat ccaccgcagg    1860

aggaagaacc agcgtgcccg ccagtccccg gaggacgttt acttctccaa gtcagaacaa    1920

ctgaagcccc tgaagacata cgtggacccc cacacatatg aggaccccaa ccaggctgtg    1980

ttgaagttca ctaccgagat ccatccatcc tgtgtcactc ggcagaaggt gatcggagca    2040

ggagagtttg gggaggtgta caagggcatg ctgaagacat cctcggggaa gaaggaggtg    2100

ccggtggcca tcaagacgct gaaagccggc tacacagaga agcagcgagt ggacttcctc    2160

ggcgaggccg gcatcatggg ccagttcagc caccacaaca tcatccgcct agagggcgtc    2220

atctccaaat acaagcccat gatgatcatc actgagtaca tggagaatgg ggccctggac    2280

aagttccttc gggagaagga tggcgagttc agcgtgctgc agctggtggg catgctgcgg    2340

ggcatcgcag ctggcatgaa gtacctggcc aacatgaact atgtgcaccg tgacctggct    2400

gcccgcaaca tcctcgtcaa cagcaacctg gtctgcaagg tgtctgactt tggcctgtcc    2460

cgcgtgctgg aggacgaccc cgaggccacc tacaccacca gtggcggcaa gatccccatc    2520

cgctggaccg ccccggaggc catttcctac cggaagttca cctctgccag cgacgtgtgg    2580

agctttggca ttgtcatgtg ggaggtgatg acctatggcg agcggcccta ctgggagttg    2640

tccaaccacg aggtgatgaa agccatcaat gatggcttcc ggctccccac acccatggac    2700

tgcccctccg ccatctacca gctcatgatg cagtgctggc agcaggagcg tgcccgccgc    2760

cccaagttcg ctgacatcgt cagcatcctg gacaagctca ttcgtgcccc tgactccctc    2820

aagaccctgg ctgactttga cccccgcgtg tctatccggc tccccagcac gagcggctcg    2880

gagggggtgc ccttccgcac ggtgtccgag tggctggagt ccatcaagat gcagcagtat    2940

acggagcact tcatggcggc cggctacact gccatcgaga aggtggtgca gatgaccaac    3000

gacgacatca agaggattgg ggtgcggctg cccggccacc agaagcgcat cgcctacagc    3060

ctgctgggac tcaaggacca ggtgaacact gtggggatcc ccatc                    3105

<210>20

<211>1035

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the fusion of prediction

<400>20

Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu Val Ser Leu

1               5                   10                  15

Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala Ser Ala phe Asn Lys

            20                  25                  30

Glu Asn Ser Ile Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser

        35                  40                  45

Pro Lys Thr pro Ile Glu Lys Lys His Ala Asp Leu Glu Leu Gln Ala

    50                  55                  60

Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys Ala Leu Ala Ala Ala

65                  70                  75                  80

Ala Ala Ala Gln Gly Lys Glu Val Val Lau Leu Asp Phe Ala Ala Ala

                85                  90                  95

Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp

            100                 105                 110

Leu Met Gln Asn Ile Met Asn Asp Met Pro Ile Tyr Met Tyr Ser Val

        115                 120                 125

Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp

    130                 135                 140

Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr

145                 150                 155                 160

Val Arg Asp Cys Asn Ser phe Pro Gly Gly Ala Ser Ser Cys Lys Glu

                165                 170                 175

Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn

            180                 185                 190

Phe Gln Lys Arg Leu Phe Thr Lys Ile Asp Thr Ile Ala Pro Asp Glu

        195                 200                 205

Ile Thr Val Ser Ser Asp Phe Glu Ala Arg His Val Lys Leu Asn Val

    210                 215                 220

Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala

225                 230                 235                 240

phe Gln Asp Ile Gly Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr

                245                 250                 255

Tyr Lys Lys Cys Pro Glu Leu Leu Gln Gly Leu Ala His Phe Pro Glu

            260                 265                 270

Thr Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala Gly Thr

        275                 280                 285

Cys Val Asp His Ala Val Val Pro Pro Gly Gly Glu Glu Pro Arg Met

    290                 295                 300

His Cys Ala Val Asp Gly Glu Trp Leu Val Pro Ile Gly Gln Cys Leu

305                 310                 315                 320

Cys Gln Ala Gly Tyr Glu Lys Val Glu Asp Ala Cys Gln Ala Cys Ser

                325                 330                 335

Pro Gly Phe Phe Lys phe Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys

            340                 345                 350

Pro Glu His Thr Leu Pro Ser pro Glu Gly Ala Thr Ser Cys Glu Cys

        355                 360                 365

Glu Glu Gly Phe Phe Arg Ala Pro Gln Asp Pro Ala Ser Met Pro Cys

    370                 375                 380

Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly Met Gly

385                 390                 395                 400

Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gln Asp Ser Gly Gly Arg

                405                 410                 415

Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Gln Cys Trp Pro Glu Ser

            420                 425                 430

Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro

        435                 440                 445

His Gly Leu Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu Pro His

    450                 455                 460

Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn Gly Val Ser Gly Leu

465                 470                 475                 480

Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val Ser Ile Asn Gln Thr

                485                 490                 495

Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser

            500                 505                 510

Val Ser Trp Ser Ile Pro Pro Pro Gln Gln Ser Arg Val Trp Lys Tyr

        515                 520                 525

Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg

    530                 535                 540

Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro Asp Thr

545                 550                 555                 560

Thr Tyr Leu Val Gln Val Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala

                565                 570                 575

Gly Ser Arg Val His Glu Phe Gln Thr Leu Ser Pro Glu Gly Ser Gly

            580                 585                 590

Asn Leu Ala Val Ile Gly Gly Val Ala Val Gly Val Val Leu Leu Leu

        595                 600                 605

Val Leu Ala Gly Val Gly Phe Phe Ile His Arg Arg Arg Lys Asn Gln

    610                 615                 620

Arg Ala Arg Gln Ser Pro Glu Asp Val Tyr Phe Ser Lys Ser Glu Gln

625                 630                 635                 640

Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His Thr Tyr Glu Asp Pro

                645                 650                 655

Asn Gln Ala Val Leu Lys Phe Thr Thr Glu Ile His Pro Ser Cys Val

            660                 665                 670

Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe Gly Glu Val Tyr Lys

        675                 680                 685

Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu Val Pro Val Ala Ile

    690                 695                 700

Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gln Arg Val Asp Phe Leu

705                 710                 715                 720

Gly Glu Ala Gly Ile Met Gly Gln Phe Ser His His Asn Ile Ile Arg

                725                 730                 735

Leu Glu Gly Val Ile Ser Lys Tyr Lys Pro Met Met Ile Ile Thr Glu

            740                 745                 750

Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu Arg Glu Lys Asp Gly

        755                 760                 765

Glu Phe Ser Val Leu Gln Leu Val Gly Met Leu Arg Gly Ile Ala Ala

    770                 775                 780

Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val His Arg Asp Leu Ala

785                 790                 795                 800

Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp

                805                 810                 815

Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr

            820                 825                 830

Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile

        835                 840                 845

Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val Trp Ser Phe Gly Ile

    850                 855                 860

Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg Pro Tyr Trp Glu Leu

865                 870                 875                 880

Ser Asn His Glu Val Met Lys Ala Ile Asn Asp Gly Phe Arg Leu Pro

                885                 890                 895

Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr Gln Leu Met Met Gln Cys

            900                 905                 910

Trp Gln Gln Glu Arg Ala Arg Arg Pro Lys Phe Ala Asp Ile Val Ser

        915                 920                 925

Ile Leu Asp Lys Leu Ile Arg Ala pro Asp Ser Leu Lys Thr Leu Ala

    930                 935                 940

Asp Phe Asp Pro Arg Val Ser Ile Arg Leu Pro Ser Thr Ser Gly Ser

945                 950                 955                 960

Glu Gly Val pro Phe Arg Thr Val Ser Glu Trp Leu Glu Ser Ile Lys

                965                 970                 975

Met Gln Gln Tyr Thr Glu His Phe Met Ala Ala Gly Tyr Thr Ala Ile

            980                 985                 990

Glu Lys Val Val Gln Met Thr Asn Asp Asp Ile Lys Arg Ile Gly Val

        995                 1000                1005

Arg Leu Pro Gly His Gln Lys Arg Ile Ala Tyr Ser Leu Leu Gly

    1010                1015                1020

Leu Lys Asp Gln Val Asn Thr Val Gly Ile Pro Ile

    1025                1030                1035

<210>21

<211>1506

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>21

cagggcaagg aagtggtact gctggacttt gctgcagctg gaggggagct cggctggctc    60

acacacccgt atggcaaagg gtgggacctg atgcagaaca tcatgaatga catgccgatc    120

tacatgtact ccgtgtgcaa cgtgatgtct ggcgaccagg acaactggct ccgcaccaac    180

tgggtgtacc gaggagaggc tgagcgtatc ttcattgagc tcaagtttac tgtacgtgac    240

tgcaacagct tccctggtgg cgccagctcc tgcaaggaga ctttcaacct ctactatgcc    300

gagtcggacc tggactacgg caccaacttc cagaagcgcc tgttcaccaa gattgacacc    360

attgcgcccg atgagatcac cgtcagcagc gacttcgagg cacgccacgt gaagctgaac    420

gtggaggagc gctccgtggg gccgctcacc cgcaaaggct tctacctggc cttccaggat    480

atcggtgcct gtgtggcgct gctctccgtc cgtgtctact acaagaagtg ccccgagctg    540

ctgcagggcc tggcccactt ccctgagacc atcgccggct ctgatgcacc ttccctggcc    600

actgtggccg gcacctgtgt ggaccatgcc gtggtgccac cggggggtga agagccccgt    660

atgcactgtg cagtggatgg cgagtggctg gtgcccattg ggcagtgcct gtgccaggca    720

ggctacgaga aggtggagga tgcctgccag gcctgctcgc ctggattttt taagtttgag    780

gcatctgaga gcccctgctt ggagtgccct gagcacacgc tgccatcccc tgagggtgcc    840

acctcctgcg agtgtgagga aggcttcttc cgggcacctc aggacccagc gtcgatgcct    900

tgcacacgac ccccctccgc cccacactac ctcacagccg tgggcatggg tgccaaggtg    960

gagctgcgct ggacgccccc tcaggacagc gggggccgcg aggacattgt ctacagcgtc    1020

acctgcgaac agtgctggcc cgagtctggg gaatgcgggc cgtgtgaggc cagtgtgcgc    1080

tactcggagc ctcctcacgg actgacccgc accagtgtga cagtgagcga cctggagccc    1140

cacatgaact acaccttcac cgtggaggcc cgcaatggcg tctcaggcct ggtaaccagc    1200

cgcagcttcc gtactgccag tgtcagcatc aaccagacag agccccccaa ggtgaggctg    1260

gagggccgca gcaccacctc gcttagcgtc tcctggagca tccccccgcc gcagcagagc    1320

cgagtgtgga agtacgaggt cacttaccgc aagaagggag actccaacag ctacaatgtg    1380

cgccgcaccg agggtttctc cgtgaccctg gacgacctgg ccccagacac cacctacctg    1440

gtccaggtgc aggcactgac gcaggagggc cagggggccg gcagcagggt gcacgaattc    1500

cagacg                                                               1506

<210>22

<211>1506

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the human sequence with the employed optimization codon in Listeria

<400>22

caaggtaaag aagttgtttt attagatttt gcagcagcag gtggtgaatt aggttggtta    60

acacatccat atggtaaagg ttgggattta atgcaaaata ttatgaatga tatgccaatt    120

tatatgtata gtgtttgtaa tgttatgagt ggtgatcaag ataattggtt acgtacaaat    180

tgggtttatc gtggtgaagc agaacgtatt tttattgaat taaaatttac agttcgtgat    240

tgtaatagtt ttccaggtgg tgcaagtagt tgtaaagaaa catttaattt atattatgca    300

gaaagtgatt tagattatgg tacaaatttt caaaaacgtt tatttacaaa aattgataca    360

attgcaccag atgaaattac agttagtagt gattttgaag cacgtcatgt taaattaaat    420

gttgaagaac gtagtgttgg tccattaaca cgtaaaggtt tttatttagc atttcaagat    480

attggtgcat gtgttgcatt attaagtgtt cgtgtttatt ataaaaaatg tccagaatta    540

ttacaaggtt tagcacattt tccagaaaca attgcaggta gtgatgcacc aagtttagca    600

acagttgcag gtacatgtgt tgatcatgca gttgttccac caggtggtga agaaccacgt    660

atgcattgtg cagttgatgg tgaatggtta gttccaattg gtcaatgttt atgtcaagca    720

ggttatgaaa aagttgaaga tgcatgtcaa gcatgtagtc caggtttttt taaatttgaa    780

gcaagtgaaa gtccatgttt agaatgtcca gaacatacat taccaagtcc agaaggtgca    840

acaagttgtg aatgtgaaga aggttttttt cgtgcaccac aagatccagc aagtatgcca    900

tgtacacgtc caccaagtgc accacattat ttaacagcag ttggtatggg tgcaaaagtt    960

gaattacgtt ggacaccacc acaagatagt ggtggtcgtg aagatattgt ttatagtgtt    1020

acatgtgaac aatgttggcc agaaagtggt gaatgtggtc catgtgaagc aagtgttcgt    1080

tatagtgaac caccacatgg tttaacacgt acaagtgtta cagttagtga tttagaacca    1140

catatgaatt atacatttac agttgaagca cgtaatggtg ttagtggttt agttacaagt    1200

cgtagttttc gtacagcaag tgttagtatt aatcaaacag aaccaccaaa agttcgttta    1260

gaaggtcgta gtacaacaag tttaagtgtt agttggagta ttccaccacc acaacaaagt    1320

cgtgtttgga aatatgaagt tacatatcgt aaaaaaggtg atagtaatag ttataatgtt    1380

cgtcgtacag aaggttttag tgttacatta gatgatttag caccagatac aacatattta    1440

gttcaagttc aagcattaac acaagaaggt caaggtgcag gtagtcgtgt tcatgaattt    1500

caaaca                                                               1506

<210>23

<211>502

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>23

Gln Gly Lys Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly Glu

1               5                   10                  15

Leu Gly Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gln

            20                  25                  30

Asn Ile Met Asn Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn Val

        35                  40                  45

Met Ser Gly Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg

    50                  55                  60

Gly Glu Ala Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr Val Arg Asp

65                  70                  75                  80

Cys Asn Ser Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn

                85                  90                  95

Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gln Lys

            100                 105                 110

Arg Leu phe Thr Lys Ile Asp Thr Ile Ala Pro Asp Glu Ile Thr Val

        115                 120                 125

Ser Ser Asp Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu Arg

    130                 135                 140

Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala phe Gln Asp

145                 150                 155                 160

Ile Gly Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys

                165                 170                 175

Cys Pro Glu Leu Leu Gln Gly Leu Ala His Phe Pro Glu Thr Ile Ala

            180                 185                 190

Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val Asp

        195                 200                 205

His Ala Val Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys Ala

    210                 215                 220

Val Asp Gly Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln Ala

225                 230                 235                 240

Gly Tyr Glu Lys Val Glu Asp Ala Cys Gln Ala Cys Ser Pro Gly Phe

                245                 250                 255

Phe Lys Phe Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys pro Glu His

            260                 265                 270

Thr Leu pro Ser pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu Gly

        275                 280                 285

Phe Phe Arg Ala Pro Gln Asp Pro Ala Ser Met Pro Cys Thr Arg Pro

    290                 295                 300

Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys Val

305                 310                 315                 320

Glu Leu Arg Trp Thr Pro Pro Gln Asp Ser Gly Gly Arg Glu Asp Ile

                325                 330                 335

Val Tyr Ser Val Thr Cys Glu Gln Cys Trp Pro Glu Ser Gly Glu Cys

            340                 345                 350

Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly Leu

        355                 360                 365

Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr

    370                 375                 380

Thr Phe Thr Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr Ser

385                 390                 395                 400

Arg Ser phe Arg Thr Ala Ser Val Ser Ile Asn Gln Thr Glu Pro Pro

                405                 410                 415

Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser Trp

            420                 425                 430

Ser Ile pro Pro pro Gln Gln Ser Arg Val Trp Lys Tyr Glu Val Thr

        435                 440                 445

Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr Glu

    450                 455                 460

Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu

465                 470                 475                 480

Val Gln Val Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser Arg

                485                 490                 495

Val His Glu Phe Gln Thr

            500

<210>24

<211>1689

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: fusion protein construct

<400>24

atgaaaaaaa taatgctagt ttttattaca cttatattag ttagtctacc aattgcgcaa    60

caaactgaag caaaggatgc atctgcattc aataaagaaa attcaatttc atccatggca    120

ccaccagcat ctccgcctgc aagtcctaag acgccaatcg aaaagaaaca cgcggatctc    180

gagcagggca aggaagtggt actgctggac tttgctgcag ctggagggga gctcggctgg    240

ctcacacacc cgtatggcaa agggtgggac ctgatgcaga acatcatgaa tgacatgccg    300

atctacatgt actccgtgtg caacgtgatg tctggcgacc aggacaactg gctccgcacc    360

aactgggtgt accgaggaga ggctgagcgt atcttcattg agctcaagtt tactgtacgt    420

gactgcaaca gcttccctgg tggcgccagc tcctgcaagg agactttcaa cctctactat    480

gccgagtcgg acctggacta cggcaccaac ttccagaagc gcctgttcac caagattgac    540

accattgcgc ccgatgagat caccgtcagc agcgacttcg aggcacgcca cgtgaagctg    600

aacgtggagg agcgctccgt ggggccgctc acccgcaaag gcttctacct ggccttccag    660

gatatcggtg cctgtgtggc gctgctctcc gtccgtgtct actacaagaa gtgccccgag    720

ctgctgcagg gcctggccca cttccctgag accatcgccg gctctgatgc accttccctg    780

gccactgtgg ccggcacctg tgtggaccat gccgtggtgc caccgggggg tgaagagccc    840

cgtatgcact gtgcagtgga tggcgagtgg ctggtgccca ttgggcagtg cctgtgccag    900

gcaggctacg agaaggtgga ggatgcctgc caggcctgct cgcctggatt ttttaagttt    960

gaggcatctg agagcccctg cttggagtgc cctgagcaca cgctgccatc ccctgagggt    1020

gccacctcct gcgagtgtga ggaaggcttc ttccgggcac ctcaggaccc agcgtcgatg    1080

ccttgcacac gacccccctc cgccccacac tacctcacag ccgtgggcat gggtgccaag    1140

gtggagctgc gctggacgcc ccctcaggac agcgggggcc gcgaggacat tgtctacagc    1200

gtcacctgcg aacagtgctg gcccgagtct ggggaatgcg ggccgtgtga ggccagtgtg    1260

cgctactcgg agcctcctca cggactgacc cgcaccagtg tgacagtgag cgacctggag    1320

ccccacatga actacacctt caccgtggag gcccgcaatg gcgtctcagg cctggtaacc    1380

agccgcagct tccgtactgc cagtgtcagc atcaaccaga cagagccccc caaggtgagg    1440

ctggagggcc gcagcaccac ctcgcttagc gtctcctgga gcatcccccc gccgcagcag    1500

agccgagtgt ggaagtacga ggtcacttac cgcaagaagg gagactccaa cagctacaat    1560

gtgcgccgca ccgagggttt ctccgtgacc ctggacgacc tggccccaga caccacctac    1620

ctggtccagg tgcaggcact gacgcaggag ggccaggggg ccggcagcag ggtgcacgaa    1680

ttccagacg                                                            1689

<210>25

<211>563

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the fusion of prediction

<400>25

Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu Val Ser Leu

1               5                   10                  15

Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys

            20                  25                  30

Glu Asn Ser Ile Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser

        35                  40                  45

Pro Lys Thr pro Ile Glu Lys Lys His Ala Asp Leu Glu Gln Gly Lys

    50                  55                  60

Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp

65                  70                  75                  80

Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met

                85                  90                  95

Asn Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly

            100                 105                 110

Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala

        115                 120                 125

Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser

    130                 135                 140

Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr

145                 150                 155                 160

Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe

                165                 170                 175

Thr Lys Ile Asp Thr Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp

            180                 185                 190

Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly

        195                 200                 205

Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala

    210                 215                 220

Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu

225                 230                 235                 240

Leu Leu Gln Gly Leu Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp

                245                 250                 255

Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val Asp His Ala Val

            260                 265                 270

Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys Ala Val Asp Gly

        275                 280                 285

Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu

    290                 295                 300

Lys Val Glu Asp Ala Cys Gln Ala Cys Ser Pro Gly Phe Phe Lys phe

305                 310                 315                 320

Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro

                325                 330                 335

Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu Gly Phe phe Arg

            340                 345                 350

Ala Pro Gln Asp Pro Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala

        355                 360                 365

Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg

    370                 375                 380

Trp Thr Pro Pro Gln Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser

385                 390                 395                 400

Val Thr Cys Glu Gln Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys

                405                 410                 415

Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr

            420                 425                 430

Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr

        435                 440                 445

Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe

    450                 455                 460

Arg Thr Ala Ser Val Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg

465                 470                 475                 480

Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser Trp Ser Ile Pro

                485                 490                 495

pro Pro Gln Gln Ser Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys

            500                 505                 510

Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser

        515                 520                 525

Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val

    530                 535                 540

Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser Arg Val His Glu

545                 550                 555                 560

Phe Gln Thr

<210>26

<211>1989

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: pre-fusion protein construct

<400>26

ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac    60

atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata    120

atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg    180

gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg    240

aaaaaaataa tgctagtttt tattacactt atattagtta gtctaccaat tgcgcaacaa    300

actgaagcaa aggatgcatc tgcattcaat aaagaaaatt caatttcatc catggcacca    360

ccagcatctc cgcctgcaag tcctaagacg ccaatcgaaa agaaacacgc ggatggatcc    420

gattataaag atgatgatga taaacaaggt aaagaagttg ttttattaga ttttgcagca    480

gcaggtggtg aattaggttg gttaacacat ccatatggta aaggttggga tttaatgcaa    540

aatattatga atgatatgcc aatttatatg tatagtgttt gtaatgttat gagtggtgat    600

caagataatt ggttacgtac aaattgggtt tatcgtggtg aagcagaacg tatttttatt    660

gaattaaaat ttacagttcg tgattgtaat agttttccag gtggtgcaag tagttgtaaa    720

gaaacattta atttatatta tgcagaaagt gatttagatt atggtacaaa ttttcaaaaa    780

cgtttattta caaaaattga tacaattgca ccagatgaaa ttacagttag tagtgatttt    840

gaagcacgtc atgttaaatt aaatgttgaa gaacgtagtg ttggtccatt aacacgtaaa    900

ggtttttatt tagcatttca agatattggt gcatgtgttg cattattaag tgttcgtgtt    960

tattataaaa aatgtccaga attattacaa ggtttagcac attttccaga aacaattgca    1020

ggtagtgatg caccaagttt agcaacagtt gcaggtacat gtgttgatca tgcagttgtt    1080

ccaccaggtg gtgaagaacc acgtatgcat tgtgcagttg atggtgaatg gttagttcca    1140

attggtcaat gtttatgtca agcaggttat gaaaaagttg aagatgcatg tcaagcatgt    1200

agtccaggtt tttttaaatt tgaagcaagt gaaagtccat gtttagaatg tccagaacat    1260

acattaccaa gtccagaagg tgcaacaagt tgtgaatgtg aagaaggttt ttttcgtgca    1320

ccacaagatc cagcaagtat gccatgtaca cgtccaccaa gtgcaccaca ttatttaaca    1380

gcagttggta tgggtgcaaa agttgaatta cgttggacac caccacaaga tagtggtggt    1440

cgtgaagata ttgtttatag tgttacatgt gaacaatgtt ggccagaaag tggtgaatgt    1500

ggtccatgtg aagcaagtgt tcgttatagt gaaccaccac atggtttaac acgtacaagt    1560

gttacagtta gtgatttaga accacatatg aattatacat ttacagttga agcacgtaat    1620

ggtgttagtg gtttagttac aagtcgtagt tttcgtacag caagtgttag tattaatcaa    1680

acagaaccac caaaagttcg tttagaaggt cgtagtacaa caagtttaag tgttagttgg    1740

agtattccac caccacaaca aagtcgtgtt tggaaatatg aagttacata tcgtaaaaaa    1800

ggtgatagta atagttataa tgttcgtcgt acagaaggtt ttagtgttac attagatgat    1860

ttagcaccag atacaacata tttagttcaa gttcaagcat taacacaaga aggtcaaggt    1920

gcaggtagtc gtgttcatga atttcaaaca gaacaaaaat taattagtga agaagattta    1980

tgagagctc                                                            1989

<210>27

<211>581

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the fusion of prediction

<400>27

Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu Val Ser Leu

1               5                   10                  15

Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys

            20                  25                  30

Glu Asn Ser Ile Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser

        35                  40                  45

Pro Lys Thr pro Ile Glu Lys Lys His Ala Asp Gly Ser Asp Tyr Lys

    50                  55                  60

Asp Asp Asp Asp Lys Gln Gly Lys Glu Val Val Leu Leu Asp Phe Ala

65                  70                  75                  80

Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr Gly Lys Gly

                85                  90                  95

Trp Asp Leu Met Gln Asn Ile Met Asn Asp Met Pro Ile Tyr Met Tyr

            100                 105                 110

Ser Val Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp Leu Arg Thr

        115                 120                 125

Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile Glu Leu Lys

    130                 135                 140

Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala Ser Ser Cys

145                 150                 155                 160

Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly

                165                 170                 175

Thr Asn Phe Gln Lys Arg Leu Phe Thr Lys Ile Asp Thr Ile Ala Pro

            180                 185                 190

Asp Glu Ile Thr Val Ser Ser Asp Phe Glu Ala Arg His Val Lys Leu

        195                 200                 205

Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr

    210                 215                 220

Leu Ala Phe Gln Asp Ile Gly Ala Cys Val Ala Leu Leu Ser Val Arg

225                 230                 235                 240

Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gln Gly Leu Ala His Phe

                245                 250                 255

Pro Glu Thr Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala

            260                 265                 270

Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly Glu Glu Pro

        275                 280                 285

Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro Ile Gly Gln

    290                 295                 300

Cys Leu Cys Gln Ala Gly Tyr Glu Lys Val Glu Asp Ala Cys Gln Ala

305                 310                 315                 320

Cys Ser Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser pro Cys Leu

                325                 330                 335

Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala Thr Ser Cys

            340                 345                 350

Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gln Asp Pro Ala Ser Met

        355                 360                 365

Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly

    370                 375                 380

Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gln Asp Ser Gly

385                 390                 395                 400

Gly Arg Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Gln Cys Trp Pro

                405                 410                 415

Glu Ser Gly Glu Cys Gly pro Cys Glu Ala Ser Val Arg Tyr Ser Glu

            420                 425                 430

Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu

        435                 440                 445

Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn Gly Val Ser

    450                 455                 460

Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val Ser Ile Asn

465                 470                 475                 480

Gln Thr Glu Pro pro Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser

                485                 490                 495

Leu Ser Val Ser Trp Ser Ile Pro Pro Pro Gln Gln Ser Arg Val Trp

            500                 505                 510

Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn

        515                 520                 525

Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro

    530                 535                 540

Asp Thr Thr Tyr Leu Val Gln Val Gln Ala Leu Thr Gln Glu Gly Gln

545                 550                 555                 560

Gly Ala Gly Ser Arg Val His Glu Phe Gln Thr Glu Gln Lys Leu Ile

                565                 570                 575

Ser Glu Glu Asp Leu

            580

<210>28

<211>1989

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: fusion protein construct

<400>28

ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac    60

atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata    120

atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg    180

gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg    240

aaaaaaatta tgttagtttt tattacatta attttagtta gtttaccaat tgcacaacaa    300

acagaagcaa aagatgcaag tgcatttaat aaagaaaata gtattagtag tatggcacca    360

ccagcaagtc caccagcaag tccaaaaaca ccaattgaaa aaaaacatgc agatggatcc    420

gattataaag atgatgatga taaacaaggt aaagaagttg ttttattaga ttttgcagca    480

gcaggtggtg aattaggttg gttaacacat ccatatggta aaggttggga tttaatgcaa    540

aatattatga atgatatgcc aatttatatg tatagtgttt gtaatgttat gagtggtgat    600

caagataatt ggttacgtac aaattgggtt tatcgtggtg aagcagaacg tatttttatt    660

gaattaaaat ttacagttcg tgattgtaat agttttccag gtggtgcaag tagttgtaaa    720

gaaacattta atttatatta tgcagaaagt gatttagatt atggtacaaa ttttcaaaaa    780

cgtttattta caaaaattga tacaattgca ccagatgaaa ttacagttag tagtgatttt    840

gaagcacgtc atgttaaatt aaatgttgaa gaacgtagtg ttggtccatt aacacgtaaa    900

ggtttttatt tagcatttca agatattggt gcatgtgttg cattattaag tgttcgtgtt    960

tattataaaa aatgtccaga attattacaa ggtttagcac attttccaga aacaattgca    1020

ggtagtgatg caccaagttt agcaacagtt gcaggtacat gtgttgatca tgcagttgtt    1080

ccaccaggtg gtgaagaacc acgtatgcat tgtgcagttg atggtgaatg gttagttcca    1140

attggtcaat gtttatgtca agcaggttat gaaaaagttg aagatgcatg tcaagcatgt    1200

agtccaggtt tttttaaatt tgaagcaagt gaaagtccat gtttagaatg tccagaacat    1260

acattaccaa gtccagaagg tgcaacaagt tgtgaatgtg aagaaggttt ttttcgtgca    1320

ccacaagatc cagcaagtat gccatgtaca cgtccaccaa gtgcaccaca ttatttaaca    1380

gcagttggta tgggtgcaaa agttgaatta cgttggacac caccacaaga tagtggtggt    1440

cgtgaagata ttgtttatag tgttacatgt gaacaatgtt ggccagaaag tggtgaatgt    1500

ggtccatgtg aagcaagtgt tcgttatagt gaaccaccac atggtttaac acgtacaagt    1560

gttacagtta gtgatttaga accacatatg aattatacat ttacagttga agcacgtaat    1620

ggtgttagtg gtttagttac aagtcgtagt tttcgtacag caagtgttag tattaatcaa    1680

acagaaccac caaaagttcg tttagaaggt cgtagtacaa caagtttaag tgttagttgg    1740

agtattccac caccacaaca aagtcgtgtt tggaaatatg aagttacata tcgtaaaaaa    1800

ggtgatagta atagttataa tgttcgtcgt acagaaggtt ttagtgttac attagatgat    1860

ttagcaccag atacaacata tttagttcaa gttcaagcat taacacaaga aggtcaaggt    1920

gcaggtagtc gtgttcatga atttcaaaca gaacaaaaat taattagtga agaagattta    1980

tgagagctc                                                            1989

<210>29

<21l>581

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the fusion of prediction

<400>29

Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu Val Ser Leu

1               5                   10                  15

Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys

            20                  25                  30

Glu Asn Ser Ile Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser

        35                  40                  45

Pro Lys Thr Pro Ile Glu Lys Lys His Ala Asp Gly Ser Asp Tyr Lys

    50                  55                  60

Asp Asp Asp Asp Lys Gln Gly Lys Glu Val Val Leu Leu Asp Phe Ala

65                  70                  75                  80

Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His pro Tyr Gly Lys Gly

                85                  90                  95

Trp Asp Leu Met Gln Asn Ile Met Asn Asp Met Pro Ile Tyr Met Tyr

            100                 105                 110

Ser Val Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp Leu Arg Thr

        115                 120                 125

Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile Glu Leu Lys

    130                 135                 140

Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala Ser Ser Cys

145                 150                 155                 160

Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu Asp Tyr Gly

                165                 170                 175

Thr Asn Phe Gln Lys Arg Leu Phe Thr Lys Ile Asp Thr Ile Ala Pro

            180                 185                 190

Asp Glu Ile Thr Val Ser Ser Asp phe Glu Ala Arg His Val Lys Leu

        195                 200                 205

Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys Gly Phe Tyr

    210                 215                 220

Leu Ala Phe Gln Asp Ile Gly Ala Cys Val Ala Leu Leu Ser Val Arg

225                 230                 235                 240

Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gln Gly Leu Ala His Phe

                245                 250                 255

Pro Glu Thr Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala Thr Val Ala

            260                 265                 270

Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly Glu Glu Pro

        275                 280                 285

Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro Ile Gly Gln

    290                 295                 300

Cys Leu Cys Gln Ala Gly Tyr Glu Lys Val Glu Asp Ala Cys Gln Ala

305                 310                 315                 320

Cys Ser Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser Pro Cys Leu

                325                 330                 335

Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala Thr Ser Cys

            340                 345                 350

Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gln Asp Pro Ala Ser Met

        355                 360                 365

pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr Ala Val Gly

    370                 375                 380

Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gln Asp Ser Gly

385                 390                 395                 400

Gly Arg Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Gln Cys Trp pro

                405                 410                 415

Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg Tyr Ser Glu

            420                 425                 430

Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser Asp Leu Glu

        435                 440                 445

Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn Gly Val Ser

    450                 455                 460

Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val Ser Ile Asn

465                 470                 475                 480

Gln Thr Glu Pro pro Lys Val Arg Leu Glu Gly Arg Ser Thr Thr Ser

                485                 490                 495

Leu Ser Val Ser Trp Ser Ile Pro Pro Pro Gln Gln Ser Arg Val Trp

            500                 505                 510

Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn Ser Tyr Asn

        515                 520                 525

Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp Leu Ala Pro

    530                 535                 540

Asp Thr Thr Tyr Leu Val Gln Val Gln Ala Leu Thr Gln Glu Gly Gln

545                 550                 555                 560

Gly Ala Gly Ser Arg Val His Glu Phe Gln Thr Glu Gln Lys Leu Ile

                565                 570                 575

Ser Glu Glu Asp Leu

            580

<210>30

<211>1968

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: pre-fusion protein construct

<400>30

ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac    60

atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata    120

atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg    180

gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg    240

gcatacgaca gtcgttttga tgaatgggta cagaaactga aagaggaaag ctttcaaaac    300

aatacgtttg accgccgcaa atttattcaa ggagcgggga agattgcagg actttctctt    360

ggattaacga ttgcccagtc ggttggggcc tttggatccg attataaaga tgatgatgat    420

aaacaaggta aagaagttgt tttattagat tttgcagcag caggtggtga attaggttgg    480

ttaacacatc catatggtaa aggttgggat ttaatgcaaa atattatgaa tgatatgcca    540

atttatatgt atagtgtttg taatgttatg agtggtgatc aagataattg gttacgtaca    600

aattgggttt atcgtggtga agcagaacgt atttttattg aattaaaatt tacagttcgt    660

gattgtaata gttttccagg tggtgcaagt agttgtaaag aaacatttaa tttatattat    720

gcagaaagtg atttagatta tggtacaaat tttcaaaaac gtttatttac aaaaattgat    780

acaattgcac cagatgaaat tacagttagt agtgattttg aagcacgtca tgttaaatta    840

aatgttgaag aacgtagtgt tggtccatta acacgtaaag gtttttattt agcatttcaa    900

gatattggtg catgtgttgc attattaagt gttcgtgttt attataaaaa atgtccagaa    960

ttattacaag gtttagcaca ttttccagaa acaattgcag gtagtgatgc accaagttta    1020

gcaacagttg caggtacatg tgttgatcat gcagttgttc caccaggtgg tgaagaacca    1080

cgtatgcatt gtgcagttga tggtgaatgg ttagttccaa ttggtcaatg tttatgtcaa    1140

gcaggttatg aaaaagttga agatgcatgt caagcatgta gtccaggttt ttttaaattt    1200

gaagcaagtg aaagtccatg tttagaatgt ccagaacata cattaccaag tccagaaggt    1260

gcaacaagtt gtgaatgtga agaaggtttt tttcgtgcac cacaagatcc agcaagtatg    1320

ccatgtacac gtccaccaag tgcaccacat tatttaacag cagttggtat gggtgcaaaa    1380

gttgaattac gttggacacc accacaagat agtggtggtc gtgaagatat tgtttatagt    1440

gttacatgtg aacaatgttg gccagaaagt ggtgaatgtg gtccatgtga agcaagtgtt    1500

cgttatagtg aaccaccaca tggtttaaca cgtacaagtg ttacagttag tgatttagaa    1560

ccacatatga attatacatt tacagttgaa gcacgtaatg gtgttagtgg tttagttaca    1620

agtcgtagtt ttcgtacagc aagtgttagt attaatcaaa cagaaccacc aaaagttcgt    1680

ttagaaggtc gtagtacaac aagtttaagt gttagttgga gtattccacc accacaacaa    1740

agtcgtgttt ggaaatatga agttacatat cgtaaaaaag gtgatagtaa tagttataat    1800

gttcgtcgta cagaaggttt tagtgttaca ttagatgatt tagcaccaga tacaacatat    1860

ttagttcaag ttcaagcatt aacacaagaa ggtcaaggtg caggtagtcg tgttcatgaa    1920

tttcaaacag aacaaaaatt aattagtgaa gaagatttat gagagctc                 1968

<210>31

<211>574

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the fusion of prediction

<400>31

Met Ala Tyr Asp Ser Arg Phe Asp Glu Trp Val Gln Lys Leu Lys Glu

1               5                   10                  15

Glu Ser Phe Gln Asn Asn Thr Phe Asp Arg Arg Lys phe Ile Gln Gly

            20                  25                  30

Ala Gly Lys Ile Ala Gly Leu Ser Leu Gly Leu Thr Ile Ala Gln Ser

        35                  40                  45

Val Gly Ala Phe Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Gln Gly

    50                  55                  60

Lys Glu Val Val Leu Leu Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly

65                  70                  75                  80

Trp Leu Thr His Pro Tyr Gly Lys Gly Trp Asp Leu Met Gln Asn Ile

                85                  90                  95

Met Asn Asp Met Pro Ile Tyr Met Tyr Ser Val Cys Asn Val Met Ser

            100                 105                 110

Gly Asp Gln Asp Asn Trp Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu

        115                 120                 125

Ala Glu Arg Ile Phe Ile Glu Leu Lys Phe Thr Val Arg Asp Cys Asn

    130                 135                 140

Ser Phe Pro Gly Gly Ala Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr

145                 150                 155                 160

Tyr Ala Glu Ser Asp Leu Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu

                165                 170                 175

phe Thr Lys Ile Asp Thr Ile Ala Pro Asp Glu Ile Thr Val Ser Ser

            180                 185                 190

Asp Phe Glu Ala Arg His Val Lys Leu Asn Val Glu Glu Arg Ser Val

        195                 200                 205

Gly Pro Leu Thr Arg Lys Gly Phe Tyr Leu Ala phe Gln Asp Ile Gly

    210                 215                 220

Ala Cys Val Ala Leu Leu Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro

 225                230                 235                 240

Glu Leu Leu Gln Gly Leu Ala His Phe Pro Glu Thr Ile Ala Gly Ser

                245                 250                 255

Asp Ala Pro Ser Leu Ala Thr Val Ala Gly Thr Cys Val Asp His Ala

            260                 265                 270

Val Val Pro Pro Gly Gly Glu Glu Pro Arg Met His Cys Ala Val Asp

        275                 280                 285

Gly Glu Trp Leu Val Pro Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr

    290                 295                 300

Glu Lys Val Glu Asp Ala Cys Gln Ala Cys Ser Pro Gly Phe phe Lys

305                 310                 315                 320

Phe Glu Ala Ser Glu Ser Pro Cys Leu Glu Cys Pro Glu His Thr Leu

                325                 330                 335

Pro Ser Pro Glu Gly Ala Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe

            340                 345                 350

Arg Ala Pro Gln Asp Pro Ala Ser Met Pro Cys Thr Arg Pro Pro Ser

        355                 360                 365

Ala Pro His Tyr Leu Thr Ala Val Gly Met Gly Ala Lys Val Glu Leu

    370                 375                 380

Arg Trp Thr Pro Pro Gln Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr

385                 390                 395                 400

Ser Val Thr Cys Glu Gln Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro

                405                 410                 415

Cys Glu Ala Ser Val Arg Tyr Ser Glu Pro Pro His Gly Leu Thr Arg

            420                 425                 430

Thr Ser Val Thr Val Ser Asp Leu Glu Pro His Met Asn Tyr Thr phe

        435                 440                 445

Thr Val Glu Ala Arg Asn Gly Val Ser Gly Leu Val Thr Ser Arg Ser

    450                 455                 460

Phe Arg Thr Ala Ser Val Ser Ile Asn Gln Thr Glu Pro Pro Lys Val

465                 470                 475                 480

Arg Leu Glu Gly Arg Ser Thr Thr Ser Leu Ser Val Ser Trp Ser Ile

                485                 490                 495

Pro Pro Pro Gln Gln Ser Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg

            500                 505                 510

Lys Lys Gly Asp Ser Asn Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe

        515                 520                 525

Ser Val Thr Leu Asp Asp Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln

    530                 535                 540

Val Gln Ala Leu Thr Gln Glu Gly Gln Gly Ala Gly Ser Arg Val His

545                 550                 555                 560

Glu Phe Gln Thr Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

                565                 570

<210>32

<211>1254

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>32

caccgcagga ggaagaacca gcgtgcccgc cagtccccgg aggacgttta cttctccaag    60

tcagaacaac tgaagcccct gaagacatac gtggaccccc acacatatga ggaccccaac    120

caggctgtgt tgaagttcac taccgagatc catccatcct gtgtcactcg gcagaaggtg    180

atcggagcag gagagtttgg ggaggtgtac aagggcatgc tgaagacatc ctcggggaag    240

aaggaggtgc cggtggccat caagacgctg aaagccggct acacagagaa gcagcgagtg    300

gacttcctcg gcgaggccgg catcatgggc cagttcagcc accacaacat catccgccta    360

gagggcgtca tctccaaata caagcccatg atgatcatca ctgagtacat ggagaatggg    420

gccctggaca agttccttcg ggagaaggat ggcgagttca gcgtgctgca gctggtgggc    480

atgctgcggg gcatcgcagc tggcatgaag tacctggcca acatgaacta tgtgcaccgt    540

gacctggctg cccgcaacat cctcgtcaac agcaacctgg tctgcaaggt gtctgacttt    600

ggcctgtccc gcgtgctgga ggacgacccc gaggccacct acaccaccag tggcggcaag    660

atccccatcc gctggaccgc cccggaggcc atttcctacc ggaagttcac ctctgccagc    720

gacgtgtgga gctttggcat tgtcatgtgg gaggtgatga cctatggcga gcggccctac    780

tgggagttgt ccaaccacga ggtgatgaaa gccatcaatg atggcttccg gctccccaca    840

cccatggact gcccctccgc catctaccag ctcatgatgc agtgctggca gcaggagcgt    900

gcccgccgcc ccaagttcgc tgacatcgtc agcatcctgg acaagctcat tcgtgcccct    960

gactccctca agaccctggc tgactttgac ccccgcgtgt ctatccggct ccccagcacg    1020

agcggctcgg agggggtgcc cttccgcacg gtgtccgagt ggctggagtc catcaagatg    1080

cagcagtata cggagcactt catggcggcc ggctacactg ccatcgagaa ggtggtgcag    1140

atgaccaacg acgacatcaa gaggattggg gtgcggctgc ccggccacca gaagcgcatc    1200

gcctacagcc tgctgggact caaggaccag gtgaacactg tggggatccc catc          1254

<210>33

<211>1254

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the sequence with the employed optimization codon in Listeria

<400>33

cacagacgta gaaaaaatca acgtgctcga caatccccag aagatgtgta tttttcgaaa    60

agtgaacaat taaaaccatt aaaaacttat gttgatccgc atacgtacga agacccaaat    120

caagcagtat taaaatttac aacagaaata cacccaagtt gtgttacaag acaaaaagtt    180

attggagcag gtgaattcgg agaggtatat aaaggtatgt taaaaacatc atcaggtaaa    240

aaagaagttc cggttgcaat taaaacctta aaggcaggat atacagaaaa acagcgagtt    300

gattttttag gtgaagcagg aattatgggt caatttagcc atcataatat tattcgtttg    360

gaaggagtaa taagtaaata taaaccaatg atgattatta cagaatacat ggaaaacggt    420

gctttagata aatttttacg tgaaaaggat ggtgaattta gtgttttaca attggttggt    480

atgttaagag gaattgctgc aggtatgaaa tatttagcta atatgaatta tgttcaccgt    540

gatttggcag caagaaatat cctagtcaat tccaatttag tatgtaaagt tagtgatttt    600

ggtttaagca gagtattaga agacgatcca gaggcaacct atacaacatc gggaggtaaa    660

attcctattc gttggacagc accagaagct atcagttacc gtaaatttac aagtgcatca    720

gacgtgtgga gttttgggat tgtaatgtgg gaagttatga catatggaga aagaccatat    780

tgggaattaa gtaatcatga agttatgaaa gcaattaacg atggatttag attaccaact    840

ccgatggatt gtccatctgc catttatcaa ctaatgatgc aatgttggca acaagaaaga    900

gcacgacgtc caaaatttgc agatattgtt agtattttag acaaattaat tcgtgcacca    960

gatagtttaa aaactttagc agactttgat cctcgtgtta gtattcgatt accaagtacg    1020

tcaggttccg aaggagttcc atttcgcaca gtctccgaat ggttggaatc aattaaaatg    1080

caacaataca ccgaacactt tatggcagca ggttacacag caatcgaaaa agttgttcaa    1140

atgacaaatg atgatattaa acgtattgga gttagattac caggccacca gaaacgtatt    1200

gcatattctt tattaggttt aaaagatcaa gttaataccg tgggaattcc aatt          1254

<210>34

<211>456

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>34

Val His Glu Phe Gln Thr Leu Ser Pro Glu Gly Ser Gly Asn Leu Ala

1               5                   10                  15

Val Ile Gly Gly Val Ala Val Gly Val Val Leu Leu Leu Val Leu Ala

            20                  25                  30

Gly Val Gly Phe Phe Ile His Arg Arg Arg Lys Asn Gln Arg Ala Arg

        35                  40                  45

Gln Ser Pro Glu Asp Val Tyr Phe Ser Lys Ser Glu Gln Leu Lys Pro

    50                  55                  60

Leu Lys Thr Tyr Val Asp pro His Thr Tyr Glu Asp Pro Asn Gln Ala

65                  70                  75                  80

Val Leu Lys Phe Thr Thr Glu Ile His Pro Ser Cys Val Thr Arg Gln

                85                  90                  95

Lys Val Ile Gly Ala Gly Glu Phe Gly Glu Val Tyr Lys Gly Met Leu

            100                 105                 110

Lys Thr Ser Ser Gly Lys Lys Glu Val Pro Val Ala Ile Lys Thr Leu

        115                 120                 125

Lys Ala Gly Tyr Thr Glu Lys Gln Arg Val Asp Phe Leu Gly Glu Ala

    130                 135                 140

Gly Ile Met Gly Gln Phe Ser His His Asn Ile Ile Arg Leu Glu Gly

145                 150                 155                 160

Val Ile Ser Lys Tyr Lys Pro Met Met Ile Ile Thr Glu Tyr Met Glu

                165                 170                 175

Asn Gly Ala Leu Asp Lys Phe Leu Arg Glu Lys Asp Gly Glu Phe Ser

            180                 185                 190

Val Leu Gln Leu Val Gly Met Leu Arg Gly Ile Ala Ala Gly Met Lys

        195                 200                 205

Tyr Leu Ala Asn Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn

    210                 215                 220

Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu

225                 230                 235                 240

Ser Arg Val Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr Thr Ser Gly

                245                 250                 255

Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ser Tyr Arg

            260                 265                 270

Lys Phe Thr Ser Ala Ser Asp Val Trp Ser Phe Gly Ile Val Met Trp

        275                 280                 285

Glu Val Met Thr Tyr Gly Glu Arg Pro Tyr Trp Glu Leu Ser Asn His

    290                 295                 300

Glu Val Met Lys Ala Ile Asn Asp Gly Phe Arg Leu Pro Thr pro Met

305                 310                 315                 320

Asp Cys Pro Ser Ala Ile Tyr Gln Leu Met Met Gln Cys Trp Gln Gln

                325                 330                 335

Glu Arg Ala Arg Arg Pro Lys Phe Ala Asp Ile Val Ser Ile Leu Asp

            340                 345                 350

Lys Leu Ile Arg Ala Pro Asp Ser Leu Lys Thr Leu Ala Asp Phe Asp

        355                 360                 365

Pro Arg Val Ser Ile Arg Leu Pro Ser Thr Ser Gly Ser Glu Gly Val

    370                 375                 380

Pro Phe Arg Thr Val Ser Glu Trp Leu Glu Ser Ile Lys Met Gln Gln

385                 390                 395                 400

Tyr Thr Glu His Phe Met Ala Ala Gly Tyr Thr Ala Ile Glu Lys Val

                405                 410                 415

Val Gln Met Thr Asn Asp Asp Ile Lys Arg Ile Gly Val Arg Leu Pro

            420                 425                 430

Gly His Gln Lys Arg Ile Ala Tyr Ser Leu Leu Gly Leu Lys Asp Gln

        435                 440                 445

Val Asn Thr Val Gly Ile Pro Ile

    450                 455

<210>35

<211>1437

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: fusion

<400>35

atgaaaaaaa taatgctagt ttttattaca cttatattag ttagtctacc aattgcgcaa    60

caaactgaag caaaggatgc atctgcattc aataaagaaa attcaatttc atccatggca    120

ccaccagcat ctccgcctgc aagtcctaag acgccaatcg aaaagaaaca cgcggatctc    180

gagcaccgca ggaggaagaa ccagcgtgcc cgccagtccc cggaggacgt ttacttctcc    240

aagtcagaac aactgaagcc cctgaagaca tacgtggacc cccacacata tgaggacccc    300

aaccaggctg tgttgaagtt cactaccgag atccatccat cctgtgtcac tcggcagaag    360

gtgatcggag caggagagtt tggggaggtg tacaagggca tgctgaagac atcctcgggg    420

aagaaggagg tgccggtggc catcaagacg ctgaaagccg gctacacaga gaagcagcga    480

gtggacttcc tcggcgaggc cggcatcatg ggccagttca gccaccacaa catcatccgc    540

ctagagggcg tcatctccaa atacaagccc atgatgatca tcactgagta catggagaat    600

ggggccctgg acaagttcct tcgggagaag gatggcgagt tcagcgtgct gcagctggtg    660

ggcatgctgc ggggcatcgc agctggcatg aagtacctgg ccaacatgaa ctatgtgcac    720

cgtgacctgg ctgcccgcaa catcctcgtc aacagcaacc tggtctgcaa ggtgtctgac    780

tttggcctgt cccgcgtgct ggaggacgac cccgaggcca cctacaccac cagtggcggc    840

aagatcccca tccgctggac cgccccggag gccatttcct accggaagtt cacctctgcc    900

agcgacgtgt ggagctttgg cattgtcatg tgggaggtga tgacctatgg cgagcggccc    960

tactgggagt tgtccaacca cgaggtgatg aaagccatca atgatggctt ccggctcccc    1020

acacccatgg actgcccctc cgccatctac cagctcatga tgcagtgctg gcagcaggag    1080

cgtgcccgcc gccccaagtt cgctgacatc gtcagcatcc tggacaagct cattcgtgcc    1140

cctgactccc tcaagaccct ggctgacttt gacccccgcg tgtctatccg gctccccagc    1200

acgagcggct cggagggggt gcccttccgc acggtgtccg agtggctgga gtccatcaag    1260

atgcagcagt atacggagca cttcatggcg gccggctaca ctgccatcga gaaggtggtg    1320

cagatgacca acgacgacat caagaggatt ggggtgcggc tgcccggcca ccagaagcgc    1380

atcgcctaca gcctgctggg actcaaggac caggtgaaca ctgtggggat ccccatc       1437

<210>36

<211>479

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the protein sequence of prediction

<400>36

Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu Val Ser Leu

1               5                   10                  15

Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys

            20                  25                  30    Glu Asn Ser Ile Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser

        35                  40                  45

Pro Lys Thr Pro Ile Glu Lys Lys His Ala Asp Leu Glu His Arg Arg

    50                  55                  60

Arg Lys Asn Gln Arg Ala Arg Gln Ser Pro Glu Asp Val Tyr Phe Ser

65                  70                  75                  80

Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His Thr

                85                  90                  95

Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys Phe Thr Thr Glu Ile His

            100                 105                 110

Pro Ser Cys Val Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe Gly

        115                 120                 125

Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu Val

    130                 135                 140

Pro Val Ala Ile Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gln Arg

145                 150                 155                 160

Val Asp Phe Leu Gly Glu Ala Gly Ile Met Gly Gln Phe Ser His His

                165                 170                 175

Asn Ile Ile Arg Leu Glu Gly Val Ile Ser Lys Tyr Lys pro Met Met

            180                 185                 190

Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu Arg

        195                 200                 205

Glu Lys Asp Gly Glu Phe Ser Val Leu Gln Leu Val Gly Met Leu Arg

    210                 215                 220

Gly Ile Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val His

225                 230                 235                 240

Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val Cys

                245                 250                 255

Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro Glu

            260                 265                 270

Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile pro Ile Arg Trp Thr Ala

        275                 280                 285

Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val Trp

    290                 295                 300

Ser Phe Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg Pro

305                 310                 315                 320

Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala Ile Asn Asp Gly

                325                 330                 335

Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr Gln Leu

            340                 345                 350

Met Met Gln Cys Trp Gln Gln Glu Arg Ala Arg Arg Pro Lys Phe Ala

        355                 360                 365

Asp Ile Val Ser Ile Leu Asp Lys Leu Ile Arg Ala Pro Asp Ser Leu

    370                 375                 380

Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser Ile Arg Leu Pro Ser

385                 390                 395                 400

Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp Leu

                405                 410                 415

Glu Ser Ile Lys Met Gln Gln Tyr Thr Glu His Phe Met Ala Ala Gly

            420                 425                 430

Tyr Thr Ala Ile Glu Lys Val Val Gln Met Thr Asn Asp Asp Ile Lys

        435                 440                 445

Arg Ile Gly Val Arg Leu Pro Gly His Gln Lys Arg Ile Ala Tyr Ser

    450                 455                 460

Leu Leu Gly Leu Lys Asp Gln Val Asn Thr Val Gly Ile Pro Ile

465                 470                 475

<210>37

<211>1737

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: fusion sequence

<400>37

ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac    60

atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata    120

atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg    180

gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg    240

aaaaaaataa tgctagtttt tattacactt atattagtta gtctaccaat tgcgcaacaa    300

actgaagcaa aggatgcatc tgcattcaat aaagaaaatt caatttcatc catggcacca    360

ccagcatctc cgcctgcaag tcctaagacg ccaatcgaaa agaaacacgc ggatggatcc    420

gattataaag atgatgatga taaacacaga cgtagaaaaa atcaacgtgc tcgacaatcc    480

ccagaagatg tgtatttttc gaaaagtgaa caattaaaac cattaaaaac ttatgttgat    540

ccgcatacgt acgaagaccc aaatcaagca gtattaaaat ttacaacaga aatacaccca    600

agttgtgtta caagacaaaa agttattgga gcaggtgaat tcggagaggt atataaaggt    660

atgttaaaaa catcatcagg taaaaaagaa gttccggttg caattaaaac cttaaaggca    720

ggatatacag aaaaacagcg agttgatttt ttaggtgaag caggaattat gggtcaattt    780

agccatcata atattattcg tttggaagga gtaataagta aatataaacc aatgatgatt    840

attacagaat acatggaaaa cggtgcttta gataaatttt tacgtgaaaa ggatggtgaa    900

tttagtgttt tacaattggt tggtatgtta agaggaattg ctgcaggtat gaaatattta    960

gctaatatga attatgttca ccgtgatttg gcagcaagaa atatcctagt caattccaat    1020

ttagtatgta aagttagtga ttttggttta agcagagtat tagaagacga tccagaggca    1080

acctatacaa catcgggagg taaaattcct attcgttgga cagcaccaga agctatcagt    1140

taccgtaaat ttacaagtgc atcagacgtg tggagttttg ggattgtaat gtgggaagtt    1200

atgacatatg gagaaagacc atattgggaa ttaagtaatc atgaagttat gaaagcaatt    1260

aacgatggat ttagattacc aactccgatg gattgtccat ctgccattta tcaactaatg    1320

atgcaatgtt ggcaacaaga aagagcacga cgtccaaaat ttgcagatat tgttagtatt    1380

ttagacaaat taattcgtgc accagatagt ttaaaaactt tagcagactt tgatcctcgt    1440

gttagtattc gattaccaag tacgtcaggt tccgaaggag ttccatttcg cacagtctcc    1500

gaatggttgg aatcaattaa aatgcaacaa tacaccgaac actttatggc agcaggttac    1560

acagcaatcg aaaaagttgt tcaaatgaca aatgatgata ttaaacgtat tggagttaga    1620

ttaccaggcc accagaaacg tattgcatat tctttattag gtttaaaaga tcaagttaat    1680

accgtgggaa ttccaattga acaaaaatta atttccgaag aagacttata agagctc       1737

<210>38

<211>497

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the fusion of prediction

<400>38

Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu Val Ser Leu

1               5                   10                  15

Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys

            20                  25                  30

Glu Asn Ser Ile Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser

        35                  40                  45

Pro Lys Thr Pro Ile Glu Lys Lys His Ala Asp Gly Ser Asp Tyr Lys

    50                  55                  60

Asp Asp Asp Asp Lys His Arg Arg Arg Lys Asn Gln Arg Ala Arg Gln

65                  70                  75                  80

Ser Pro Glu Asp Val Tyr Phe Ser Lys Ser Glu Gln Leu Lys Pro Leu

                85                  90                  95

Lys Thr Tyr Val Asp Pro His Thr Tyr Glu Asp Pro Asn Gln Ala Val

            100                 105                 110

Leu Lys Phe Thr Thr Glu Ile His Pro Ser Cys Val Thr Arg Gln Lys

        115                 120                 125

Val Ile Gly Ala Gly Glu Phe Gly Glu Val Tyr Lys Gly Met Leu Lys

    130                 135                 140

Thr Ser Ser Gly Lys Lys Glu Val Pro Val Ala Ile Lys Thr Leu Lys

145                 150                 155                 160

Ala Gly Tyr Thr Glu Lys Gln Arg Val Asp Phe Leu Gly Glu Ala Gly

                165                 170                 175

Ile Met Gly Gln Phe Ser His His Asn Ile Ile Arg Leu Glu Gly Val

            180                 185                 190

Ile Ser Lys Tyr Lys Pro Met Met Ile Ile Thr Glu Tyr Met Glu Asn

        195                 200                 205

Gly Ala Leu Asp Lys Phe Leu Arg Glu Lys Asp Gly Glu Phe Ser Val

    210                 215                 220

Leu Gln Leu Val Gly Met Leu Arg Gly Ile Ala Ala Gly Met Lys Tyr

225                 230                 235                 240

Leu Ala Asn Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile

                245                 250                 255

Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu Ser

            260                 265                 270

Arg Val Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr Thr Ser Gly Gly

        275                 280                 285

Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ser Tyr Arg Lys

    290                 295                 300

Phe Thr Ser Ala Ser Asp Val Trp Ser Phe Gly Ile Val Met Trp Glu

305                 310                 315                 320

Val Met Thr Tyr Gly Glu Arg Pro Tyr Trp Glu Leu Ser Asn His Glu

                325                 330                 335

Val Met Lys Ala Ile Asn Asp Gly Phe Arg Leu Pro Thr Pro Met Asp

            340                 345                 350

Cys Pro Ser Ala Ile Tyr Gln Leu Met Met Gln Cys Trp Gln Gln Glu

        355                 360                 365

Arg Ala Arg Arg Pro Lys Phe Ala Asp Ile Val Ser Ile Leu Asp Lys

    370                 375                 380

Leu Ile Arg Ala Pro Asp Ser Leu Lys Thr Leu Ala Asp Phe Asp Pro

385                 390                 395                 400

Arg Val Ser Ile Arg Leu Pro Ser Thr Ser Gly Ser Glu Gly Val Pro

                405                 410                 415

Phe Arg Thr Val Ser Glu Trp Leu Glu Ser Ile Lys Met Gln Gln Tyr

            420                 425                 430

Thr Glu His phe Met Ala Ala Gly Tyr Thr Ala Ile Glu Lys Val Val

        435                 440                 445

Gln Met Thr Asn Asp Asp Ile Lys Arg Ile Gly Val Arg Leu Pro Gly

    450                 455                 460

His Gln Lys Arg Ile Ala Tyr Ser Leu Leu Gly Leu Lys Asp Gln Val

465                 470                 475                 480

Asn Thr Val Gly Ile Pro Ile Glu Gln Lys Leu Ile Ser Glu Glu Asp

                485                 490                 495

Leu

<210>39

<211>1737

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: pre-fusion protein construct

<400>39

ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac    60

atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata    120

atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg    l80

gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg    240

aaaaaaatta tgttagtttt tattacatta attttagtta gtttaccaat tgcacaacaa    300

acagaagcaa aagatgcaag tgcatttaat aaagaaaata gtattagtag tatggcacca    360

ccagcaagtc caccagcaag tccaaaaaca ccaattgaaa aaaaacatgc agatggatcc    420

gattataaag acgatgatga taaacacaga cgtagaaaaa atcaacgtgc tcgacaatcc    480

ccagaagatg tgtatttttc gaaaagtgaa caattaaaac cattaaaaac ttatgttgat    540

ccgcatacgt acgaagaccc aaatcaagca gtattaaaat ttacaacaga aatacaccca    600

agttgtgtta caagacaaaa agttattgga gcaggtgaat tcggagaggt atataaaggt    660

atgttaaaaa catcatcagg taaaaaagaa gttccggttg caattaaaac cttaaaggca    720

ggatatacag aaaaacagcg agttgatttt ttaggtgaag caggaattat gggtcaattt    780

agccatcata atattattcg tttggaagga gtaataagta aatataaacc aatgatgatt    840

attacagaat acatggaaaa cggtgcttta gataaatttt tacgtgaaaa ggatggtgaa    900

tttagtgttt tacaattggt tggtatgtta agaggaattg ctgcaggtat gaaatattta    960

gctaatatga attatgttca ccgtgatttg gcagcaagaa atatcctagt caattccaat    1020

ttagtatgta aagttagtga ttttggttta agcagagtat tagaagacga tccagaggca    1080

acctatacaa catcgggagg taaaattcct attcgttgga cagcaccaga agctatcagt    1140

taccgtaaat ttacaagtgc atcagacgtg tggagttttg ggattgtaat gtgggaagtt    1200

atgacatatg gagaaagacc atattgggaa ttaagtaatc atgaagttat gaaagcaatt    1260

aacgatggat ttagattacc aactccgatg gattgtccat ctgccattta tcaactaatg    1320

atgcaatgtt ggcaacaaga aagagcacga cgtccaaaat ttgcagatat tgttagtatt    1380

ttagacaaat taattcgtgc accagatagt ttaaaaactt tagcagactt tgatcctcgt    1440

gttagtattc gattaccaag tacgtcaggt tccgaaggag ttccatttcg cacagtctcc    1500

gaatggttgg aatcaattaa aatgcaacaa tacaccgaac actttatggc agcaggttac    1560

acagcaatcg aaaaagttgt tcaaatgaca aatgatgata ttaaacgtat tggagttaga    1620

ttaccaggcc accagaaacg tattgcatat tctttattag gtttaaaaga tcaagttaat    1680

accgtgggaa ttccaattga acaaaaatta atttccgaag aagacttata agagctc       1737

<210>40

<211>497

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the fusion of prediction

<400>40

Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu Val Ser Leu

1               5                   10                  15

pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys

            20                  25                  30

Glu Asn Ser Ile Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser

        35                  40                  45

Pro Lys Thr Pro Ile Glu Lys Lys His Ala Asp Gly Ser Asp Tyr Lys

    50                  55                  60

Asp Asp Asp Asp Lys His Arg Arg Arg Lys Asn Gln Arg Ala Arg Gln

65                  70                  75                  80

Ser Pro Glu Asp Val Tyr Phe Ser Lys Ser Glu Gln Leu Lys Pro Leu

                85                  90                  95

Lys Thr Tyr Val Asp Pro His Thr Tyr Glu Asp Pro Asn Gln Ala Val

            100                 105                 110

Leu Lys Phe Thr Thr Glu Ile His Pro Ser Cys Val Thr Arg Gln Lys

        115                 120                 125

Val Ile Gly Ala Gly Glu Phe Gly Glu Val Tyr Lys Gly Met Leu Lys

    130                 135                 140

Thr Ser Ser Gly Lys Lys Glu Val Pro Val Ala Ile Lys Thr Leu Lys

145                 150                 155                 160

Ala Gly Tyr Thr Glu Lys Gln Arg Val Asp Phe Leu Gly Glu Ala Gly

                165                 170                 175

Ile Met Gly Gln Phe Ser His His Asn Ile Ile Arg Leu Glu Gly Val

            180                 185                 190

Ile Ser Lys Tyr Lys Pro Met Met Ile Ile Thr Glu Tyr Met Glu Asn

        195                 200                 205

Gly Ala Leu Asp Lys Phe Leu Arg Glu Lys Asp Gly Glu Phe Ser Val

    210                 215                 220

Leu Gln Leu Val Gly Met Leu Arg Gly Ile Ala Ala Gly Met Lys Tyr

225                 230                 235                 240

Leu Ala Asn Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile

                245                 250                 255

Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp phe Gly Leu Ser

            260                 265                 270

Arg Val Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr Thr Ser Gly Gly

        275                 280                 285

Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ser Tyr Arg Lys

    290                 295                 300

Phe Thr Ser Ala Ser Asp Val Trp Ser Phe Gly Ile Val Met Trp Glu

305                 310                 315                 320

Val Met Thr Tyr Gly Glu Arg Pro Tyr Trp Glu Leu Ser Asn His Glu

                325                 330                 335

Val Met Lys Ala Ile Asn Asp Gly Phe Arg Leu Pro Thr Pro Met Asp

            340                 345                 350

Cys Pro Ser Ala Ile Tyr Gln Leu Met Met Gln Cys Trp Gln Gln Glu

        355                 360                 365

Arg Ala Arg Arg Pro Lys Phe Ala Asp Ile Val Ser Ile Leu Asp Lys

    370                 375                 380

Leu Ile Arg Ala Pro Asp Ser Leu Lys Thr Leu Ala Asp Phe Asp Pro

385                 390                 395                 400

Arg Val Ser Ile Arg Leu Pro Ser Thr Ser Gly Ser Glu Gly Val Pro

                405                 410                 415

Phe Arg Thr Val Ser Glu Trp Leu Glu Ser Ile Lys Met Gln Gln Tyr

            420                 425                 430

Thr Glu His phe Met Ala Ala Gly Tyr Thr Ala Ile Glu Lys Val Val

        435                 440                 445

Gln Met Thr Asn Asp Asp Ile Lys Arg Ile Gly Val Arg Leu Pro Gly

    450                 455                 460

His Gln Lys Arg Ile Ala Tyr Ser Leu Leu Gly Leu Lys Asp Gln Val

465                 470                 475                 480

Asn Thr Val Gly Ile pro Ile Glu Gln Lys Leu Ile Ser Glu Glu Asp

                485                 490                 495

Leu

<210>41

<211>1716

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: pre-fusion protein construct

<400>41

ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac    60

atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata    120

atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg    180

gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg    240

gcatacgaca gtcgttttga tgaatgggta cagaaactga aagaggaaag ctttcaaaac    300

aatacgtttg accgccgcaa atttattcaa ggagcgggga agattgcagg actttctctt    360

ggattaacga ttgcccagtc ggttggggcc tttggatccg attataaaga tgatgatgat    420

aaacacagac gtagaaaaaa tcaacgtgct cgacaatccc cagaagatgt gtatttttcg    480

aaaagtgaac aattaaaacc attaaaaact tatgttgatc cgcatacgta cgaagaccca    540

aatcaagcag tattaaaatt tacaacagaa atacacccaa gttgtgttac aagacaaaaa    600

gttattggag caggtgaatt cggagaggta tataaaggta tgttaaaaac atcatcaggt    660

aaaaaagaag ttccggttgc aattaaaacc ttaaaggcag gatatacaga aaaacagcga    720

gttgattttt taggtgaagc aggaattatg ggtcaattta gccatcataa tattattcgt    780

ttggaaggag taataagtaa atataaacca atgatgatta ttacagaata catggaaaac    840

ggtgctttag ataaattttt acgtgaaaag gatggtgaat ttagtgtttt acaattggtt    900

ggtatgttaa gaggaattgc tgcaggtatg aaatatttag ctaatatgaa ttatgttcac    960

cgtgatttgg cagcaagaaa tatcctagtc aattccaatt tagtatgtaa agttagtgat    1020

tttggtttaa gcagagtatt agaagacgat ccagaggcaa cctatacaac atcgggaggt    1080

aaaattccta ttcgttggac agcaccagaa gctatcagtt accgtaaatt tacaagtgca    1140

tcagacgtgt ggagttttgg gattgtaatg tgggaagtta tgacatatgg agaaagacca    1200

tattgggaat taagtaatca tgaagttatg aaagcaatta acgatggatt tagattacca    1260

actccgatgg attgtccatc tgccatttat caactaatga tgcaatgttg gcaacaagaa    1320

agagcacgac gtccaaaatt tgcagatatt gttagtattt tagacaaatt aattcgtgca    1380

ccagatagtt taaaaacttt agcagacttt gatcctcgtg ttagtattcg attaccaagt    1440

acgtcaggtt ccgaaggagt tccatttcgc acagtctccg aatggttgga atcaattaaa    1500

atgcaacaat acaccgaaca ctttatggca gcaggttaca cagcaatcga aaaagttgtt    1560

caaatgacaa atgatgatat taaacgtatt ggagttagat taccaggcca ccagaaacgt    1620

attgcatatt ctttattagg tttaaaagat caagttaata ccgtgggaat tccaattgaa    1680

caaaaattaa tttccgaaga agacttataa gagctc                              1716

<210>42

<211>490

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the fusion of prediction

<400>42

Met Ala Tyr Asp Ser Arg Phe Asp Glu Trp Val Gln Lys Leu Lys Glu

1               5                   10                  15

Glu Ser Phe Gln Asn Asn Thr Phe Asp Arg Arg Lys Phe Ile Gln Gly

            20                  25                  30

Ala Gly Lys Ile Ala Gly Leu Ser Leu Gly Leu Thr Ile Ala Gln Ser

        35                  40                  45

Val Gly Ala Phe Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys His Arg

    50                  55                  60

Arg Arg Lys Asn Gln Arg Ala Arg Gln Ser Pro Glu Asp Val Tyr Phe

65                  70                  75                  80

Ser Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His

                85                  90                  95

Thr Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys Phe Thr Thr Glu Ile

            100                 105                 110

His Pro Ser Cys Val Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe

        115                 120                 125

Gly Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu

    130                 135                 140

Val Pro Val Ala Ile Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gln

145                 150                 155                 160

Arg Val Asp Phe Leu Gly Glu Ala Gly Ile Met Gly Gln Phe Ser His

                165                 170                 175

His Asn Ile Ile Arg Leu Glu Gly Val Ile Ser Lys Tyr Lys Pro Met

            180                 185                 190

Met Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu

        195                 200                 205

Arg Glu Lys Asp Gly Glu Phe Ser Val Leu Gln Leu Val Gly Met Leu

    210                 215                 220

Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val

225                 230                 235                 240

His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val

                245                 250                 255

Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro

            260                 265                 270

Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp Thr

        275                 280                 285

Ala Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val

    290                 295                 300

Trp Ser Phe Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg

305                 310                 315                 320

Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala Ile Asn Asp

                325                 330                 335

Gly Phe Arg Leu pro Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr Gln

            340                 345                 350

Leu Met Met Gln Cys Trp Gln Gln Glu Arg Ala Arg Arg Pro Lys Phe

        355                 360                 365

Ala Asp Ile Val Ser Ile Leu Asp Lys Leu Ile Arg Ala pro Asp Ser

    370                 375                 380

Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser Ile Arg Leu Pro

385                 390                 395                 400

Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp

                405                 410                 415

Leu Glu Ser Ile Lys Met Gln Gln Tyr Thr Glu His Phe Met Ala Ala

            420                 425                 430

Gly Tyr Thr Ala Ile Glu Lys Val Val Gln Met Thr Asn Asp Asp Ile

        435                 440                 445

Lys Arg Ile Gly Val Arg Leu Pro Gly His Gln Lys Arg Ile Ala Tyr

    450                 455                 460

Ser Leu Leu Gly Leu Lys Asp Gln Val Asn Thr Val Gly Ile Pro Ile

465                 470                 475                 480

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

                485                 490

<210>43

<211>9808

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: pre-fusion protein construct

<400>43

ctttaaacgt ggatcatttt ctttaaattt atgctgacga cctttgaatt tgcctttttt    60

cttagcaatt tcgattcctt gtgcctgacg ttccttaatt ttttttcgtt ctgattctgc    120

ttgatacttg tacaattcaa tgacaaggct attaatcaaa cgccttaaat tttcatcttc    180

aataccattc attgagggta aatttaagac ttccagggtt gcccccttaa tttgaatttg    240

attcatcaat tctgttaatt ctttattatt tcgtcctaat cgatctaatt cagtaacaat    300

aacaatatcc ccttcacgaa tatagttaag catagcttgt aattgtgggc gttcgaccga    360

ttgaccgctt aatttgtctg aaaagacctt agaaacgccc tgtaacgctt gtaattgccg    420

atctaagttc tgttctttgc tactgacacg tgcataacca attttagcca ttttcaacca    480

acctctaaaa ttctctcggt tgcaataacc aatcagcaat atctactttt tcaatttcaa    540

attgcttatc agaaattgtc ttttcgtaag cgataaaatc ttgcgcatat tgttgctcat    600

taaaaatagc caccacttcg tcattttcta aaactcgata aataaatttt ttcattttac    660

tcctcctatt atgcccaact taaatgacct attcaccaag tcaattatac tgctaaaatc    720

atattaggac aaataggtat actctattga cctataaatg atagcaactt aaaagatcaa    780

gtgttcgctt cgctctcact gcccctcgac gttttagtag cctttccctc acttcgttca    840

gtccaagcca actaaaagtt ttcgggctac tctctccttc tccccctaat aattaattaa    900

aatcttactc tgtatatttc tgctaatcat tcactaaaca gcaaagaaaa acaaacacgt    960

atcatagata taaatgtaat ggcatagtgc gggttttatt ttcagcctgt atcgtagcta    1020

aacaaatcga gttgtgggtc cgttttgggg cgttctgcca atttgtttag agtttcttga    1080

ataaatgtac gttctaaatt aaacgaagct gtcagcgcct ttatatagct ttctcgttct    1140

tcttttttta atttaatgat cgatagcaac aatgatttaa cactagcaag ttgaatgcca    1200

ccatttcttc ctggtttaat cttaaagaaa atttcctgat tcgccttcag taccttcagc    1260

aatttatcta atgtccgttc aggaatgcct agcacttctc taatctcttt tttggtcgtc    1320

gctaaataag gcttgtatac atcgcttttt tcgctaatat aagccattaa atcttctttc    1380

cattctgaca aatgaacacg ttgacgttcg cttctttttt tcttgaattt aaaccaccct    1440

tgacggacaa ataaatcttt actggttaaa tcacttgata cccaagcttt gcaaagaatg    1500

gtaatgtatt ccctattagc cccttgatag ttttctgaat aggcacttct aacaattttg    1560

attacttctt tttcttctaa gggttgatct aatcgattat taaactcaaa catattatat    1620

tcgcacgttt cgattgaata gcctgaacta aagtaggcta aagagagggt aaacataacg    1680

ctattgcgcc ctactaaacc cttttctcct gaaaatttcg tttcgtgcaa taagagatta    1740

aaccagggtt catctacttg ttttttgcct tctgtaccgc ttaaaaccgt tagacttgaa    1800

cgagtaaagc ccttattatc tgtttgtttg aaagaccaat cttgccattc tttgaaagaa    1860

taacggtaat tgggatcaaa aaattctaca ttgtccgttc ttggtatacg agcaatccca    l920

aaatgattgc acgttagatc aactggcaaa gactttccaa aatattctcg gatattttgc    1980

gagattattt tggctgcttt gacagattta aattctgatt ttgaagtcac atagactggc    2040

gtttctaaaa caaaatatgc ttgataacct ttatcagatt tgataattaa cgtaggcata    2100

aaacctaaat caatagctgt tgttaaaata tcgcttgctg aaatagtttc tttttccgtg    2160

tgaatatcaa aatcaataaa gaaggtattg atttgtctta aattgttttc agaatgtcct    2220

ttagtgtatg aacggttttc gtctgcatac gtaccataac gataaacgtt tggtgtccaa    2280

tgcgtaaatg tatcttgatt ttcgtgaatc gcttcttcgg aagtcagaac aacgccacgt    2340

ccgccaatca tgcttttttt tgagcgatac gcaaaaatag cccctttact tttacctggc    2400

ttggtagtga ttgagcgaat tttactattt ttaaatttgt actttaacaa gccgtcatga    2460

agcacagttt ctacaacaaa agggatattc attcagctgt tctcctttct tacgaaaatt    2520

aattagttag aagctacgat caaagttgaa tcacaacaaa aaaggcaatc aactaagttt    2580

ttcttaattg attgcctggt atcttcttaa agacttgaaa tcccctcaaa aacccgatat    2640

aatgggttta cagatattta agtatctgat taataaagta attaaatact ttaccaaatt    2700

ttgggtctcg acttctttaa ttgattggtg gtaatcaatt aaggctcgca gttaaaattt    2760

ctcaggcttt aactggtcgt ggctcttttt ttgtattctt tattcagttc gttgtttcgt    2820

tatatctagt atatcgcttt ttaaaaaaat aagcaatgat ttcgtgcatt attcacacga    2880

aatcattgct tttttcttct tccatttcta actccaatgt tacttgttct gtttctggtt    2940

ctggttctgt tggctcattt gggattaaat ccactactag cgttgagtta gttccgtctc    3000

taatagccgg ttaagtaata gccggttaag tggtcaaact ttgggaaaat ctcaacccgc    3060

attaagtttt gatgccatga caatcgttgg aaatttgaac aaaactaatg ctaaaaagct    3120

atctgacttt atgagtgtag agccacaaat acgactttgg gatatacttc aaacaaagtt    3180

taaagctaag gcacttcaag aaaaagttta tatcgaatat gacaaagtaa aagcagatac    3240

ttgggataga cgtaatatgc gtgttgaatt taatcccaat aaactcacac atgaagaaat    3300

gatttggtta aaacaaaata ttatcgacta catggaagat gacggtttta caagattaga    3360

cttagctttt gattttgaag atgatttgag cgattactat gcaatgactg ataaagcagt    3420

taagaaaact gttttttatg gtcgtaatgg caagccagaa acaaaatatt ttggtgtccg    3480

tgatagtgat agatttatta gaatttataa taaaaaacaa gaacgtaaag ataacgcaga    3540

tgttgaagtt gtgtttgaac atttatggcg tgtagaagtt gaattaaaaa gagatatggt    3600

tgattactgg aatgattgtt ttaatgattt acacatcttt gaaacctgcg tgggctactt    3660

tagaaaaaat taatgagcaa gctatggttt atactttgtt gcatgaagaa agtatgtggg    3720

gaaagctaag taagaatact aagactaaat ttaaaaaatt gattagagaa atatctccaa    3780

ttgatttaac ggaattaatg aaatcgactt taaaagcgaa cgaaaaacaa ttgcaaaagc    3840

agattgattt ttggcaacgt gaatttaggt tttggaagta aaataagttt tatttgataa    3900

aaattgctaa ttcagtataa ttaatattta cgaggtgaca taacgtatga aaaaatcaga    3960

ggattattcc tcctaaatat aaaaatttaa aatttaggag gaagttatat atgactttta    4020

atattattga attagaaaat tgggatagaa aagaatattt tgaacactat tttaatcagc    4080

aaactactta tagcattact aaagaaattg atattacttt gtttaaagat atgataaaaa    4140

agaaaggata tgaaatttat ccctctttaa tttatgcaat tatggaagtt gtaaataaaa    4200

ataaagtgtt tagaacagga attaatagtg agaataaatt aggttattgg gataagttaa    4260

atcctttgta tacagttttt aataagcaaa ctgaaaaatt tactaacatt tggactgaat    4320

ctgataaaaa cttcatttct ttttataata attataaaaa tgacttgctt gaatataaag    4380

ataaagaaga aatgtttcct aaaaaaccga tacctgaaaa caccataccg atttcaatga    4440

ttccttggat tgattttagt tcatttaatt taaatattgg taacaatagc agctttttat    4500

tgcctattat tacgataggt aaattttata gtgagaataa taaaatttat ataccagttg    4560

ctctgcaact tcatcattct gtatgtgatg gttaccatgc ttcactattt atgaatgaat    4620

ttcaagatat aattcatagg gtagatgatt ggatttagtt tttagatttt gaaagtgaat    4680

ttaattttat acacgtaagt gatcataaaa tttatgaacg tataacaacc acattttttg    4740

gttgcttgtg gttttgattt tgaatttggt tttgaactta tggactgatt tattcagtcc    4800

attttttgtg cttgcacaaa aactagcctc gcagagcaca cgcattaatg acttatgaaa    4860

cgtagtaaat aagtctagtg tgttatactt tacttggaag atgcaccgaa taaaaaatat    4920

tgaagaacaa ctagcaaaag attttaaaga gttattttat tttaagtctt tataacatga    4980

gtgaagcgaa tttttaaatt tcgatagaaa tttttacatc aaaaagcccc ctgtcaaaat    5040

tgacgaaggg ggttttttgg cgcacgcttt tcgttagaaa tatacaagat tgaaaatcgt    5100

gtataagtgc gccctttgtt ttgaacttag cacgttacat caatttttta aaatgatgta    5160

taagtgcgcc cttttaaatt ttgagtgatt atattttttg agttagaaaa agggattggg    5220

aaaatttccc aaaataattt aaaaaataag caaaaatttt cgatagagaa tgtgctattt    5280

tttgtcaaag gtgtatacct tgactgtgct tgctgttaca ttaagtttat ttttaagtta    5340

ttaaaaaaga aatagctttt aaagtttggc tcgctgtcgc tttataaagc tgattgactt    5400

ttgattgcaa actacttaaa gaaaacaaac tcggactatt cgttttcttc tctttggttt    5460

gaacatcagc aattatcccc tcttgattgc ctattttagc ttgtttagaa gaaacaaaag    5520

ctaaaagctc ctcttgggtt ttaaaacgct gtgtggggct tagaacgccc ttaaacgacc    5580

cttggtttac ttttatacta gcttccacct cgaaaaaagg ttctttttta aaattctcta    5640

tggcttcctg gcgctgaaaa aataaggtat aaggtgggcg tttgaacacg tcctagtgaa    5700

aatgtacctt gtacgcccct tctgttgtaa atttaacgta tacaaagggc ttgcgttcat    5760

gccgatcaac caatcggcaa tttggcgtgt ttgcgcttct tgataaaagg gatagtaatt    5820

cattccaggt tgcaaatttt gaaaaccgct tcggattaca tctttttcta agctattgat    5880

ccatagtctt ttaaatgttt tatcttttga aaaggcattt gctttatgga taatcgacca    5940

ggcgatattt tcaccttctc tgtcgctatc tgttgcaaca ataattgtat ttgccttttt    6000

gagaagttct gcaacaattt taaactgctt tcccttatct tttgcaactt caaaatcgta    6060

tcgatcagga aaaatcggca aagattcaag tttccaattt tgccactttt cgtcataatg    6120

acctggttct gctaattcca ctaaatgccc aaaaccaaag gtgataaacg tttcatctgt    6180

aaatagtggg tctttgatct caaaataacc gtcttttttg gtgctttgtt ttaaagcact    6240

tgcgtaggct aatgcctggc ttggtttttc agctaaaata accgtactca ttaactatcc    6300

ctcttttcat tgttttttct ttgatcgact gtcacgttat atcttgctcg ataccttcta    6360

aacgttcggc gattgattcc agtttgttct tcaacttctt tatcggataa accattcaaa    6420

aacaaatcga aagcatggat gcgccgcgtg cggctgctgg agatggcgga cgcgatggat    6480

atgttctgcc aagggttggt ttgcgcattc acagttctcc gcaagaattg attggctcca    6540

attcttggag tggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg    6600

cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta    6660

caatccatgc caacccgttc catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca    6720

gcggtccagt gatcgaagtt aggctggtaa gagccgcgag cgatccttga agctgtccct    6780

gatggtcgtc atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc    6840

cggaagcgag aagaatcata atggggaagg ccatccagcc tcgcgtcgca atacgactca    6900

ctatagggcg aattgggtac cgggcccccc ctcgaggtcg acggtatcga taagcttgat    6960

atcgaattcc tgcagcccgg gggatccact agttctagag cggccgccac cgcggtggag    7020

ctccagcttt tgttcccttt agtgagggtt aatgctagaa atattttatc tgattaataa    7080

gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa    7140

ccgccttgca gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta    7200

actggcttgg aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc    7260

atgacttcaa gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt    7320

gcatgtcttt ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac    7380

tgaacggggg gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt    7440

gtcaggcgtg gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa    7500

ggcaggaaca ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag    7560

tcctgtcggg tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg    7620

gcggagccta tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc    7680

ctggcatctt ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga    7740

acgaccgagc gtagcgagtc agtgagcgag gaagcggaat atatcctgta tcacatattc    7800

tgctgacgca ccggtgcagc cttttttctc ctgccacatg aagcacttca ctgacaccct    7860

catcagtgcc aacatagtaa gccagtatac actccgctag cgctgatgtc cggcggtgct    7920

tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag ggacagctga tagaaacaga    7980

agccactgga gcacctcaaa aacaccatca tacactaaat cagtaagttg gcagcatcac    8040

ccgacgcact ttgcgccgaa taaatacctg tgacggaaga tcacttcgca gaataaataa    8100

atcctggtgt ccctgttgat accgggaagc cctgggccaa cttttggcga aaatgagacg    8160

ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg    8220

tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca    8280

ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc    8340

agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa    8400

agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc    8460

tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg    8520

atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct    8580

ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt    8640

gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct    8700

cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact    8760

tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc    8820

cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta    8880

atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt    8940

attggtgccc ttaaacgcct ggtgctacgc ctgaataagt gataataagc ggatgaatgg    9000

cagaaattcg aaagcaaatt cgacccggtc gtcggttcag ggcagggtcg ttaaatagcc    9060

gcttatgtct attgctggtt taccggttta ttgactaccg gaagcagtgt gaccgtgtgc    9120

ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat aattgacgat    9180

atgatcattt attctgcctc ccagagcctg ataaaaacgg ttagcgcttc gttaatacag    9240

atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg    9300

tgcagggcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg    9360

gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga    9420

cgatagtcat gccccgcgcc caccggaagg agctaccgga cagcggtgcg gactgttgta    9480

actcagaata agaaatgagg ccgctcatgg cgttgactct cagtcatagt atcgtggtat    9540

caccggttgg ttccactctc tgttgcgggc aacttcagca gcacgtaggg gacttccgcg    9600

tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag    9660

acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac    9720

cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca    9780

cccgtggcca ggacccaacg ctgcccga                                       9808

<210>44

<211>26

<212>PRT

＜213〉listeria monocytogenes (Listeria monocytogenes)

<400>44

Met Lys Lys Ile Met Leu Val phe Ile Thr Leu Ile Lau Val Ser Leu

1               5                   10                  15

Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp

            20                  25

<210>45

<211>59

<212>PRT

＜213〉listeria monocytogenes (Listeria monocytogenes)

<400>45

Met Thr Asp Lys Lys Ser Glu Asn Gln Thr Glu Lys Thr Glu Thr Lys

1               5                   10                  15

Glu Asn Lys Gly Met Thr Arg Arg Glu Met Leu Lys Leu Ser Ala Val

            20                  25                  30

Ala Gly Thr Gly Ile Ala Val Gly Ala Thr Gly Leu Gly Thr Ile Leu

        35                  40                  45

Asn Val Val Asp Gln Val Asp Lys Ala Leu Thr

    50                  55

<210>46

<211>53

<212>PRT

<213>Bacillus subtillus

<400>46

Met Ala Tyr Asp Ser Arg Phe Asp Glu Trp Val Gln Lys Leu Lys Glu

1               5                   10                  15

Glu Ser Phe Gln Asn Asn Thr phe Asp Arg Arg Lys Phe Ile Gln Gly

            20                  25                  30

Ala Gly Lys Ile Ala Gly Leu Ser Leu Gly Leu Thr Ile Ala Gln Ser

        35                  40                  45

Val Gly Ala Phe Gly

    50

<210>47

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>47

gtcaaaacat acgctcttat c                                              21

<210>48

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>48

acataatcag tccaaagtag atgc                                           24

<210>49

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>49

ctctggtacc tcctttgatt agtatattc                                      29

<210>50

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>50

ctctggatcc atccgcgtgt ttcttttcg                                      29

<210>51

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the former determinant insert of monoclonal antibody

<400>51

gattataaag atgatgatga taaa                                           24

<210>52

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the former determinant of monoclonal antibody

<400>52

Asp Tyr Lys Asp Asp Asp Asp Lys

1               5

<210>53

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the former determinant insert of monoclonal antibody

<400>53

gaacaaaaat taattagtga agaagattta                                     30

<210>54

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the former determinant of monoclonal antibody

<400>54

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1               5                   10

<210>55

<211>9

<212>PRT

<213>Mus sp.

<400>55

Ser Pro Ser Tyr Val Tyr His Gln Phe

1               5

<210>56

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the former determinant of monoclonal antibody

<400>56

Ser Pro Ser Tyr Ala Tyr His Gln Phe

1               5

<210>57

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>57

ctctggtacc tcctttgatt agtatattc                                      29

<210>58

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>58

caatggatcc ctcgagatca taatttactt catccc                              36

<210>59

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>59

atttctcgag tccatggggg gttctcatca tc                                  32

<210>60

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>60

ggtgctcgag tgcggccgca agctt                                          25

<210>61

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>61

cgattcccct agttatgttt accaccaatt tgctgca                             37

<210>62

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>62

gcaaattggt ggtaaacata actaggggaa t                                   31

<210>63

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the former determinant insert of monoclonal antibody

<400>63

agtccaagtt atgcatatca tcaattt                                        27

<210>64

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>64

cgatagtcca agttatgcat atcatcaatt tgc                                 33

<210>65

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>65

gtcgcaaatt gatgatatgc ataacttgga ctat                                34

<210>66

<211>8

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: consensus sequence

<220>

<221>misc_feature

<222>(1)..(1)

<223>n is a，c，g，or u

<400>66

naggaggu                                                             8

<210>67

<211>19

<212>DNA

＜213〉listeria monocytogenes (Listeria monocytogenes)

<400>67

aaggagagtg aaacccatg                                                 19

<210>68

<211>240

<212>DNA

＜213〉listeria monocytogenes (Listeria monocytogenes)

<400>68

ggtacctcct ttgattagta tattcctatc ttaaagtgac ttttatgttg aggcattaac    60

atttgttaac gacgataaag ggacagcagg actagaataa agctataaag caagcatata    120

atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg    180

gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg    240

<210>69

<211>240

<212>DNA

＜213〉listeria monocytogenes (Listeria monocytogenes)

<400>69

ggtacctcct ttgattagta tattcctatc ttaaagttac ttttatgtgg aggcattaac    60

atttgttaat gacgtcaaaa ggatagcaag actagaataa agctataaag caagcatata    120

atattgcgtt tcatctttag aagcgaattt cgccaatatt ataattatca aaagagaggg    180

gtggcaaacg gtatttggca ttattaggtt aaaaaatgta gaaggagagt gaaacccatg    240

<210>70

<2ll>5

<212>PRT

＜213〉listeria monocytogenes (Listeria monocytogenes)

<400>70

Thr Glu Ala Lys Asp

1               5

<210>71

<211>5

<212>PRT

＜213〉listeria monocytogenes (Listeria monocytogenes)

<400>71

Asp Lys Ala Leu Thr

1               5

<210>72

<211>5

<212>PRT

<213>Bacillus subtillus

<400>72

Val Gly Ala Phe Gly

1               5

Claims (39)

1, in individual, causes antagonism EphA2 and express the immunoreactive method of cell, described method comprises to the composition administration of individual body and function effective dose expresses the immune response of cell to cause antagonism EphA2, and said composition comprises the Listeria of expressing the EphA2 antigenic peptide.

2, the method described in according to claim 1, wherein said Listeria is listeria monocytogenes.

3, the method described in according to claim 2, wherein said Listeria is attenuation.

4, method according to claim 1, the nucleic acid of the EphA2 antigenic peptide of wherein encoding comprises the nucleotide sequence of the secretion signal of encoding, and this nucleotide sequence is connected with the series of operations of coding EphA2 antigenic peptide.

5, method according to claim 1, wherein said individuality suffers from cancer.

6, method according to claim 5, wherein said cancer are the epithelial cell sources.

7, method according to claim 5, wherein said cancer are the T cell sources.

8, method according to claim 6, wherein said cancer are cutaneum carcinoma, lung cancer, colon cancer, breast cancer, prostate cancer, carcinoma of urinary bladder or cancer of pancreas, or clear-cell carcinoma or melanoma.

9, method according to claim 7, wherein said cancer are leukaemia or lymthoma.

10, method according to claim 1, wherein said individuality suffer from the excessive proliferative disorders of Non-cancerous.

11, method according to claim 10, wherein said excessive proliferative disorders is the epithelial cell disease.

12, method according to claim 11, wherein said excessive proliferative disorders are asthma, chronic pulmonary obstruction disease, pulmonary fibrosis, bronchus overreaction, psoriasis and seborrhea.

13, treatment suffers from the method that EphA2 expresses the individual human of the excessive proliferative diseases of cell, described method comprises to the composition administration of this body and function effective dose expresses the excessive proliferative disorders of cell with treatment EphA2, and this said composition comprises the Listeria of expressing the EphA2 antigenic peptide.

14, method according to claim 13, wherein said Listeria is listeria monocytogenes.

15, method according to claim 13, wherein said individuality suffers from cancer.

16, method according to claim 15, wherein said cancer are the epithelial cell sources.

17, method according to claim 15, wherein said cancer are the endothelial cell sources.

18, method according to claim 15, wherein said cancer are the T cell sources.

19, method according to claim 15, wherein said cancer comprise the cell with respect to non-cancer cell overexpression EphA2, and this non-cancer cell has the types of organization of described cancer cell.

20, method according to claim 16, wherein said cancer are cutaneum carcinoma, lung cancer, colon cancer, breast cancer, prostate cancer, carcinoma of urinary bladder or cancer of pancreas, or clear-cell carcinoma or melanoma.

21, method according to claim 18, wherein said cancer are leukaemia or lymthoma.

22, method according to claim 13, wherein said individuality suffer from the excessive proliferative disorders of Non-cancerous.

23, method according to claim 22, wherein said excessive proliferative disorders is the epithelial cell disease.

24, method according to claim 23, wherein said excessive proliferative disorders are asthma, chronic pulmonary obstruction disease, pulmonary fibrosis, bronchus overreaction, psoriasis and seborrhea.

25, according to claim 1 or 13 described methods, wherein said EphA2 polypeptide comprises total length EphA2.

26, according to claim 1 with 13 each described methods, wherein said EphA2 polypeptide comprises the ectodomain of EphA2.

27, according to claim 1 with 13 methods described in each, wherein said EphA2 polypeptide is chimeric polyeptides, this chimeric polyeptides comprises at least antigenic portions and the second polypeptide of EphA2.

28, according to claim 1 or 13 described methods, wherein said composition comprises the Listeria of multiple expression EphA2 antigenic peptide.

29, according to claim 1 or 13 described methods, multiple EphA2 antigenic peptide is expressed in the Listeria of wherein said expression EphA2 antigenic peptide.

30, according to claim 1 with 13 each described methods, the method also comprises and gives extra anticancer therapy.

31, method according to claim 30, wherein said extra anticancer therapy are excitability EphA2 antibody.

32, method according to claim 30, wherein said extra anticancer therapy are the anti-idiotypes of excitability EphA2 antibody.

33, method according to claim 30, wherein said extra anticancer therapy are chemotherapy, biology treatment, immunization therapy, radiotherapy, hormone therapy or operation.

34, according to claim 1 with 13 each described methods, wherein said administration is in the mucous membrane, parenteral, muscle, in the abdominal cavity, intravenous or oral administration.

35, according to claim 1 or 13 described methods, wherein said administration causes CD4⁺-T cell response, CD8⁺-t cell response, innate immune response, antibody response or one or more aforementioned combinations of replying.

36, method according to claim 35, wherein said administration causes CD4⁺-t cell response and CD8⁺-t cell response.

37, treatment suffers from the method that the individual human of relevant disease occurs with abnormal vascular, described method comprises to the composition administration of individual body and function effective dose and with abnormal vascular relevant disease occurs with treatment that said composition comprises the Listeria of expression EphA2 antigenic peptide.

38, method according to claim 1, wherein said individuality suffer from the disease relevant with the abnormal vascular generation.

39, according to claim 37 or 38 described methods, wherein said disease is macular degeneration, DRP, retinopathy of prematurity, reangiostenosis, infantile hemangioma, verruca vulgaris, psoriasis, Kaposi sarcoma, neural line fibroma, recessive atrophic type bubble epithelium dissociate disease before disease, rheumatic arthritis, stiff spondylitis, systemic lupus, chronic eczema arthropathy, Lay Te Shi syndrome and house Ge Lunshi syndrome, mullerianosis, the pregnancy period eclampsia, arteriosclerosis and coronary artery disease.

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