CN101094686A - Use of flagellin in tumor immunotherapy - Google Patents

Abstract

The invention provides a fusion protein comprising a flagellin adjuvant and a tumor antigen. Also provided are compositions comprising a flagellin adjuvant and a tumor antigen. The invention also provides pharmaceutical formulations for inducing an immune response against tumor antigen and methods thereof, and a method for treating tumor in tester.

Description

The purposes of flagellin in tumour immunotherapy

Related application data

The application requires the U.S. Provisional Application sequence number 60/636 of December in 2004 submission on the 16th, the U.S. Provisional Application sequence number 60/709 that on August 19th, 635 and 2005 submitted to, 609 priority is incorporated this paper into as a reference with the disclosure of these provisional application is complete by reference.

Federal government's support statement

The present invention obtains supporting from the government according to grant number R01-AI051319 and T32-AI007401 of National Institute of Health (National Institutes of Health).U.S. government has some right of the present invention.

Invention field

The present invention relates to the purposes of the immunne response that flagellin adjuvant, tumor antigen and its fusion rotein be used to produce tumor (for example, in prevention or Therapeutic Method).

Background of invention

The treatment of the treatment breast carcinoma of current acceptance comprises operation, chemotherapy and X-ray therapy.The therapy that immunotherapy representative is new relatively, people have produced very big interest (people (2003) J.Clin.Oncol.21:2415-2432 such as Ribas to it; People such as Scanlan (2001) BreastCancer Res.3:95-98; People such as Jager (2002) Curr.Opin.Immunol.14:178-182; People such as Ko (2003) Clin.Cancer Res.9:3222-3234).The major advantage of immunological method is that it is not invasive and its selectivity target is decided breast cancer cell, does not injure normal cell.In recent years, research worker determined many sudden changes or by the inappropriate expressed protein of breast cancer cell, they can be used as the useful target of immunotherapy.For example, used mucin (MUC-I), HER-2/neu, CEA and hTERT vaccine to start I/II clinical trial phase (summary is seen people such as Ko (2003) Clin.Cancer Res.9:3222-3234).Although many these researchs have disclosed enhanced immunoreactive example, for example, T cell proliferation or cytotoxic t cell activity, clinical effectiveness have been confined to relative fraction patient.

It may be because the combination of several factors that tumor vaccine is lacked strong clinical response.Because most target tumor antigens are crossed expression, but do not suddenly change, possible self tolerance has hindered strongly replys.Yet,, may not be such situation for these antigenic mutant forms.Alternatively, the scheme that has been used for immune patients can promote to produce low-affinity but not the T cell (people such as Ko (2003) Clin.Cancer Res.9:3222-3234 is seen in discussion) of high affinity (avidity); The high affinity cell is the most effective in tumor cell is removed.At last, the excessive generation transforming growth factor of tumor cell (TGF-β) can significantly limit the effectively effectiveness of vaccination strategies.

The value of adjuvant in strengthening weak immunne response has been set up in work in other systems.Thereby, may need effective adjuvant to allow immune system not only to overcome the immunosuppressive action of the TGF β in tumor source, and promote to produce high affinity antitumor T cell, NK cell or antibody.Should be noted that many vaccine schemes have used multiple adjuvant: from each cytokine (for example, IL-2, IL-12, IFN-or GM-CSF) to microbial cell component (for example, Detox B or BCG).Although each cytokine has certain adjuvanticity, their effectiveness may be subjected to the restriction of their biological activity scopes separately.The significant need development﹠ testing promotes the new adjuvant that more effective antitumor is replied.

People are desirable to provide improved reagent, pharmaceutical preparation and the method that is used to produce at the immunne response of cancer.

Summary of the invention

A first aspect of the present invention is a fusion rotein, and it consists of or is essentially: (a) flagellin adjuvant, this flagellin adjuvant consist of or are essentially the terminal constant region of (i) flagellin N-; The (ii) terminal constant region of flagellin C-; (b) tumor antigen between terminal constant region of N-and the terminal constant region of C-(for example, be inserted into or replace the some or all of of flagellin hypervariable region, this flagellin hypervariable region is optional partly or completely to be lacked).

Another aspect of the present invention is the nucleic acid of coding fusion rotein as indicated above.In some embodiments, the nucleic acid promoter that is operably connected.

Another aspect of the present invention is the carrier that comprises nucleic acid as indicated above.

Another aspect of the present invention is to comprise the nucleic acid as indicated above or the host cell of carrier.In some embodiments, the coded fusion rotein of host cell expression.

Another aspect of the present invention is the method for preparation fusion rotein as indicated above, and this method comprises that the host cell of the nucleic acid that will comprise coding fusion rotein as indicated above enough cultivates under the condition of the described fusion rotein of generation in proper culture medium.Randomly, collect fusion rotein from host cell or culture medium.

Another aspect of the present invention is a compositions, it comprises, consists of or form and is essentially: (a) flagellin adjuvant, (b) (tumor antigen and flagellin adjuvant separate tumor antigen, perhaps interconnective, it is the form of fusion rotein, for example, fusion rotein as described herein).

Another aspect of the present invention is pharmaceutical preparation, and it comprises fusion rotein as indicated above or compositions in pharmaceutically suitable carrier.

Another aspect of the present invention is to the immunne response of tumor antigen (for example to produce in the experimenter, the immunne response of generation antibody and/or inducing cell mediation) method, it comprises to induce in described experimenter uses fusion rotein as indicated above, compositions or pharmaceutical preparation to the immunne response effective dose of tumor antigen to described experimenter.

Of the present invention is the method for treatment experimenter's tumor more on the one hand, and this method comprises fusion rotein as indicated above, compositions or the pharmaceutical preparation to described experimenter's administering therapeutic effective dose.

In the specific embodiments of the inventive method, the experimenter is that mammal is subjected to examination, primates experimenter or human experimenter.

Another aspect of the present invention is the purposes that fusion rotein as described herein or compositions are used to prepare the medicine that carries out Therapeutic Method as described herein.

These and other method of the present invention provides in the description of the invention below.

The accompanying drawing summary

TNF alpha expression in the lung of the BALB/c mouse of the flagellin 229 that Fig. 1 has described to give flagellin or sudden change by instiling in the sheath.

Fig. 2 has described the influence of flagellin to tumor growth.The empty circles representative gives the mice of the flagellin of Fra-1 antigen and inactivation form.The solid circles representative gives the mice of the flagellin of Fra-1 antigen and activity form.

Fig. 3 show with flagellin of Yersinia pestis and F1 antigen immune cause a large amount of anti--F1 antibody produces.(figure is a) with (i.t.) or the immune female BALB/c mouse of intranasal (i.n) in 10 μ g F1+1 μ g flagellin (FliC) tracheas.Control animal is only with immunity in the flagellin trachea of 10 μ g F1 or 1 μ g, 229 sudden changes.4 whens week mice is strengthened in a like fashion and 2 week the back collect blood plasma by elisa assay.The ratio of the numeral IgG1/IgG2a isotype in the bar shaped. ^*Point out significance,statistical with respect to contrast, ^*Point out that intranasal tires statistically greater than (p＜0.007) in the trachea.(figure b) anti--F1 antibody titer from obtaining with 10 μ gF1+1 μ g flagellin intranasal mice immunized.Every on behalf of a mice and arrow, line point out reinforced immunological.(figure c) with in the FliC of 10 μ gF1 and recruitment or the 5 μ g229 tracheas to the in addition immunity of female BALB/c mouse, and reinforcement when the 4th week.After reinforcement, measured in 2 weeks blood plasma anti--F1IgG tires.(figure d) only uses one group of female BALB/c mouse of 5 μ g flagellin intranasal immunity, and strengthens in an identical manner when the 4th week.Measure in 2 week backs anti--Flic antibody titer (average anti--Flic tires=8.5 * 10 ⁵) then with 10 μ gF1+1 μ g Flic to carrying out intranasal immunity through the flagellin mice immunized and strengthening.Strengthen 2 weeks of back, measure that anti--F1 tires and with the potency ratio of the animal that does not contact flagellin of 10 μ gF1+1 μ gFlic or 229 immunity.On behalf of average antibody, bar shaped tire ± s.e.m.Each immune group is used 7 female BALB/c mouse.

Fig. 4 shows that flagellin stimulator antigen specificity replys and need the T cell.(figure is a) with the group of 7 female BALB/c mouse of 10 μ gF1 antigens+1 μ g flagellin (Flic) intranasal immunity, and when the 4th week with PBS, only 1 μ gFlic, only 10 μ gF1 or 10 μ g F1+1 μ g FliC reinforcement. ^*Point out with respect to PBS or the significance,statistical of the FliC animal of strengthening only, ^*Pointing out to strengthen with F1+FliC causes antibody titer statistically greater than F1 antigen (p＜0.01) only.On behalf of average antibody, bar shaped tire ± s.e.m.(figure b) carries out the intranasal immunity with 10 μ g F1+1 μ g FliC to one group 7 athymic nude mices (BALB/cAnNCr-nu/nu) and strengthens.2 weeks were collected blood plasma after reinforcement, were used for carrying out the analysis that anti-F1 IgG tires by ELISA. ^*Point out the significance,statistical (p＜0.001) compared of normal BALB/c mouse with immunity in the same manner.

Fig. 5 has described the needs for the adjuvant effect of flagellin.With 10 μ gF1 antigens+1 μ g flagellin (FliC) or the sudden change flagellin (229) to TNFR ^-/-(scheme a) or IL6 ^-/-(figure b) and wild type B6; 129 control mice are carried out immunity in the trachea. ^*Point out TNFR ^-/-Tire statistically less than B6; 129 contrasts (p＜0.001).With 10 μ gF1+1 μ gFliC or 229 (figure c) immune C3H/HeJ (Tlr4 P712H mutant) and wild type C3H/HeN mice.With 10 μ gF1+1 μ gFHC or 229 couples of IFN α/β R ^-/-(figure d) and IFN γ ^-/-The intranasal immunity is carried out in (figure e) mice and corresponding wild-type contrast.In each immune group with 7 female mices.Strengthen mice in an identical manner in 4 weeks, and collect blood plasma in 2 week backs, be used for by elisa assay anti--F1 tires.On behalf of average antibody, bar shaped tire ± s.e.m.

Fig. 6 shows that flagellin promotes for the protective response with Yersinia pestis CO92 intranasal infection.With 10 μ gF1 antigens+1 μ g flagellin (FliC) or 15 female BALB/c mouse of PBS immunity (figure group a), and strengthen in a like fashion only in 4 weeks.Strengthen 2 weeks of back and collect blood plasma, be used for by the elisa assay antibody titer (average anti--F1 tires=9.4 * 10 ⁵).1 Zhou Houyong equals 100xLD ₅₀Yersinia pestis CO92 dosage mice carried out intranasal attack (challenge).Attack the back and monitor mice 30 days.With 10 μ gF1+1 μ gFliC or only PBS to the IgH of 10 antibody deficiencies ^-/-Mice (figure b) group is carried out the intranasal immunity, and strengthens in an identical manner when 4 weeks.2 Zhou Houyong equal 155xLD ₅₀Yersinia pestis dosage carry out intranasal and attack.With 10 μ gF1+1 μ g FliC to 10 wild type C57BL/6 mices (group c) and female IFN γ ^-/-The group of mice (figure d) is carried out the intranasal immunity and is strengthened.Strengthen 2 weeks of back and collect blood plasma, be used for by the elisa assay antibody titer (anti--F1 tires 〉=1 * 10 ⁶).1 Zhou Houyong equals 150xLD ₅₀Yersinia pestis dosage carry out that intranasal is attacked and monitor 16 days after attack.

Fig. 7 shows that flagellin is effective adjuvant of inhuman primate.With 150 μ gF1/V fusion rotein+50 μ g flagellin intranasal (n=6) or the immune female macaque of intramuscular (n=6) (Macaca fascicularis).Only control animal (n=3) is carried out intranasal or intramuscular immunity with PBS.Body temperature does not significantly change in 12 hours, and 4h, 12h and 24h collect TNF-α in the blood plasma after immunity.In an identical manner animal is strengthened in 4 weeks, and, be used for analyzing by ELISA at 2 weeks back collection blood plasma.Average anti--F1/V antibody titer ± s.e.m is pointed out in bar shaped, ^*Expression is with respect to the significance,statistical (p＜0.006) of intranasal immunity.

Fig. 8 shows that the fusion rotein that contains flagellin and Yersinia pestis F1 and V protein matter keeps the flagellin biologic activity.

DESCRIPTION OF THE PREFERRED

The present invention partly is based on and finds that flagellin and its fragment can be used as adjuvant and be used for strengthening the anti-tumor immune response that produces in the experimenter.

Unless otherwise noted, all technology used herein and scientific terminology all have the identical implication with one of ordinary skill in the art's common sense of the present invention.The term that uses in the description of the present invention only is used to describe specific embodiment and be not intended to restriction the present invention.The all complete by reference this paper that incorporates into of all publications mentioned in this article, patent application, patent and other list of references.

Unless spell out difference in the context, " a " of the singulative that uses in description of the present invention and appending claims, " an " and " the " are intended to comprise plural form.

" form substantially and be " (consisting essentially of) used herein refers to that pointed peptide, protein, fusion rotein, nucleic acid, chemical compound, compositions or the like do not comprise any other material composition (that is, influencing the composition of the structure and/or the function of described peptide, protein, fusion rotein, nucleic acid, chemical compound or compositions in fact).

In representative embodiment more of the present invention, peptide of the present invention, protein, fusion rotein, nucleic acid and/or cell are " separated "." separated " refers to that peptide, protein, fusion rotein, nucleic acid and/or cell are at least from other component partial purification.

1. Tumor antigen

The molecule that term used herein " tumor antigen " refers to tumor cells expression (for example, protein or peptide), and (a) different with the copy (counterpart) of expressing in its normal cell in nature, perhaps (b) in tumor cell with than horizontal expression higher in normal cell.Thereby, tumor antigen can different with its copy in normal cell (for example, when molecule be protein) by one or more aminoacid or it can be identical with described copy.Some tumor antigens are not expressed by normal cell, perhaps in tumor cell with at least 2 times in the counter part of tumor cell high horizontal expressions (for example, about 2 times, 3 times, 5 times, 10 times, 20 times, 40 times, 100 times, 500 times, 1000 times, 5000 times or 15000 times high).

Any suitable tumor antigen can be used to implement the present invention.Tumor antigen includes but not limited to: the modified forms of naturally occurring tumor antigen and its induce immune response in the experimenter, also comprise and the bonded antigen of tumor cell, with to the special antigen of tumor cell and aforementioned in the experimenter the modified form of induce immune response.The term tumor antigen also comprises corresponding to the proteinic antigen relevant with inducing of tumor, as oncogene virus antigen (for example, human papillomavirus antigen).Exemplary oncologic antigen includes but not limited to, the HER2/neu of breast carcinoma and BRCA1 antigen, MART-1/MelanA (melanoma antigen), Fra-1 (breast carcinoma), NY-BR62, NY-BR85, hTERT, gp100, tryrosinase, TRP-1, TRP-2, NY-ESO-1, CDK-4, beta-catenin is white, MUM-1, Caspase-8, KIAA0205, SART-1, PRAME, with p15 antigen, MAGE family (melanoma antigen), BAGE family (melanoma antigen), DAGE/PRAME (as DAGE-1), GAGE family (melanoma antigen), RAGE family (as RAGE-1), the member of SMAGE family, NAG, TAG-72, CA125, the proto-oncogene of sudden change, as p21ras, the tumor suppressor gene of sudden change is as p53, the virus antigen (as HPV E6 and E7) that tumor is relevant, the HOM-MEL-55 of SSX family, NY-COL-2, HOM-HD-397, HOM-RCC-1.14, HOM-HD-21, HOM-NSCLC-11, HOM-MEL-2.4, HOM-TES-11, RCC-3.1.3, NY-ESO-1, member with SCP family.The member of MAGE family includes, but not limited to MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-11 and MAGE-12.The member of GAGE family includes, but not limited to GAGE-1, GAGE-6.For example see Van den Eynde and van der Bruggen, (1997) Curr.Opin.Immunol.9:684-693; With people such as Sahin, the summary of (1997) Curr.Opin.Immunol.9:709-716.

Tumor antigen can also be but be not limited to, HEP's mucin (Muc-1; 20 aminoacid cores of Muc-1 glycoprotein repeat, and are present on breast cancer cell and the pancreatic cancer cell), MUC-2, MUC-3, MUC-18, carcinoembryonic antigen (CEA), raf oncoprotein, CA-125, GD2, GD3, GM2, TF, sTn, gp75, EBV-LMP1﹠amp; 2, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), GnT-V intron V sequence (N-acetyl-glucosamine transferase V intron V sequence), alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, β-HCG, gp43, HSP-70, p17mel, HSP-70, gp43, HMW, HOJ-1, the melanoma ganglioside, TAG-72, the proto-oncogene of sudden change, as p21ras, the tumor suppressor gene of sudden change, as p53, estrogen receptor, the butterfat globulin, telomerase, nuclear matrix protein, prostate acid phosphatase, protein MZ2-E, multiform epithelium mucin (PEM), folic acid-binding protein LK26, the epithelial growth factor receptor of truncate (EGFR), Thomsen-Friedenreich (T) antigen, GM-2 and GD-2 ganglioside, multiform epithelium mucin, folic acid-binding protein LK26, human chorionic gonadotropin (HCG), pancreatic oncofetal antigen, cancer antigen 1 5-3,19-9,549,195, squamous cell carcinoma antigen (SCCA), ovarian cancer antigen (OCA), pancreas cancer-associated antigen (PaA), EBNA (eb nuclear antigen) 1-6, gp75, chimeric protein P210BCR-ABL, lung resistance protein (LRP) Bcl-2 and Ki-67.For example see U.S. Patent number 6,537,552; Also see U.S. Patent number 6,815,531,6,773,707,6,682,928 and 6,623,739.

Tumor antigen can also be B cell tumour (for example a, B cell lymphoma; B cell leukemia; Myeloma; Hairy cell) antibody of Chan Shenging, the fragment of this antibody, it comprises epi-position, Malignant B cell antigen receptor, Malignant B cell immunoglobulin idiotype, the variable region of immunoglobulin, the hypervariable region of immunoglobulin or complementary determining region (CDR), malignant T cell receptor (TCR), the variable region of TCR and/or the hypervariable region of TCR of variable region of the idiotype (idiotype) of antibody.In one embodiment, tumor antigen of the present invention can be single-chain antibody (scFv), and it comprises the VH and the VL domain of connection, and it has kept the conformation and the specific bond activity of the natural idiotype of antibody.

Some is called " cancer/testis " antigenic tumor antigen and preponderates relatively on multiple different tumor.Thereby, use the present invention to use one or more cancer/testis antigens and can cause replying at kinds of tumors.For example, comprise the flagellin adjuvant and a series of (for example, two or more, 6-12 for example) mixture of cancer/testis antigen (for example, the form of fusion rotein of the present invention) kinds of tumors can be used for the treatment of, the needs of the particular tumor antigens that experimenter's tumor is expressed may be avoided identifying based on individuality.The non-limitative example of cancer/testis antigen includes but not limited to MAGE-A, MAGE-B, BAGE, GAGE-A, SSX-2, NY-ESO-I, SCP-I, CT7/MAGE-C1 HOM-TES-85, CT/BRDT, CTIO, CTpI 1/SPAN-X-C1, SAGE, OY-TES-1, cT AGE-1, CT15/Fertilin β, CT16, CT17, MMA-1 and CAGE, and they can use separately or make up mutually and/or be used in combination with other tumor antigen.

The present invention also comprises the modified form of above-mentioned tumor antigen, and it is induce immune response in the experimenter.The modified form of naturally occurring tumor antigen is compared immunogenicity and/or the enhanced immunogenicity with reduction with naturally occurring antigen.

The fragment of induce immune response in the experimenter of naturally occurring tumor antigen can be used according to the invention.Fragment can comprise one or more epi-positions, and can also comprise 6,10,15,20,30,40,50,75,100,250,500 or more continuous amino acids of full length protein.In embodiments of the invention, fragment comprise proteinic all or basically all (for example, almost about 1,2,3,5,10,15,20,25 or 50 aminoacid) extracellular domain is not randomly striden film and/or Cytoplasm part.In some other embodiment, fragment comprise proteinic extracellular part at least about 6,10,15,20,30,40,50,75,100,250,500 or more continuous amino acid, optional film or the Cytoplasm part of not striding.

In addition, quote (recitation) to " NY-ESO-1 antigen " or any other specified tumor antigen includes but not limited to, naturally occurring Her2/neu antigen (perhaps other specified tumor antigen) and its modified forms, this modified forms induce immune response in the experimenter.

For example, any suitable NY-ESO-1 antigen can be used for the present invention, comprises full length protein and its fragment.For example, antigen can comprise one or more epi-positions, and can also comprise NY-ESO-1 proteinic 6,10,15,20,30,40,50,75,100,250,500 or more continuous amino acids.In some specific embodiments, described in NY-ESO-1 antigen such as the U.S. Patent Publication 2004/0203051A1.In some other embodiment, NY-ESO-1 antigen comprise this proteinic all or all are (for example basically, almost about 1,2,3,5,10,15,20,25 or 50 aminoacid) extracellular domain is not randomly striden film and/or Cytoplasm part.In some other embodiment, NY-ESO-1 antigen comprise the proteinic extracellular of NY-ESO-1 part at least about 6,10,15,20,30,40,50,75,100,250,500 or more continuous amino acid, optional film or the Cytoplasm part of not striding.As will be apparent to those skilled in the art, NY-ESO-1 antigen can exist with the form of fusogenic peptide (separately or as warm proteic part of the present invention).For example, can send the fusogenic peptide that comprises NY-ESO-1 antigen and cytokine (for example, IFN-, IL-2[comprise the IL-2 immunotoxin, as the IL-2-diphtheria toxin, diphtherotoxin], IL-12 or GM-CSF).(referring to, for example, Dela Cruz et al., (2003) Vaccine21:1317) the present invention also comprises any modified forms of front, it is induce immune response in the experimenter.

Any appropriate H er2/neu antigen can be used for the present invention, comprises full length protein and its fragment.For example, antigen can comprise one or more epi-positions, and can also comprise Her2/neu proteinic 6,10,15,20,30,40,50,75,100,250,500 or more continuous amino acids.In some specific embodiments, described in Her2/neu antigen such as the U.S. Patent Publication numbers 20040241686.In some other embodiment, Her2/neu antigen comprises the epi-position of describing as in the U.S. Patent Publication numbers 20040121946.In some other embodiment, Her2/neu antigen comprise this proteinic all or all are (for example basically, almost about 1,2,3,5,10,15,20,25 or 50 aminoacid) extracellular domain is not randomly striden film and/or Cytoplasm part.For example see U.S. Patent number 6,333,169.In some other embodiment, Her2/neu antigen comprise the proteinic extracellular of Her2/neu part at least about 6,10,15,20,30,40,50,75,100,250,500 or more continuous amino acid, optional film or the Cytoplasm part of not striding.As will be apparent to those skilled in the art, Her2/neu antigen can exist with the form of fusogenic peptide (separately or as warm proteic part of the present invention).For example, can send comprise Her2/neu antigen and cytokine (for example, IL-12, IL-2[comprise the IL-2 immunotoxin, as the IL-2-diphtheria toxin, diphtherotoxin] or fusogenic peptide GM-CSF).The present invention also comprises any modified forms of front, and it is induce immune response in the experimenter.

Tumor antigen that can be used according to the invention never is limited to the tumor antigen that this paper lists.By method as known in the art, as U.S. Patent number 4,514, other tumor antigen can be identified, separates and be cloned to disclosed method in 506.

Shown that recently modulability (regulatory) T cell plays a major role in the effectiveness of restriction anti-tumor vaccine.The strategy of blocking-up (generally temporarily) regulatory T cells activity (for example, by the IL-2 immunotoxin, as IL-2-diphtheria toxin, diphtherotoxin, perhaps IFN-) can be used to strengthen the effectiveness of fusion rotein of the present invention, compositions and method.Can prepare IL-1 immunotoxin or IFN-, and it is used with fusion rotein of the present invention and immunogenic composition.Alternatively, they can be prepared separately, and optional and protein of the present invention and immunogenic composition are used simultaneously.Term used herein " simultaneously " refers to that the time is upward enough near with the generation combined effect (promptly, simultaneously can be simultaneously, perhaps it can be the two or more incidents that take place in [for example, a few minutes or hour] before mutually and during the short time afterwards).In some specific embodiments, flagellin/tumor antigen fusion rotein comprises IL-2 immunotoxin or IFN-.In some other embodiment that flagellin adjuvant and tumor antigen do not provide as fusion rotein, IL-2 immunotoxin or IFN-and flagellin or tumor antigen merge.

Term " tumor " is understood that in the art: for example, and the unusual agglomerate of undifferentiated cell in multicellular organism.Tumor can be pernicious or benign.Usually, method of the present invention disclosed herein can be used for the treatment of malignant tumor.To treat or (promptly its immunity, prophylactic treatment) tumor can be but be not limited to, the tumor that exists in the myeloma, the blood cancer, as leukemia and lymphoma (as B cell lymphoma, t cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma), hemopoietic vegetation (hematopoietic neoplasmas), thymoma, head and neck cancer, sarcoma, pulmonary carcinoma, hepatocarcinoma, the apparatus urogenitalis cancer, bladder cancer, carcinoma of testis, carcinoma of prostate or carcinoma of penis), adenocarcinoma, breast carcinoma, cancer of pancreas, pulmonary carcinoma, renal carcinoma, hepatocarcinoma, former or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, neural tumor, comprise cerebroma, as neuroastrocytoma and glioblastoma, angiosarcoma, angiosarcoma, head and neck cancer, thyroid carcinoma, soft tissue sarcoma, osteocarcinoma, as osteosarcoma, the blood vessel cancer, human primary gastrointestinal cancers is (as gastric cancer, stomach or colon cancer), (see with present any other tumor known or later evaluation, for example, Rosenberg (1996) Ann.Rev.Med.47:481-491).

2. Flagellin

The definite flagellin of inventor can be brought into play the function of adjuvant, replys with the active immunity to tumor antigen that strengthens host's generation.Term used herein " adjuvant " has those skilled in the art's common sense implication.For example, adjuvant can be defined as enhancement antigen at the material of experimenter's moderate stimulation at the ability of this antigenic immunne response.In some specific embodiments, adjuvant will at antigenic immunne response strengthen at least about 2,3,4,5,10,15,20,30,40,50,60,75,100,150,500,1000 times or more than.In some other embodiment, adjuvant reduces the required antigenic amount of immunne response (cell and/or body fluid and/or mucosa) that realizes specified level, for example, be reduced by at least about 15%, 25%, 35%, 50%, 65%, 75%, 80%, 85%, 90%, 95%, 98% or more than.Adjuvant can also be prolong time that immunne response continues, the material of time of continuing of protective immune response (for example, at least about 2 times, 3 times, 5 times, 10 times, 20 times longer times or more than) randomly.In some cases, do not exist under the situation of adjuvant, can not cause remarkable immunne response among the host.

Flagellin is known, and for example at U.S. Patent number 6,585,980,6130,082,5,888,810,5,618,533,4,886,748 and people, (2002) J Biol.Chem.43:40456 such as U.S. Patent Publication US 2003/0044429 A1 and Donnelly in describe to some extent.Most gram negative bacteria are expressed flagellin, and it provides mobility's surface texture.Form flagellin from matrix, filament (filament) and the hook that is connected both (hook).Albumen--the polymer of the length of flagellin forms filament by single kind, has little medicated cap albumen endways.The polymerization of flagellin is by the mediation of the conserved region of N and C-terminal, and flagellin interleave district (intervening region) unusual difference between species.

In illustrative embodiment more of the present invention, fusion rotein is provided, it comprises flagellin adjuvant and one or more tumor antigens.Usually, fusion rotein of the present invention comprises, forms and be essentially or consist of: (a) flagellin adjuvant, this adjuvant comprise the terminal constant region of (i) flagellin N-; The (ii) terminal constant region of flagellin C-; (b) tumor antigen, wherein this tumor antigen is between terminal constant region of N-and the terminal constant region of C-.In some embodiments, the flagellin hypervariable region between the constant region is lacked (all or part of); In some other embodiment, there is the hypervariable region.When having hypervariable region (all or part of), antigen can be inserted in (i) hypervariable region, (ii) between terminal constant region of flagellin N-and the hypervariable region, perhaps (iii) between terminal constant region of flagellin C-and the hypervariable region.

In addition, the terminal constant region of N-can be connected by hinge region with the terminal constant region of C-.Hypervariable region or tumor antigen can be used as hinge region and play a role.In addition, perhaps alternatively, about 2,3,4,6,8,10,15,20,30,50 or more the section of amino acids can be used as hinge region and play a role.

The conserved region of flagellin is as known in the art and for example people such as Mimori-Kiyosue, (1997) J.Mol.Virol.270:222-237; People such as lino, (1977) Ann.Rev.Genet.11:161-182; With people such as Schoenhals, describe to some extent among (1993) J Bacteriol175:5395-5402.As understood by a person skilled in the art, the big young pathbreaker of constant region depends on the proteinic source of flagellin to a certain extent and becomes.Usually, the terminal constant region of N-comprises proteinic about 170 or 180-terminal amino acids, and the terminal constant region of C-is crossed over about 85 to 100 C-end amino acids usually.The hypervariable region, center is along with the size in the antibacterial and sequence and great changes have taken place, and is the main cause of molecular weight difference.From proteinic N-of the flagellin of various bacteria and the terminal constant region of C-is known, other can use known comparison technology easily to identify by those skilled in the art, illustrating of the monomeric crystal structure of flagellin made things convenient for described comparison technology (people such as Samatey, (2001) Nature41:331).

Term used herein " the terminal constant region of flagellin N-" and " the terminal constant region of flagellin C-" comprise that active fragment (for example, long be at least about 50,100 or 120 amino acid whose fragments) and aforementioned any strengthen modification (for example, by activation TLR5 approach) to the immunne response of tumor antigen.For example, can be modified, to strengthen safety and/or immunne response natural flagellin district.In some embodiments, the terminal constant region of flagellin N-end and/or C-comprises total length zone or alternatively, can only comprise one or two regional fragment.

In some specific embodiments, the terminal constant region of N-end and/or C-comprises the TLR5 recognition site and can activate the TLR5 approach.In some representative embodiment, the terminal constant region of N-comprises the terminal RINSA domain (the aminoacid 31-52 of S.dublin flagellin) of the N-that describes as people such as Eaves-Pyles (2001) J Immunology 167:7009-7016, perhaps the immunogenic modified forms of its congener or enhancing tumor antigen.In some other embodiment, the terminal constant region of N-comprises D1 and D2 domain, and the terminal constant region of C-comprises D1 and D2 domain people (2001) JImmunology 167:7009-7016 such as () Eaves-Pyles or it strengthens the immunogenic modified forms of tumor antigen.

In some other embodiment, the terminal constant region of flagellin N-end and/or C-comprises, consists of or forms and is essentially: the peptide GAVQNRFNSAIT (SEQ ID NO:4) that describes as people such as U.S. Patent Publication US 2003/0044429A1 or Alderem, the perhaps immunogenic modified forms of its congener or enhancing tumor antigen.

In some embodiments again, the terminal constant region of N-comprises people such as Kanneganti, (2004) the motif N that identifies of J Biol.Chem.279:5667-5676 " (for example; the aminoacid 98-108 of S.muenchen flagellin) and/or the terminal constant region of C-comprise " motif C " (for example; the aminoacid 441-449 of S.muenchen flagellin), perhaps its congener or strengthen the immunogenic modified forms of tumor antigen.

In some other property illustrated embodiment, the terminal constant region of N-comprises aminoacid 88-97 (for example seeing people such as Verma, (2005) Infect.Immun.73:8237-8246) or its congener of Pseudomonas aeruginosa (P.aeruginosa) flagellin or strengthens the immunogenic modified forms of tumor antigen.

People such as Smith (2003) Nat.Immunol.4:1247-1253 has identified the zone (for example, aminoacid 78-129, the 135-173 of S.typhimurium flagellin and 394-444 or its congener or modified forms) that the participation TLR5 signal of flagellin transmits.

The terminal constant region of flagellin N-, the terminal constant region of C-and hypervariable region can be from the flagellins in any suitable source, some of these zones or all from identical biology or from different biologies.Cloned and many flagellin genes that checked order (are for example seen people such as Kuwajima, (1986) J Bact.168:1479; People such as Wei, (1985) J Mol.Biol.186:791-803; With people such as Gill, (1983) J Biol.Chem.258:7395-7401).The non-limiting source of flagellin includes but not limited to, S.enteritidis, S.typhimurium, S.dublin, helicobacter pylori (H.pylori), V.cholera, S.marcesens, S.flexneri, S.enterica, T.pallidum, L.pneumophila, B.burgdorferei, C.difficile, A.tumefaciens, R.meliloti, B.clarridgeiae, R.lupine, P.mirabilis, bacillus subtilis (B.subtilis), Pseudomonas aeruginosa (P.aeruginosa) and escherichia coli (E.coli).

The non-limitative example of fusion rotein of the present invention is provided in the work embodiment of this paper.

Randomly, fusion rotein can comprise any other peptide or protein.For example, fusion rotein can also comprise one or more antigens from other biology.In some representative embodiment, fusion rotein also comprises immunomodulatory compounds.For example, known in the art can enhance immunity replying by immune regulative cytokine or chemotactic factor (as alpha-interferon, beta-interferon, gamma interferon, ω-interferon, τ-interferon, il-1 α, il-1 β, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, IL-10 INTERLEUKIN-10, interleukin-11, il-1 2, interleukin-13, il-1 4, il-1 8, Bcell growth factor, the CD40 part, tumor necrosis factor-alpha, tumor necrosis factor-β, monocyte chemoattractant protein-1, granulocyte macrophage colony stimulating factor, lymphocytotoxin, CCL25[MECK] and CCL28[TECH]) enhance immunity replys.

In specific embodiments, fusion rotein comprises gamma interferon and/or IL-2 immunotoxin (for example, IL-2-diphtheria toxin, diphtherotoxin).The present invention also provides the compositions that comprises flagellin adjuvant and tumor antigen.According to this embodiment, the flagellin adjuvant can be the total length flagellin or can be to comprise as mentioned the flagellin peptide of the terminal constant region of N-end and/or C-in greater detail.In addition, also described above, the flagellin adjuvant can with tumor antigen coupling (for example, merge), to form fusion rotein.In some exemplary, tumor antigen is tumor F1 protein, tumor V protein matter or its fusion rotein.Compositions can comprise one or more tumor antigens that merge with the flagellin adjuvant, and randomly, one or more tumor antigens that exist in the compositions do not merge with the flagellin adjuvant.In other embodiments, the flagellin adjuvant is not coupled to tumor antigen.

Unless otherwise noted, fusion rotein itself is used as protein (the perhaps nucleic acid of coded protein), and not as live, part that kill or recombinant bacteria or vector-viral vaccine uses.

4. The generation of recombinant nucleic acid and fusion rotein

Except pointing out in addition, can standard method well known by persons skilled in the art can be used for clone gene, amplification and detection nucleic acid, produce fusion constructs, in host cell or biology expression of peptides, or the like.This type of technology is well known by persons skilled in the art.For example see people such as Sambrook, " Molecular Cloning " A Laboratory Manual second edition (Cold SpringHarbor, NY, 1989); People Current Protocols in MolecularBiology such as F.M.Ausubel (Green Publishing Associates, Inc.and John Wiley ﹠amp; Sons, Inc., New York).

As used herein, " nucleic acid " comprises RNA and DNA, comprises the chimera of cDNA, genomic DNA, synthetic (for example, chemosynthesis) DNA and RNA and DNA.Nucleic acid can be two strands or strand.Can use oligonucleotide analogs or derivant (for example, trophicardyl or thiophosphate nucleotide) nucleic acid.This class oligonucleotide can for example be used to prepare base pairing ability with change or to the nucleic acid of the toleration of the increase of nuclease.

Can (for example be used for multiple purpose at the expression coding, produce immunogenic composition, as diagnosis or research reagent or the like) the cultured cells of heterologous nucleic acids of fusion rotein or biology in produce fusion rotein of the present invention, and optional from described cell or biological it is carried out purification.

In some embodiments, can collect and choose wantonly purified fusion protein from host cell.For example, can collect fusion rotein from conditioned medium.According to this embodiment, the fusion rotein that expression can be operatively connected secretory signal sequence may be favourable.Alternatively, can divide isolated fusion protein (for example, can the cracking host cell and therefrom divide isolated fusion protein) from host cell.

In some other embodiment, collect host cell and therefrom do not divide isolated fusion protein.

Usually, heterologous nucleic acids is incorporated in the expression vector (virus or non-virus).Suitable expression vector includes but not limited to, plasmid, phage, bacterial artificial chromosome (bacs), yeast artificial chromosome (yacs), cosmid, viral vector or the like.The expression vector compatible with multiple host cell is as known in the art, and contains the appropriate members of transcribing and translating that is useful on nucleic acid.Typically, expression vector contains " expression cassette ", it comprises the coded sequence of promoter, coding and the exercisable fusion rotein that is connected of promoter in 5 ' to 3 ' direction, randomly, terminator sequence, it comprises the polyadenylation signal at the termination signal of RNA polymerase and polyadenylic acid enzyme (polyadenylase).

Can be designed expression vector, be used at protokaryon or eukaryotic cell express polypeptide.For example, can be in bacterial cell (as escherichia coli), insect cell (for example, baculovirus expression system), yeast cells, mammalian cell or plant cell express polypeptide.The example that is used for the carrier of expressing at saccharomyces cerevisiae comprises pYepSecl (people such as Baldari, (1987) EMBO J.6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell30:933-943), pJRY88 (people such as Schultz, (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).The insect cell that is used in cultivation (for example, the Sf9 cell) express nucleic acid comprises pAc series (people such as Smith to produce proteinic baculovirus vector, Mol.Cell.Biol.3:2156-2165) and pVL series (Luckiow (1983), V.A., and Summers, M.d. (1989) Virology 170:31-39).

In addition, expression vector will generally include expression control sequenc (for example, transcribing/translate control signal and polyadenylation signal), the nucleotide sequence of its coding fusion rotein of the present invention that is operably connected.Should be appreciated that and depend on and desired horizontal and tissue specific expression can use multiple promoter/enhancer element.Promoter can be (for example, metallothionein promoter or hormone induction type promoter) composing type or induction type, and this depends on desirable expression pattern.Promoter can be original natural that have or external source and can be natural or synthetic sequence.External source refers to that promoter do not find in the wild type host that this promoter imported.Select promoter to make it in the target cell, bring into play function.In addition, the protein coding sequence that generally provides the specificity initial signal to insert with effective translation.These translation control sequences (can comprise ATG start codon and flanking sequence) can be multiple sources, can be natural and synthetic.Wherein expression vector comprises in the embodiment of the present invention with two open reading-frames that will be transcribed, open reading-frame can be operably connected independent promoter or single upstream promoter and one or more downstreams internal ribosome entry sites (IRES) sequence (for example, picorna virus EMC IRES sequence).

The example of mammalian expression vector comprises pCDM8 (Seed, (1987) Nature 329:840) and pMT2PC (people (1987) such as Kaufman, EMBO J.6:187-195).When being used for mammalian cell, the control function of expression vector provides by viral regulating element usually.For example, Chang Yong promoter is from polyoma, adenovirus 2, cytomegalovirus and simian virus 40.

The present invention also provides (temporary transient or stable) to comprise the host cell of the nucleic acid of the fusion rotein of the present invention of encoding.Proper host cell is as known in the art, and comprises protokaryon and eukaryotic cell.See, for example, Goeddel, Gene Expression Technology:Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990).Known: protein can be at bacterial cell such as escherichia coli, insect cell (for example, baculovirus expression system), express in yeast cells, plant cell or the mammalian cell (for example, people, rat, mice, hamster, cattle, pig, sheep, goat, horse, cat, Canis familiaris L., Lagomorph, ape or the like).Host cell can be a cultured cells, as the cell of primary cell or immortalized cell line.Host cell can be the cell that is used as microorganism, animal or the plant of bioreactor basically.In specific embodiments more of the present invention, host cell is the insect cell that allows expression vector to duplicate.For example, host cell can be from fall army worm (Spodoptera frugiperda), as Sf9 or or Sf21 cell line, drosophila cell system, perhaps mosquito cells system, for example, the cell line in Aedes albopictus (Aedes albopictus) source.The purposes of insect cell expression heterologous protein, and with nucleic acid, as carrier, for example, the compatible carrier (as baculovirus vector) of insect cell imports the method in this type of cell and cultivates and keeps the method for this type of cell to be documented.For example see Methods inMolecular Biology, ed.Richard, Humana Press, NJ (1995); People such as O ' Reilly, Baculoviras Expression Vectors, A Laboratory Manual, OxfordUniv.Press (1994); People such as Samulski, J.Vir.63:3822-8 (1989); People such as Kajigaya, Proc.Natl Acad.Sci USA 88:4646-50 (1991); People such as Ruffing, J.Vir.66:6922-30 (1992); People such as Kimbauer, Vir.219:37-44 (1996); People such as Zhao, Vir.272:382-93 (2000); U.S. Patent number 6,204,059 with people such as Samulski.In specific embodiments more of the present invention, insect cell is the Sf9 cell.

Can in protokaryon or eukaryotic cell, introduce carrier by routine conversion or rotaring dyeing technology.Term used herein " conversion " and " transfection " refer to the multiple several different methods that exogenous nucleic acid (for example DNA) is imported host cell as known in the art, transfection, fat transfection, electroporation, microinjection, the liposome of loading DNA, the fat that comprises calcium phosphate or calcium chloride co-precipitation, the mediation of DEAE-glucosan changes soft amine (lipofectamine)-dna complex, cell supersound process, uses high speed particle to carry out gene bombardment and virus-mediated transfection.Be used to transform or the suitable method of transfection host cell can be seen people (MolecularCloning:A Laboratory Manual such as Sambrook, second edition, Cold Spring HarborLaboratory press (1989)) and other laboratory manual.In other embodiments of the present invention, host cell can be used the heterologous nucleic acid sequence stable conversion of encoding fusion protein." stable conversion " used herein is often referred in the genome that heterologous nucleic acid sequence is incorporated into host cell, and this is opposite with " instantaneous conversion ", and in the instantaneous conversion, the heterologous nucleic acids unconformity that imports host cell is in the genome of host cell.Term " stable conversion " can also refer to the stable maintenance of episome (episome) (for example, Epstein-Barr virus (EBV)) in cell.

When producing the cell of stable conversion, only sub-fraction cell (especially mammalian cell) is incorporated into foreign cell in their genome usually.In order to identify and select these integrate bodies, the nucleic acid of coding selected marker (for example, antibiotic resistance) can be imported host cell with purpose nucleic acid.Preferred selected marker comprises those selected markers of giving the resistance of medicine (as G418, hygromycin and methotrexate).Can import in the host cell on the nucleic acid of coding selected marker and the same vehicle of carrier that comprises purpose nucleic acid, perhaps can import on the independent carrier.Can identify with the nucleic acid stability cells transfected that imports (for example, comprised that into the cell of selectable marker gene will be survived, and other cell death) by medicament selection.

Can also produce fusion rotein in transgenic plant, in the described plant, the isolating nucleic acid of encoding fusion protein has been inserted in the genome of nucleus or plastid.Plant Transformation is as known in the art; Generally speaking, Methods in Enzymology Vol.153 (" Recombinant DNA Part D ") 1987, Wu and Grossman Eds., AcademicPress and European patent application EP 693554.

Can exogenous nucleic acid be imported in plant cell or the protoplast by several method.For example, can come mechanically transfer nucleic acid by using the direct microinjection of micropipettor in plant cell.Can also also can change exogenous nucleic acid in plant cell by using Polyethylene Glycol, Polyethylene Glycol forms the precipitation complex with hereditary material, and it is by cell absorption people (1984) EMBO such as (J.3:2712-22) Paszkowski.Can in plant cell, import exogenous nucleic acid (people (1985) Proc.Natl.Acad.Sci USA 82:5824 such as Fromm) by electroporation.In this technology, under the situation of the existence of plasmid that contains relevant genetic constructs or nucleic acid, plant protoplast is carried out electroporation.The electric pulse of high field intensity is reversibly changed biomembrane thoroughly, and this allows the importing of plasmid.Plant protoplast through electroporation forms cell wall, division again and forms plant callus.Use phenotypic markers can finish to comprising the selection through the plant transformed cell of exogenous nucleic acid.

Cauliflower mosaic virus (CaMV) can be as the carrier that imports exogenous nucleic acid in plant cell (people (1982) " Molecular Biology of Plant Tumors, " Academic Press such as Hohn, New York, pp.549-560; Howell, U.S. Patent number 4,407,956).CaMV viral DNA genome is inserted in parent's bacterial plasmid, the recombinant DNA molecules that generation can be bred in antibacterial.Can further modify recombiant plasmid by importing the DNA sequence of wishing.The modified virus part of downcutting recombiant plasmid from parent's bacterial plasmid and is used to inoculate plant cell or plant then.

The high-velocity projectiles that is undertaken by granule penetrates and can be used for importing exogenous nucleic acid to plant cell.Nucleic acid is disposed in globule or the particulate substrate, perhaps from the teeth outwards (people (1987) Nature 327:70-73 such as Klein).Although typically only need single to import new nucleic acid segment, this method also provides repeatedly and imports.

By using Agrobacterium tumefaciems (Agrobacterium tumefaciens) transfection of plant cells, explant, separate living tissue or the seed that transform through nucleic acid can in plant cell, import nucleic acid.Under appropriate condition, can cultivate through the plant transformed cell forming branch, root, and further develop into plant.For example, can in plant cell, import nucleic acid by the Ti-plasmids of Agrobacterium tumefaciems.When agrobacterium tumefaciens infection, Ti-plasmids shifts into plant cell, and stable integration (people such as Horsch, (1987) Science 227:1229-1231 in Plant Genome; People such as Fraley (1983) Proc.Natl.Acad.Sci.USA 80:4803).

Can be from other peptide or the protein of being operably connected, as the nucleic acid of the purification signal that is operably connected (as poly His) or as with other protein (for example, cytokine is as gamma interferon and/or IL-2-immunotoxin, as the IL-2-diphtheria toxin, diphtherotoxin) the chimera expressed fusion protein.

5. Use fusion rotein of the present invention, flagellin adjuvant and method for compositions

In order to treat and prevent purpose, can implement the present invention according to known technology (for example seeing that people PCT such as Pizzo apply for WO 2004/101737).Usually, preventative enforcement the present invention forms to reduce tumor.Yet, in some other embodiment, implement method of the present invention, produced the experimenter of tumor with treatment.The immunogenic composition that is used for the inventive method is described hereinafter.Can in the time course of several weeks, several months or several years, further use reinforcement dosage.For existing tumor, it can be useful then using reinforcement dosage behind the initial high dose.

Can implement the present invention has the experimenter or the prevention of existing tumor or postpones the tumor generation with treatment.In addition, the inventive method can be used for the treatment of primary tumor and prevention transfer.In addition, the inventive method can be advantageously used in and reduce or the nodular growth of prevention transitivity (after for example, primary tumo(u)r is removed in operation).Method of the present invention can also be preventative, for example, is used for the treatment of and thinks and be in experimenter in the tumor danger.

The seriousness of experimenter's patient's condition that refers to term " treatment " (the perhaps term that is equal on the grammer) reduces or improves or alleviate to small part, and/or realized certain alleviating, relax or go down of at least a clinical symptoms, and/or the progress of situation is delayed, and/or to the prevention or the delay of disease or disease outbreak.Term " treatment " etc. also comprises the prophylactic treatment (for example, the outbreak of prevention infection or cancer) to the experimenter.Term used herein " prevention " (with its grammer equivalent) be not intended to the elimination fully that implies disease and, it comprises the prophylactic treatment of any kind, it reduces the sickness rate of situation, postpones the outbreak and/or the progress of situation, and/or alleviates the symptom relevant with this situation.Thereby term " treatment " (the perhaps term that is equal on the grammer) refers to prevention and therapeutic scheme.

As used herein, the method for " tumor among the treatment experimenter " comprises existing tumor treatment method (comprise and reduce metastasis rate and/or seriousness) of treatment and prevention or reduces the prophylactic methods that tumor forms.

Term " inoculation " or " immunity " are as known in the art, and unless otherwise, it exchanges use in this article, unless otherwise noted.For example, term inoculation or immunity can be understood that to increase the biological method of therefore antigenic immunne response also being resisted or overcome infection.In situation of the present invention, at the immunne response of the inoculation of tumor antigen or immunostimulant biology with to the resistance of tumor.

" treatment effective dose " used herein is enough treatments (as defined herein) experimenter's amount.

The feature of " active immunity is replied " or " active immunity " be " after running into immunogen, the participation of host tissue and cell.It relates to the differentiation or the propagation of immunologically competent cell in the lymphoreticular tissue, and this causes synthetic or cell-mediated reactive development of antibody, and perhaps both are all caused ".Herbert B.Herscowitz, Immunophysiology:Cell Functionand Cellular Interactions in Antibody Formation, in IMMUNOLOGY:BASIC PROCESSES 117 (Joseph A.Bellanti ed., 1985). perhaps we can say, by infecting or after inoculation was exposed to immunogen, the host started active immunity to reply.Active immunity is opposite with passive immunity, in the passive immunity, obtains passive immunity by " the preformed material (antibody, transfer factor, thymic graft, interleukin-2) from the host of active immunity is transferred to non-immune host ".

" protectiveness " immunne response or " protectiveness " immunity refer to as used herein: this immunne response is given certain benefit to the experimenter, because it prevents or reduces disease incidence rate and/or seriousness.Alternatively, protective immune response or protective immunity can be used for the therapeutic treatment to present illness.

Can be for medical science or veterinary's purpose enforcement the present invention.The medicable experimenter of the inventive method (for example comprises birds and mammalian subject, (mammalian subject includes but not limited to the people), non-human primates, monkey, ape, baboon and chimpanzee), Canis familiaris L., cat, rabbit, goat, horse, pig, cattle, sheep or the like (comprise experimenter male and female subject and institute's has age, comprise baby, teenager, youth and adult experimenter).Can be for any therapeutic interest experimenter, for example, in order to cause protective immune response; In order to cause the generation of antibody in experimenter's (typically, animal subjects), can collect antibody and be used for other purpose, as diagnostic purpose or be applied to other experimenter producing passive immunity therein, or the like.In some specific embodiments, the experimenter is an animal model.In other embodiments, the experimenter has tumor and/or is to think to be in experimenter in the tumor danger.

Randomly, the experimenter can be the experimenter of " needing fusion rotein of the present invention, compositions, pharmaceutical preparation and method ".

In some embodiments, the experimenter is old experimenter, for example, 50 or 60 years old or older human experimenter, wherein the common effect of other adjuvant such as Alumen is lower.

Therefore, in some specific embodiments, the invention provides the experimenter (as mammalian subject, for example people or primate) in induce method at the immunne response of tumor antigen, this method comprises uses fusion rotein of the present invention or its pharmaceutical composition to the experimenter with the immunogenicity effective dose.In some representative embodiment, implementation method is as treating the experimenter (as mammalian subject, for example people or primate) in the method for tumor, this method comprises can use fusion rotein of the present invention or its pharmaceutical composition by effective dose to the experimenter with treatment.Randomly, by sending fusion rotein to mucomembranous surface (for example, using) or pharmaceutical composition is implemented these methods by intranasal or suction.

The present invention also provides the experimenter (as mammalian subject, for example people or primate) in induce method to the immunne response of tumor antigen, this method comprises uses flagellin adjuvant or tumor antigen to the experimenter with the immunogenicity effective dose, perhaps its pharmaceutical composition.In some representative embodiment, implement this method as treating the experimenter (as mammalian subject, for example people or primate) in the method for tumor, this method comprises uses flagellin adjuvant or tumor antigen to the experimenter can treat effective dose, perhaps its pharmaceutical composition.Randomly, by sending fusion rotein to mucomembranous surface (for example, using) or pharmaceutical composition is implemented this method by intranasal or suction.Can in identical or independent compositions, use flagellin adjuvant and tumor antigen.If as dividing other compositions to use, randomly, they can be used simultaneously.

Use and to be undertaken by any approach as known in the art.As non-limitative example, route of administration can be by (for example sucking, per os and/or snuffing are gone into), per os, (for example suck, the Sublingual), rectum, vagina, part (comprise and be applied to respiratory tract), ophthalmic, percutaneous, (for example by parenteral, in intramuscular [comprise and be applied to skeletal muscle, cardiac muscle and/or diaphram], intravenous, subcutaneous, Intradermal, the pleura, in the brain and in intra-arterial and the sheath) approach, and directly muscle or organ injection, perhaps by being applied to central nervous system's (for example, stereotaxis (stereotactic) is applied to brain).

In specific embodiments, be administered to mucomembranous surface, for example, by in intranasal, suction, the trachea, per os, rectum or vaginal application, or the like.Usually, mucosal administration refers to be delivered to mucomembranous surface, as the surface of respiratory tract, gastrointestinal tract, urinary tract, reproductive tract or the like.

The method that is applied to the respiratory tract surface includes but not limited to, uses in through mucous membrane, intranasal, suction or the trachea or is applied to lung.Other mucosal administration method comprises per os, sucks (for example, Sublingual), in the trachea, rectum, vagina and ophthalmic use.

In some embodiments of the present invention, in tumor locus or near use.In other embodiments, be applied directly in the lymphsystem (for example, in the lymph node or near).Can produce described protein by sending that code book is invented proteinic nucleic acid intermediates and in the host, expressing, send protein of the present invention, as describing in people's such as Feigner the U.S. Patent number 5,589,466.

Immunomodulatory compounds can be applied to the experimenter jointly as immune regulative chemotactic factor and cytokine (preferably, CTL induction type cytokine).Can be by any method administer cytokines as known in the art.The cytokine of external source can be applied to the experimenter, perhaps alternatively, use suitable carriers the nucleotide sequence of the Codocyte factor can be delivered to the experimenter, and produce this cytokine in vivo.In some specific embodiments, cytokine provides as the fusion rotein with flagellin adjuvant and/or tumor antigen.For example, can use the fusion rotein that comprises flagellin adjuvant, tumor antigen and immune regulative cytokine (for example, gamma interferon or IL-2-immunotoxin).Alternatively, can use the fusion rotein that comprises cytokine and tumor antigen or flagellin adjuvant.

Except they be used to prevent or the purposes of therapeutic purposes, can use fusion rotein of the present invention and compositions to the experimenter, be used to produce the antibody at tumor antigen, this antibody can be used for humans and animals experimenter's diagnosis or treatment/prevention purpose again.

6. Pharmaceutical composition

The present invention also provides the pharmaceutical composition that is in the fusion rotein of the present invention in pharmaceutically suitable carrier (for example, immunogenic composition).In some specific embodiments, the compounding pharmaceutical compositions is used for mucosal delivery." pharmaceutically useful " refers to not to be deleterious or undesirable material.

In some representative embodiment, fusion rotein exists with " immunogenicity is effective " amount in pharmaceutical composition." immunogenicity effective dose " is enough to cause in having used the experimenter that pharmaceutical composition uses that active immunity replys the amount of (that is, cell and/or body fluid).Randomly, dosage enough produces protective immune response (it is preventative or curative to infect the generation back).The degree of protection of giving needs not to be completely or is persistent, as long as the benefit of drug administration compositions surpasses its any shortcoming.The immunology effective dose depends on stage and seriousness and experimenter's the body weight and general health state and prescription doctor's the judgement of the disease that protein, method of application, quilt are treated.

Can determine the dosage of pharmaceutically active compound by methods known in the art, for example see, and Remington ' s Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa).In some specific embodiments, for typical (for example, 70kg) experimenter, the dosage of fusion rotein of the present invention is the scopes of about 0.1,0.5,1,10,25,250,100,150 or 250 μ g to about 300,500,1000,2500,5000 or 10,000 μ g fusion rotein.In some specific embodiments, for typical experimenter, dosage is about 50 to 2000 μ g, about 100 to 1500 μ g, the scope of perhaps about 250 to 1000 μ g.After the initial dosage, can carry out the reinforcement dosage of about 1 μ g to 250,500 or 1000 μ g in several weeks, several months or several years, this depends on experimenter's replying initial dosage.

The present invention also provides following pharmaceutical composition, and it comprises: (a) flagellin adjuvant; (b) tumor antigen.In some specific embodiments, the compounding pharmaceutical compositions is used for mucosal administration.Randomly, tumor antigen and the coupling of flagellin adjuvant are the form with the antigenic fusion rotein of flagellin.According to this embodiment, described compositions can also comprise one or more additionally, not with the tumor antigen of flagellin adjuvant coupling (that is, be not with the fusion rotein of flagellin adjuvant part).

Randomly, tumor antigen exists with as defined herein immunogenicity effective dose.In addition, in some embodiments, the flagellin adjuvant exists with " adjuvant effective dose "." adjuvant effective dose " is the amount of the flagellin adjuvant of following amount, and this amount is enough to strengthen or the active immunity of the pin tumor antigen that stimulation of host is initiated is replied (cell and/or body fluid), optional mucosal immune response initiatively.In specific embodiments, host's active immunity reply (for example, mucosal immune response) be enhanced about at least 2,3,4,5,10,15,20,30,40,50,60,75,100,150,500,1000 times or more than.In some other embodiment, " adjuvant effective dose " is the amount of the flagellin adjuvant of following amount, this amount can reduce the immunity (cell and/or body fluid) that realizes specified level, the optional required antigenic amount of mucosal immunity, for example, antigenic amount be reduced by at least about 15%, 25%, 35%, 50%, 65%, 75%, 80%, 85%, 90%, 95%, 98% or more than.As another selection, " adjuvant effective dose " can refer to the amount of the flagellin adjuvant of following amount, and this amount can be quickened the needs of inducing and/or reducing reinforced immunological is protected with realization of immunne response among the host.As another selection, " adjuvant effective dose " can be to prolong immunne response, the amount of the time bar (for example, at least about 2 times, 3 times, 5 times, 10 times, 20 times longer time bars or longer) that continues of protective immune response randomly.

Those skilled in the art can determine the dosage of flagellin adjuvant and tumor antigen (if not the form of fusion rotein).In some specific embodiments, for typical (for example 70kg) experimenter, the dosage of flagellin adjuvant is the scopes of about 0.1,0.5,1,10,25,50,100 or 150 μ g to about 200,250,300,500,1000 or 2500 μ g.In specific embodiments, for typical experimenter, dosage is about 10 to 1000 μ g, perhaps about 50 to 500 μ g, perhaps about 150 to 300 μ g. are for typical (for example 70kg) experimenter, and the proper dosage of tumor antigen can arrive about 200,300,500,1000,1500,2000,2500 or 5000 or 10000 μ g for about 0.1,0.5,1,10,25,50,100 or 150 μ g.In some specific embodiments, for typical experimenter, the dosage of tumor antigen is about 50 to 2000 μ g, about 150 to about 1500 μ g, perhaps about 300 to about 1000 μ g.Can carry out the reinforcement dosage of about 1 μ g to about 1000 μ g after the initial dosage in several weeks, several months or several years, this depends on experimenter's replying initial dosage.

Pharmaceutical composition of the present invention can be chosen wantonly and comprise other medicinal agent, pharmacy agent, stabilizing agent, buffer agent, carrier, diluent, salt, tension regulator, humidizer or the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, Arlacel-20, Emulphor FM, or the like.

For injection, carrier will typically be liquid.For other application process, carrier can be solid or liquid.Use for suction, carrier will be breathed, and be generally solid or liquid particle form.

Although do not need the adjuvant outside the flagellin usually, compositions can be chosen wantonly and comprise extra adjuvant, as complete or incomplete Freund's adjuvant, aluminum phosphate, aluminium hydroxide, Alumen, cytokine, TLR part or the like.

Proteinic concentration can alter a great deal in the pharmaceutical composition, for example, from by weight less than about 0.01% or 0.1% at least about 2% to much 20% to 50% or more than, and will mainly select it according to selected concrete method of application by fluid volume, viscosity or the like.

Can prepare protein with pharmaceutical carrier according to known technology, use being used to.For example see Remington, The Science And Practice of Pharmacy (the 9th edition, 1995).In to production, usually protein (comprising its physiologically acceptable salt) is mixed with acceptable carrier according to pharmaceutical composition of the present invention.Carrier can be solid or liquid or both, and optionally is formulated as unit dose formulations with chemical compound, for example tablet.Can use multiple pharmaceutically useful aqueous carrier, as water, phosphate buffered saline(PBS), bacteriostatic water or Cremophor EL[R through buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, the water of no pyrogen, no pyrogen] (BASF, Parsippany, N.J.), or the like.Can be by routine techniques to these compositions sterilizations.One or more protein can be mixed in the preparation of the present invention, can prepare preparation of the present invention by any known technology of pharmaceutics.

Pharmaceutical composition can former state use after packing, and perhaps by lyophilizing, freeze dried preparation makes up with aseptic aqueous solution usually with before using.Compositions can also the unit of being packaged in/dosage or multi-dose container, in the ampoule and bottle as sealing.

Can be according to the routine techniques compounding pharmaceutical compositions of pharmaceutics, use by any known method of this area being used for.For example, can compositions formulated carry out intranasal administration, by (for example sucking, per os sucks), per os, (for example suck, the Sublingual), rectum, vagina, local, in the sheath, ophthalmic, applied dermally, by parenteral administration (for example, intramuscular (comprises and is applied to skeletal muscle, cardiac muscle and/or diaphram], intravenous, subcutaneous, Intradermal, in the pleura, interior and the intra-arterial of brain, in sheath) approach, the part (for example, be applied to skin and mucomembranous surface, comprise the respiratory tract surface) and directly muscle or organ injection, perhaps use by being applied to central nervous system's (for example, stereotaxis (stereotactic) is applied to brain).

In some embodiments of the present invention, the compounding pharmaceutical compositions be used for tumor locus or near use.In other embodiments, the compounding pharmaceutical compositions is used for directly being applied near lymphsystem (for example, in the lymph node or).

In specific embodiments, pharmaceutical composition is applied to mucomembranous surface, for example, by in intranasal, suction, the trachea, per os, rectum or vaginal application, or the like.

Use for intranasal or suction, pharmaceutical composition can be formulated as aerosol (this term comprises liquid and dry powder aerosol).For example, pharmaceutical composition can provide pharmaceutical composition with surfactant and propellant with the form of segmentation.The typical percent of compositions is by weight 0.01-20%, preferred 1-10%.Surfactant is normally nontoxic, and dissolves in the propellant.The representative of this type of material is the fatty acid that contains 6 to 22 carbon atoms (as caproic acid, sad, dodecylic acid, Palmic acid, stearic acid, linoleic acid, linolenic acid, olestericacid and oleic acid) and the ester or the part ester of aliphatic polyol or its cyclic anhydride.Can use blended ester, as blended or natural glycerin ester.Surfactant can constitute the 0.1-20% of composition weight, preferred 0.25-5%.The difference of compositions is propellant normally.As needs, can also comprise carrier, as be used for the lecithin of intranasal delivery.Can be by any suitable manner, as with pressure-actuated aerosol atomizer or ultrasonic nebulizer, produce the aerosol of liquid particles, as known in the art.For example see U.S. Patent number 4,501,729.By known technology in the pharmaceutical field, similarly, can produce the aerosol of solid particle equally with any solid particle pharmaceutical aerosol generator.Also can be applied to the nose surface and also can carry out intranasal administration by droplet.

Injectable preparation can prepare with conventionally form, as liquid solution agent or suspensoid, be suitable for before injection dissolving or be suspended from solid form in the liquid, perhaps as Emulsion.Alternatively, can be with part rather than systemic fashion, as with depot formulation (depot) or extended release preparation drug administration compositions.

Can from before sterile powder, granule and the tablet of kind of description, prepare interim injection solution and suspension.For example, can in airtight container, provide injectable, stable aseptic composite of the present invention with unit dosage forms.Can provide compositions with the form of lyophilized products, can be suitable for being expelled to experimenter's fluid composition with formation with suitable pharmaceutically suitable carrier reconstruct lyophilized products.Unit dosage forms can arrive about 10g compositions of the present invention for about 1 μ g.When compositions when being water-insoluble basically, can comprise that the pharmaceutically acceptable emulsifying agent of q.s is with emulsification composition in aqueous carrier with enough amounts.A kind of this type of useful emulsifying agent is a phosphatidylcholine.

The pharmaceutical composition that is suitable for dosage forms for oral administration can be discrete unit, as capsule, cachet, dragee or tablet, as powder or granule; As solution in aqueous or the non-aqueous liquid or suspensoid; Perhaps as oil-in-water or water in oil emulsion.By chemical compound of the present invention is compound with the carrier that can resist the degraded of digestive enzyme in the animal intestine, carry out oral delivery.The example of examples of such carriers comprises plastic capsule or tablet, as known in the art.Can be by this type of preparation of prepared by any suitable process of pharmaceutics, described method comprises makes chemical compound and suitable carriers (it can contain as one or more above-mentioned auxiliary elements) associating.Usually,, then if necessary, again the gained mixture is shaped by with the solid-state carrier of chemical compound and liquid or segmentation or both are all even closely is pre-mixed, can pharmaceutical compositions.For example, can be by to compressing or molded powder or the granule that contains chemical compound, randomly compress or molded with one or more auxiliary elements, prepare tablet.Can carry out tabletting by compositions (as optional and binding agent, lubricant, inert diluent and/or blended powder of surfactant/dispersant or granule) in suitable machine to free-flowing form.Can carry out moldedly with the moistening powdered chemical compound of inert liquid binder by molded in suitable machine, prepare through molded tablet.

Be suitable for sucking the pharmaceutical composition of (Sublingual) using and comprise dragee: it comprises the substrate of flavouring, (be generally sucrose and Radix Acaciae senegalis or Calculus Bovis from Northwest of China and chemical compound in the glue (tragacanth)); And lozenge, it comprises inert base (as the lozenge of the chemical compound in gelatin and glycerol or sucrose and the Radix Acaciae senegalis).

The pharmaceutical composition that is suitable for parenteral administration of the present invention can comprise the sterile aqueous and the non-aqueous injection solution of The compounds of this invention, and said preparation preferably oozes with the experimenter's of expection blood etc.These preparations can contain antioxidant, buffer agent, bacteriostatic agent and solute, and it makes experimenter's the blood etc. of compositions and expection ooze.Aqueous and non-aqueous aseptic suspensoid, solution and Emulsion can comprise suspending agent and thickening agent.The example of non-aqueous solute is propylene glycol, Polyethylene Glycol, vegetable oil, as olive oil and injectable organic ester, as ethyl oleate.Aqueous carrier comprises water, alcohol/aqueous solution, emulsion or suspension, comprises saline and buffering medium.The parenteral carrier comprises sodium chloride solution, Lin Ge (Ringer ' s) glucose, glucose and sodium chloride, Ringer lactate solution or expressed oi (fixed oil).Intravenous vehicles comprises fluid and supplementary, electrolyte replenisher (as based on those of woods lattice glucose), or the like.Can also there be antiseptic and other additive, as antimicrobial, antioxidant, chelating agen and noble gas or the like.

The pharmaceutical composition that is suitable for rectal administration preferably exists as unit dose suppository.These can be by mixing the conventional solid-state carrier of chemical compound and one or more (as cocoa butter), then the obtained by molding mixture in addition molding prepare.

The pharmaceutical composition of the present invention that is suitable for being applied topically to skin preferably adopts the form of ointment (ointment), cream, lotion, paste, gel, spray, aerosol or oil.Operable carrier includes, but are not limited to two or more combination of vaseline, lanoline, Polyethylene Glycol, alcohol, transdermal enhancer and they.In some embodiments, for example, can be by pharmaceutical composition of the present invention and the lipophilic reagent that can pass skin (for example, DMSO), be mixed and can carry out local delivery.

The pharmaceutical composition that is suitable for applied dermally can be the form of discrete patch, and it is suitable for keeping tight the contact for a long time with experimenter's epidermis.The compositions that is suitable for applied dermally can also be used by ionotherapy (for example seeing Pharmaceutical Research 3:318 (1986)), and typically, takes the form of the optional buffered aqueous solution of chemical compound.Appropriate formulation can comprise citric acid or bis/tris buffer (pH6) or ethanol/water, and it can contain 0.1 to the 0.2M active component.

In addition, chemical compound can be formulated as Liposomal formulation.The technology that is used to form liposome turbid liquor is as known in the art.When chemical compound or its salt are water soluble salt, can use conventional Liposomal formulation, described salt can be incorporated in the lipid vesicle.In this case, because the water solublity of chemical compound or salt, chemical compound or salt will be directed in the core of hydrophilic centre or liposome substantially.The lipid layer that uses can be the compositions of any routine, and can contain cholesterol or can be no cholesterol.When purpose chemical compound or salt are water miscible, reuse conventional liposome formation technology, salt can be directed in the hydrophobic lipid bilayer basically, and this bilayer forms the structure of liposome.In either case, the liposome of generation can reduce size, as handling and the homogenate technology by the use standard ultrasound.

Can freeze-dried lipidosome preparation to produce lyophilized products, can use pharmaceutically useful carrier, as water it is reconstructed, with the liposome turbid liquor of regenerating.

Described the present invention, will explain in more detail in the following embodiments the present invention to comprise that herein into these embodiment only are used to illustrate purpose, and be not intended to restriction the present invention.Use the following GM-CSF of abbreviation among the present invention, granulocyte macrophage colony stimulating factor; IL, interleukin; I.n., intranasal; I.t., in the trachea; NK, natural killer cell; NO; Nitric oxide; S.c., subcutaneous; TLR, toll sample receptor; TGF-β, transforming growth factor.

Embodiment 1

Cancer antigen and its vaccine

Flagellin is to the influence of innate immunity in the mouse lung.The maximum generation (Fig. 1) that (i.t.) instils and enough induced TNF α after about 4 hours in the non-operation trachea of 1 μ g flagellin.After 12-24 hour, the cytokine levels in the broncho-pulmonary eluate turns back to baseline values.Attention: thus also do not lack the not inducing cell factor generation of flagellin that signal transmits active sudden change in conjunction with TLR5.Except TNF α, several other cytokines (comprising IL-6, G-CSF) are induced into relative high level with chemotactic factor MIP-2 and KC.The increase of cytokine-expressing then is the temporary transient infiltration (maximum in the time of 12-24 hour) of neutrophil cell afterwards.Emphasize that it is important that innate immune responses that flagellin causes does not cause serious tissue injury's inflammation.Inductive inflammatory response is appropriate and acute relatively in nature.The body internal latent heat of flagellin as the activator of innate immunity established in the discovery of these discoveries and other research worker together.

The adjuvant effect of flagellin in the mouse lung.Except analyzing inductive congenital the replying of flagellin, also checked the effect that the flagellin antagonist is replied.With the flagellin mutain of 10 μ gF1 antigens and 1 μ g flagellin or inactivation by the immune BALB/c mouse that instils of (i.t.) or intranasal (i.n.) in the nonoperative trachea.After 4 weeks, mice is strengthened, then the serum levels of the anti-F1 IgG of 2 week back mensuration with identical scheme.Flagellin, but not the mutant of non-activity demonstrate as by serum anti--especially intensive adjuvanticity that F1 IgG horizontal survey obtains.In the animal of i.t. immunity, antibody titer is 20,000 to greater than 100,000, and for the situation of the animal of i.n. immunity, it is 90,000 to greater than 300,000.I.n. Mian Yi result provides in table 1.Use knock-out mice that the adjuvant effect that the analysis showed that flagellin that this cytokine of replying needs or not IL-6, IL-12, also do not need TNF-α (Fig. 5).In addition, use the maximum of replying to induce the flagellin dosage of the required little 5-10x of dosage (1 μ g) to realize the maximum adjuvant effect of flagellin than innate immunity in the respiratory tract of mice.Thereby, realized maximum adjuvanticity with the flagellin dosage that produces limited inflammation.

Flagellin	Total IgG is tired
		Non-activity mutant (7 mices)	＜100
Wild-type mice 1	＞300,000
		Mice 2	＞300,000
Mice 3	＞300,000
		Mice 4	90,000
Mice 5	＞300,000
		Mice 6	300,000

Table 1. causes strong anti-F1 IgG to reply with Yersinia pestis F1 antigen and the immunity of wild type flagellin intranasal.Flagellin with 10 μ gF1 antigens and 1 μ g wild type or sudden change gives twice immunity to mice.Measuring the anti-F1 IgG of serum by ELISA tires.

Embodiment 2

Flagellin is replied to produce antigenic specificity breast carcinoma as adjuvant

The mastadenoma interior therapeutic.Inactivation form immunity BALB/c mouse with Fra-1 (in many mastadenoma, crossing the antigen of expressing), flagellin or flagellin.Before a kind of breast cancer tumour D2F2 of subcutaneous injection cell, to the mice primary reinforcement.Monitor the tumor growth of mice and measure gross tumor volume.Data show in Fig. 2.The empty circles representative gives the mice of the flagellin of Fra-1 antigen and inactivation form.The solid circles representative gives the mice of the flagellin of Fra-1 antigen and activity form.Apparent from figure, flagellin+Fra-1 antigen has appreciable impact to growth of tumor.

Whether the assessment flagellin can promote the protective immune response at D2F2 breast cancer cell in the mouse model.Use Fra-1 as antigen, estimate the ability of flagellin promotion at the guard mode of the immunity of D2F2 breast cancer cell, Fra-1 is a kind of protein that these cancerous cell are crossed expression.These experiments comprise: immunity, and attack with the D2F2 cell then, and attack the back immunity, in addition, determine the character of inductive effect antibody contrast cytolytic T lymphocyte and/or NK cell.

A. to the antigenic preparation of reorganization Fra-1 of purification.The pET22 expression vector of the cDNA of fgs encoder Mus Fra-1 (being provided by Dr.Rong Xiang (The Scripps ResearchInstitute) friendship) is used for expressing Fra-1 protein on the Rosetta-gami-pLysS antibacterial.Use the affine resin of Talon (going up the identification of His label by Fra-1) protein purification, and the protein by purification passes Detoxi-gel column people (2000) Infect.Immun.68:5525-5529 such as () McDermott and removes the contaminative endotoxin.Use this method, obtained the some protein (for example, flagellin, F1 antigen and IRAK-4[eukaryotic protein]) of mg amount before this.

B. assess flagellin whether promote with in the BALB/c mouse of Fra-1 antigen and flagellin immunity to the protective immunity of D2F2 breast cancer cell.With 10 μ g Fra-1 albumen with or not with flagellin 229 intraperitoneal (i.p.) of 1 μ g flagellin or sudden change or 7 mices groups of intramuscular (i.m.) immunity.After 2 weeks, mice is strengthened, use 1 * 10 then ⁶D2F2 attacks (subcutaneous (s.c.) gives).Tracking of knub quality and mice survived about 3 months.When not having immunity, about 50% mice is dead in 4 weeks after attack.Measure the width and the length of Subcutaneous tumor, thereby can calculate gross tumor volume.If observe the remarkable result of flagellin, determine to produce the dosage of maximum flagellin of replying so.In case set up the optimal dose of its flagellin, just determine whether flagellin can promote the removing of existing tumor.Attack 7 mices groups with the D2F2 cell by subcutaneous injection, use then Fra-1 (with or not with flagellin) immunity when attacking or after 1,3,7 and 21 day.In each case, 2 weeks back attack mice.Determine the survival of gross tumor volume and mice.

C. identify potential effector in the replying that flagellin at the D2F2 cell of expressing Fra-1 promotes.In order to identify inductive effector in the replying that flagellin promotes, determine the level of the anti-Fra-1 antibody of circulation, and the relative number of CD8+ cytolytic T lymphocyte (CTL) and NK cell.With the flagellin of Fra-1 and flagellin or sudden change, according to such scheme immunity BALB/c mouse.

1. resist-Fra-1 antibody.Attack two weeks of back with the D2F2 cell, mice is carried out euthanasia, and get blood serum sample and be used for by the anti-Fra-1 antibody of elisa assay circulation.Use suitable anti-allotypic antibody, the level of the total IgG of assessments and IgM and IgG1 and IgG2a.

2. resist-Fra-1 specific C D8+CTL.By separating Morr. cell, and discharge their cell lysis activity of algoscopy assessment, determine anti-Fra-1 specific C D8+CTL with the 51Cr of standard.The cracking of CD8+CTL mediation will be by anti--MHC I antibody-like blocking-up.Therefore, this antibody exist and not in the presence of situation under the analysis of cells sample.By changing the ratio of effector and target, obtain relative estimation with the increase of CD8+CTL in the flagellin mice immunized of Fra-1 and flagellin or sudden change.Can also measure the number of activated CD8+CTL at the ELISPOT algoscopy of IFN-(INF-γ) generation.Use anti-CD8MACS MicroBeads (Miltenyl Biotech), from mice immunized separation of C D8+ splenocyte, and with cell with through radiating D2F2 cell incubation 24 hours.At the generation of INF-γ, come analysis of cells by ELISPOT algoscopy algoscopy.If there is the cell of enough numbers, so cell within a cell factor dyeing (ICS) is used as second method.

3.NK cell.Use the YAC-I cell as target, discharge the relative number of assessing activated NK cell in the mensuration at 51Cr.Obtain splenocyte from mice, and assessment is at the cell lysis activity of YAC-I cell through immunity and D2F2 attack.Because the expression of DX5 is relevant with the NK cell, the antibody of the PE labelling that use can obtain by commercial sources, the expression of measuring this labelling by flow cytometry.Flagellin should be relevant with the increase that DX5 expresses to the effect of stimulation of the expansion of NK cell.

The reorganization flagellin that contains the Fra-1 epi-position is at the effect in the protective response of D2F2 breast cancer cell.Determine further whether flagellin can be used as vaccine carrier and adjuvant.If such, produce the flagellin of coding so from the reorganization of the multiple epi-position of different tumor antigens.

Available evidence shows: human breast carcinoma demonstrates very big heterogeneity (heterogeneity) in the tumor specific antigen that they are expressed.For example, MAGE-3 expresses in about 14% breast carcinoma, and Her2/neu about 40%, NY-BR-62 60%, NY-BR-85 expresses (Scanlan and Jager (2001) Breast CaneerRes.3:95-98) in about 90% breast carcinoma.Determine to contain the antigenic reorganization flagellin of total length Fra-1 protein and whether can bring into play function in an identical manner with these two kinds of protein.If such, so to the epitope mapping in the Fra-1 that participates in inducing protective response.Carrying out identical incident with other main breast cancer antigen target will be direct work.Can produce the flagellin protein of expression from the epi-position of a series of target antigens.Alternatively, can use the proteinic mixture-every kind flagellin of flagellin to express group's target epi-position.In addition, by foreign epitope being imported proteinic hypervariable region-do not participate in interactional zone (Donnelly and Steiner (2002) the J.Biol Chem.277:40456-40461 of flagellin and TLR5; People such as Smith (2003) Nat.Immunol.4:1247-1253; People such as Murthy (2004) J.BiolChem.279:5667-5675), can not realize the biological activity of flagellin in any significant mode.

The chimeric generation of flagellin Fra-1.The Fra-1 complete sequence is inserted into the hypervariable region of flagellin and the gained construct is cloned into pET22a expression vector, and in the Rosetta-gami-pLysS antibacterial marking protein.Use the Detoxi gel column to remove endotoxin.Use the TNF α of RAW264.7 cell to produce, assess the biological activity of chimeric protein.Titration chimera, and comparing with its effect and wild type flagellin.

Mapping to the Fra-1 epi-position need be at the protection of D2F2 cell.Having estimated chimera protects BALB/c mouse to avoid the ability of the attack of D2F2 cell.If chimera is induced protective immunity, produce the overlapping truncate of Fra-1 sequence so, and in the background of flagellin test efficacy.In order to reduce the construct number, preparation covers three kinds of overlapping fragmentses of the complete sequence of Fra-1.If one of these truncates are active, produce extra truncate so to define the required minmal sequence of protection.If three kinds of truncates do not have a kind ofly self function is arranged, so these are carried out pairwise testing, to determine that whether required sequence is in proteinic different piece.Use this method, can determine required minmal sequence.

Embodiment 3

Flagellin is at Yersinia pestis

Lethal is breathed the potent mucosal adjuvant of the immunity of attacking

Method:

Plasmid and cell culture.With the antigenic coded sequence caf1 (plasmid that contains whole caf operon of the F1 of Yersinia pestis, by Dr.J.B.Bliska (State University ofNew York, Stony Brook) friendship provides) sub-clone comes in from Novagen (EMDBiosciences, Inc., Madison is among the NdeI and XhoI site of pET29a expression vector WI).(by Drs.G.Andrews and P.Worsham, USAMRIID provides) checks order, and sub-clone advances among the pET16b to F1/V fusion constructs people (1998) Vaccine 16:1131-1137 such as () Heat of reorganization.Order-checking discloses 21 aminoacid that do not exist corresponding to the signal sequence of F1.

Reagent and antibody.(Honko and Mizel (2004) Infect.Immun.72:6676-6679 as former description; People such as McDermott (2000) Infect.Immun.68:5525-5529) preparation from Salmonella enteritidis (Salmonella enteritidis) purified, reorganization, through the flagellin of His labelling.Flagellin and the F1 and the F1/V antigen of purification 229 sudden changes in an identical manner.(EastRutherford, QCL-1000  Chromogenic LAL Test Kit NJ) is detected, level of endotoxin≤1pg/ μ g from Cambrex Corporation as passing through.Use OptEIA ELISA test kit (mono/mono) to detect TNF-α according to manufacturer's operation instruction (BD Biosciences).From ResearchDiagnostics, (Flanders, the anti--F1 mouse monoclonal IgG1 that NJ) obtains, clone YPF 19 are used as the contrast among anti--F1 ELISA to Inc..(Birmingham AL) buys goat anti-mouse IgG-HRP. from ResearchDiagnostics, and (Flanders NJ) buys the anti-monkey IgG-HRP of goat to Inc. from SouthernBiotech.

Mice.(Frederick MD) buys female BALB/cAnNCr mice from Frederick Cancer Research and Development Center.From JacksonLaboratory (Bar Harbor, ME) buy female IL-6-/-mice (B6; 129S2-I16/J), the TNFR1 mice (tm1Kopf-/-B6; 129S-Tnfrsf1a Tnfrsf1b/J), IFN γ (tm1Imx tm1Imx-/-B6.129S7-Ifngtm1Ts/J) and control mice (C57BL/6J, B6; 129SF2/J and 129/SvJ).IFN α/β R-/-mice is by Dr.C.Schindler, Columbia University, and New York (people (1994) Science264:1918-1921 such as M ü ller) provides.Mice is remained in the specific bioclean facility, and federation and mechanism's criterion of Wake Forest University Animal Care and UseCommittee regulation are all deferred in all researchs.

Interior and the intranasal immunity of the non-operation trachea of mice.For in the organ immunity, with mice with avertin (2,2,2-three bromo ethanol, Sigma; Tert-pentyl alcohol Fisher) by the peritoneal injection anesthetized mice, and suspends with the line of the certain-length preceding front tooth (front incisor) by them it in midair to it.The aseptic suction nozzle that use and to insert air inlet pipe gently, loads gel is used 50 μ L altogether and is not had the 10 μ gF1 antigens among the PBS of pyrogen and the flagellin or the flagellin mutant 229 of indicatrix.For intranasal administration, to use small size (9-12 μ L altogether) antigen and the adjuvant among the PBS through the nostril of anesthetized mice.When 4 weeks, mice is strengthened, and 2-3 week collection blood plasma is used for the analysis that antagonist is tired after reinforcement.

Immunity to monkey.According to the federation of Wake Forest University Animal Care and UseCommittee regulation and the adult female macaque (Macaca fascicularis) of 15 health of mechanism's criterion maintenance.Animal is anaesthetized animal with 7-10m/kg ketamine intramuscular, to be used for immunity and to collect blood.For the intranasal immunity, 150 μ gF1/V fusion rotein and 50 μ g flagellins are dropwise sent (100 μ L/ nostril) give animal, send with clinostatism and carry out.Use the intramuscular immunity with the 1ml volume to musculus quadriceps.Control animal intranasal and intramuscular are accepted PBS.

By enzyme-linked immunosorbent assay (ELISA) analysed for plasma antibody titer.With heparinization pipe (StatSpin; Fisher Scientific) or BD Vacutainer PST pipe carry out plasma collection.Then with the blood plasma aliquot, and freezingly with 10 μ g/mL among the aseptic PBS elisa plate is carried out the bag quilt that spends the night under 4 ℃, and seal with the 10%FCS among the PBS up to analyzing .100 μ L antigen at-70 ℃.Add duplicate or in triplicate plasma extender, and at 4 ℃ of incubation flat boards that spend the night, then itself and secondary are resisted-Ig antibody is room temperature incubation 2 hours.With 3,3 ', 5,5 '-tetramethyl benzidine (TMB) Liquid Substrate System (Sigma-Aldrich) detects peroxidase activity, and uses 2M H ₂SO ₄Cessation reaction. terminal point dilution tired to be defined as: cause absorption value (OD ₄₅₀) than the blood plasma that had not experimentized＞inverse of 0.1 high dilution.

CO92 breathes attack with Yersinia pestis.Centers for DiseaseControl (CDC) Division of Vector-Borne Infectious Diseases (FortCollins, CO) provide the original seed culture of Yersinia pestis CO92 biovar orientalis, it is from the isolating a kind of bacterial strain of the deadly case of people's primary pulmonary plague (people (1994) Am.J.Trop.Med.Hyg.51:109-114 such as Doll).Use single colony inoculation heart infusion broth, and cultivate about 1 * 10 at 28 ℃ from the subculture plate ⁹The density of individual colony-forming units (cfu)/mL.With mice in order to PBS be diluted to～1/8 * 10 ⁷Cfu/mL (equals 1.2 * 10 ⁴150 * 50% fatal dose (LD of cfu ₅₀) dosage) 10 μ L cultures, mice is carried out intranasal attacks (data not shown).By at Trypsin

(sryptose) blood agar plate upper flat plate inoculation serial dilution is determined actual cfu/mL value.All experiments are carrying out about the BSL3 of the Infectious Disease Unit of Virginia Tech and the standard operating procedure (CDC ratifies #C20031120-0016) of ABSL3 facility according to CDC approval all.

Statistical analysis.Data are with single value representation, indicate to have meansigma methods and standard error.The F check of variance neat (equality of variance) and Si Shi one-side t are checked the significance,statistical that is used to provide p＜0.05 or p＜0.01 o'clock.(Richmond CA) determines LD by nonlinear regression for SystatSoftware, Inc. with SigmaStat3.1 ₅₀Value.

The result:

With the flagellin immunological enhancement to the F1 of Yersinia pestis antigenic effective active reply.In order to determine that flagellin promotes the ability for the antigenic humoral immunoresponse(HI) of F1 of Yersinia pestis, with BALB/c mouse with 10 μ gF1 antigens and 1 μ g reorganization flagellin (FliC) trachea interior (i.t.) or intranasal (i.n.) immunity. control animal is with mutant form (the being called 229) immunity of the F1 antigen among the PBS or F1 and flagellin.After 4 weeks, strengthen mice in an identical manner and collect blood plasma and be used to analyze after reinforcement the circulating antibody of different time and tire.In matched group, do not detect the F1 specific IgG.Yet (Fig. 1 a), has high-caliber F1 specific IgG 1 and IgG2a to contain the remarkable increase that the boosting vaccine total IgG of F1 and flagellin tires.When i.t. used flagellin and F1, the average proportions of IgG1 and IgG2a (pointing out in the bar shaped) was 30-170, when the i.n. administration, is about 3.Although i.n. and i.t. immunity cause mixed type Th to reply, after the immunity, the deflection that apparent Th2 is replied is more obvious in the trachea.Notice that it is important that flagellin does not promote significant F1-specific IgE to produce.With F1 and flagellin mice immunized after twice immunity, demonstrate anti--F1IgG continue tire (Fig. 3, b).Immunity for the third time when 16 weeks has improved the antibody response that has in low two mices of tiring at first.

Show in the former research: under the flagellin dosage of 5-15 μ g, congenital the replying to flagellin in the lung is maximum (Honko and Mizel (2004) Infect.Immun.72:6676-6679).In order to determine between the amplitude of the antibody response that cytokine response and flagellin promote, whether have linear relationship, with F1 antigen and 1 μ g, 5 μ g and 15 μ g flagellins immunity BALB/c mouse (Fig. 3, c).Anti--F1 IgG tires under these dosage does not have significantly difference, shows that adjuvant effect is maximum under 1 μ g flagellin.Yet we notice: when using the flagellin that increases dosage, tend to bigger IgG1/IgG2a ratio.Thereby as if maximum adaptability is replied does not need maximum congenital replying.

When considering that flagellin is used as adjuvant, the immunity that is pre-existing in of flagellin is obviously received publicity.Therefore, we have estimated in the presence of the anti-flagellin antibody that height is tired, with the effectiveness of flagellin and F1 immunity.With immunity of 5 μ g flagellins and the female BALB/c mouse of reinforcement, and measure anti-flagellin antibody titer.Before the immunity, the flagellin specific IgG is tired and is lower than detection level, and it significantly increases, and average resisting-it is 8.5 * 10 that FliC IgG tires ⁵Then with 10 μ g F1 and the immunity of 1 μ gFliC intranasal and these mices of reinforcement.Strengthen 2 weeks of back, be not used to test and the FliC immune mouse between, it is that similar (Fig. 3 d), shows that the anti-flagellin antibody of circulation actively or does not negatively change replying flagellin that anti--FliC IgG tires.Our result has supported as drawing a conclusion: in the presence of the previous immunity to flagellin, flagellin is effective adjuvant.

The T lymphocyte is replied and depended on to the adjuvant effect stimulator antigen specificity of flagellin.Immunological memory be the basic feature of effective vaccine, this allows immune system prepare fast reaction after being exposed to antigen once more between infection period.In order to estimate in the secondary immunity needs that flagellin is stimulated, with F1 antigen and four groups of BALB/c mouse of flagellin immunity, and subsequently with PBS, only flagellin, only F1 antigen or flagellin and F1 strengthen (Fig. 4, a).In the mice that only PBS or flagellin are strengthened, the F1 specific IgG is tired and is remained on 500-1100, and it is to reply the typical value in back first.Yet, in secondary immunity, only accept the anti-F1 IgG that the antigenic mice of F1 has remarkable increase and tire.Although do not need flagellin in reinforcement, when having flagellin, anti-F1 antibody titer significantly increases.These discoveries are similar to the result that Pasare and Medzhitov (2004) Immunity 21:733-741 use LPS to report as adjuvant.Author's prompting is in case set up CD4 with LPS as adjuvant ⁺Memory, these lymphocytic activation no longer need TLR to stimulate so.Although the memory response in our system still remains to be analyzed fully,, the shortage of F1 specific IgG in the nude mouse (BALB/cAnNCr-nu/nu) of flagellin and F1 immunity, showing needs the T cell in the humoral response to this vaccine.

TNF-α, IL-6 and interferon are not that the adjuvant effect of flagellin is required.(embodiment 1 to have determined to induce high-caliber TNF-α and IL-6 by flagellin in lung before this; Honko and Mizel (2004) Infect.Immun.72,6676-6679).Therefore, assessed the effect of these cytokines in the adjuvanticity of flagellin.TNF-α is a kind of multiple-effect cytokine, its promote dendritic cell maturation (people (2000) Annu.Rev.Immunol.18 such as Banchereau, 767-811).As Fig. 5, shown in a, in these mices, it is very high that anti-F1 antibody response keeps, and shows that TNF-α is not that the adjuvant effect of flagellin is required.Yet the TNF-α that flagellin stimulates produces and seems to strengthen to F1 antigenic antibody response, because TNFR ^-/-Tiring in the mice with respect to the B6 of wild type; 129 mices reduce about twice.Use IL-6 ^-/-The effect in the adjuvanticity of flagellin that mice has been assessed IL-6---a kind of cytokine (people (2003) Rev.Physiol Biochem.Pharmacol.149:1-38 such as Kaminura) that promotes B cell proliferation and differentiation---.After F1 and FliC immunity, the generation of anti-in these mices-F1 IgG does not have defective, and (Fig. 5 b), shows that the adjuvant effect that this cytokine neither flagellin is essential.

External, by the signal transmission via functional TLR5/4 allos complex, flagellin stimulates the generation (Mizel et al. (2003) J.Immunol.170:6217-6223) of nitric oxide and interferon-(IFN-β).C3H/HeJ mouse has mutation T LR4 people (1998) Science 282:2085-2088 such as () Poltorak of not function, and this provides in the chorista TLR5/4 heteromerism (heterometric) and TLR5/5 with the model of several (hemometric) signal transmission effects.In lung, be not damaged through being created in the C3H/HeJ mouse of stimulating of flagellin to what TNF-α, IL-6, G-CSF (granulocyte colony-stimulating factor), keratinocyte were derived chemotactic factor (KC), macrophage inflammatory protein 2 (MIP-2) and MIP-1 α that (embodiment 1; Honko and Mizel (2004) Infect.Immun.72:6676-6679); Yet, interferon is not produced and is assessed.Propose: I type IFN passes through to stimulate the rise of the costimulatory molecules on MHC and the antigen-presenting cell, thereby congenital and adaptive immune response are connected (Le Bon and Tough (2002) Curr.Opin.Immunol14:432-436).In order to determine that the TLR5/4 signal transmits the influence to the adjuvant effect of flagellin, after F1 antigen and flagellin immunity, anti--F1 antibody titer in the C3H/HeJ mouse and their wild type copy C3H/HeN mice are compared (Fig. 5, c).Owing to do not have defective during antibody produces, the signal transmission by the TLR5/4 complex is not that the adjuvant effect of flagellin is required.By determining that lacking I type interferon signal transmits (IFN α/β R ^-/-) or produce IFN-(IFN γ ^/-) the mice of ability in antibody response, directly assess the effect (Fig. 5, d and e) of interferon in the adjuvanticity of flagellin.Since the mice of two kinds of strains all with to reply with the F1 antigen mode similar with the wild-type mice of flagellin immunity, so that interferon is not the adjuvant effect of flagellin is required.

Flagellin promotes the protective response to the intranasal attack of Yersinia pestis CO92.Basic test to vaccine provides the protective capability that pathogen is attacked.As model at respiratory infections, with toxicity Yersinia pestis CO92 to carrying out intranasal with control mice and attack through immunity.In order to ensure the inoculation of inducing enough protections, 50% fatal dose (LD of the intranasal infection that is Yersinia pestis is selected in the recommendation of the nearest Plague Vaccine Workshop that initiates based on NIAID and FDA (13-14 day in October, 2004) for use ₅₀) about 150 times challenge dose.With equaling 100 * LD ₅₀Dosage attack after, have 93% survival rate with the BALB/c mouse of flagellin and immunity of F1 intranasal and reinforcement, by contrast, in the matched group only be 7% (Fig. 6, a).IgH with the B cell defect ^-/-Mice estimate protective response B cell/antibody dependent (Fig. 6, b).At about 150 * LD ₅₀Dosage under, all contrasts and all die from Yersinia pestis infection through immune mouse show that the protection under this challenge dose is that B is cell-mediated, thereby and may be antibody-mediated.In the past, people such as Elvin used the Stat4 of the cellullar immunologic response of IL-12 with defective and IFN-γ mediation ^-/-Animal has been checked the effect (Elvin and Williamson (2004) Microb.Patho.37:177-184) of 1 type effector function in the immune Yersinia pestis infection of protection.And the Stat4 of immunity ^-/-Mice and their wild type copy produce the anti--F1 and the anti-VIgG of similar level, and these animals are attacked a little less than the protection that is subjected to for high dose.For the effect of the protection of intranasal attack back IFN-γ mediation in the system that handles us, use F1 antigen and flagellin to IFN-γ ^-/-After carrying out immunity and reinforcement with wild type C57BL/6 mice group, with 150 * LD ₅₀Yersinia pestis is attacked.Wild type C57BL/6 mice is subjected to the protection fully of the intranasal immunity of flagellin and F1, by contrast, only 10% the contrast be protected (Fig. 6, c).Through the IFN-of immunity γ ^-/-Mice has 80% survival after attack (Fig. 6 d), shows that replying of IFN-γ mediation is not that protection is required.Because these animals have anti--F1 IgG that height tires, avoid importance in the Yersinia pestis infection so these results have confirmed the F1 specific antibody in protection, and supported to utilize circulation IgG to tire as the purposes of the correlative of protection effect.Two IFN-γ ^-/-Mice dies from and infects this observation prompting: IFN-γ may increase the antibody-mediated protection to Yersinia pestis, and this may be by promoting the respiratory burst in the phagocyte to realize.

Flagellin is the effective adjuvant in the inhuman primates.Consider that flagellin promotes the ability of adaptive immune response in the mouse model, next we assessed the effectiveness of flagellin as adjuvant in the non-human primates.Use the female macaque of forming by F1 and the V antigen of Yersinia pestis of recombination fusion protein immunity.With 150 μ gF1/V fusion rotein and 50 μ g flagellins 6 macaque groups are carried out intranasal or intramuscular (i.m.) immunity.Extra control animal (n=3) is accepted PBS by two kinds of approach.Before the immunity, monkey demonstrates about 9.8 * 10 ⁴Anti-flagellin antibody titer.In preceding 24 hours after immunity, do not demonstrate the change of body temperature or Plasma TNF-alpha levels, and observable inflammation does not take place in the injection site with the monkey of flagellin immunity.Strengthen animal in an identical manner in 4 weeks, and determine in 2 week backs blood plasma anti--F1/V IgG tire (Fig. 7).Demonstrate the remarkable increase of F1/V specific antibody titres through the monkey of immunity.Do not detect antigenic specificity IgE.These results clearly illustrate that flagellin is effective adjuvant that antibody response takes place in non-human primate, also be like this even exist under the situation of the anti-flagellin antibody of circulation.

Embodiment 4

Yersinia pestis antigen and vaccine

In the mode similar,, produced the fusion rotein that is used to induce to the immunne response of Yersinia pestis with Yersinia pestis V antigen, Yersinia pestis F1 antigen or its fusogenic peptide to embodiment 2.This type of fusion rotein can be used for induce immune response, and randomly protective immune response is as described herein.Randomly, replying is mucosal immune response.The specific non-limitative example of suitable fusion rotein is:

Example A:fliC/F1/V aminoacid sequence (SEQ ID NO:1)

1 MAQVINTNSL SLLTQNNLNK SQSSLSSAIE RLSSGLRINS AKDDAAGQAI

51 ANRFTSNIKG LTQASRNAND GISIAQTTEG ALNEINNNLQ RVRELSVQAT

101 NGTNSDSDLK SIQDEIQQRL EEIDRVSNQT QFNGVKVLSQ DNQMKIQVGA

151 NDGETITIDL QKIDVKSLGL DGFNVNGPKE ATVGDLKSSF KNVTGRSMAD

201 LTASTTATAT LVEPARITLT YKEGAPITIM DNGNIDTELL VGTLTLGGYK

251 TGTTSTSVNF TDAAGDPMYL TFTSQDGNNH QFTTKVIGKD SRDFDISPKV

301 NGENLVGDDV VLATGSQDFF VRSIGSKGGK LAAGKYTDAV TVTVSNQGSI

351 EGRNRAYEQN PQHFIEDLEK VRVEQLTGHG SSVLEELVQL VKDKNIDISI

401 KYDPRKDSEV FANRVITDDI ELLKKILAYF LPEDAILKGG HYDNQLQNGI

451 KRVKEFLESS PNTQWELRAF MAVMHFSLTA DRIDDDILKV IVDSMNHHGD

501 ARSKLREELA ELTAELKIYS VIQAEINKHL SSSGTINIHD KSINLMDKNL

551 YGYTDEEIFK ASAEYKILEK MPQTTIQVDG SEKKIVSIKD FLGSENKRTG

601 ALGNLKNSYS YNKDNNELSH FATTCSDKSR PLNDLVSQKT TQLSDITSRF

651 NSAIEALNRF IQKYDSVMQR LLDDTSGK RS ATGDKITLAG KTMFIDKTAS

701 GVSTLINEDA AAAKKSTANP LASIDSALSK VDAVRSSLGA IQNRFDSAIT

751 NLGNTVTNLN SARSRIEDAD YATEVSNMSK AQILQQAGTS VLAQANQVPQ

801 NVLSLLRLEH HHHHH ^*

Example B:FliC/F1 aminoacid sequence (SEQ ID NO:2)

1 MAQVINTNSL SLLTQNNLNK SQSSLSSAIE RLSSGLRINS AKDDAAGQAI

51 ANRFTSNIKG LTQASRNAND GISIAQTTEG ALNEINNNLQ RVRELSVQAT

101 NGTNSDSDLK SIQDEIQQRL EEIDRVSNQT QFNGVKVLSQ DNQMKIQVGA

151 NDGETITIDL QKIDVKSLGL DGFNVNGPKE ATVGDLKSSF KNVTGRSMAD

201 LTASTTATAT LVEPARITLT YKEGAPITIM DNGNIDTELL VGTLTLGGYK

251 TGTTSTSVNF TDAAGDPMYL TFTSQDGNNH QFTTKVIGKD SRDFDISPKV

301 NGENLVGDDV VLATGSQDFF VRSIGSKGGK LAAGKYTDAV TVTVSNQ RSA

351 TGDKITLAGK TMFIDKTASG VSTLINEDAA AAKKSTANPL ASIDSALSKV

401 DAVRSSLGAI QNRFDSAITN LGNTVTNLNS ARSRIEDADY ATEVSNMSKA

451 QILQQAGTSV LAQANQVPQN VLSLLRLEHH HHHH ^*

Example C:FliC/V aminoacid sequence (SEQ ID NO:3)

1 MAQVINTNSL SLLTQNNLNK SQSSLSSAIE RLSSGLRINS AKDDAAGQAI

51 ANRFTSNIKG LTQASRNAND GISIAQTTEG ALNEINNNLQ RVRELSVQAT

101 NGTNSDSDLK SIQDEIQQRL EEIDRVSNQT QFNGVKVLSQ DNQMKIQVGA

151 NDGETITIDL QKIDVKSLGL DGFNVNGPKE ATVGDLKSSF KNVTGRSMIR

201 AYEQNPQHFI EDLEKVRVEQ LTGHGSSVLE ELVQLVKDKN IDISIKYDPR

251 KDSEVFANRV ITDDIELLKK ILAYFLPEDA ILKGGHYDNQ LQNGIKRVKE

301 FLESSPNTQW ELRAFMAVMH FSLTADRIDD DILKVIVDSM NHHGDARSKL

351 REELAELTAE LKIYSVIQAE INKHLSSSGT INIHDKSINL MDKNLYGYTD

401 EEIFKASAEY KILEKMPQTT IQVDGSEKKI VSIKDFLGSE NKRTGALGNL

451 KNSYSYNKDN NELSHFATTC SDKSRPLNDL VSQKTTQLSD ITSRFNSAIE

501 ALNRFIQKYD SVMQRLLDDT SGK RSATGDK ITLAGKTMFI DKTASGVSTL

551 INEDAAAAKK STANPLASID SALSKVDAVR SSLGAIQNRF DSAITNLGNT

601 VTNLNSARSR IEDADYATEV SNMSKAQILQ QAGTSVLAQA NQVPQNVLSL

651 LRLEHHHHHH ^*

Note: FliC obtains from Salmonella enteritidis.In each of these fusion rotein, the terminal constant region of the N-of FliC finishes at amino acid residue 198 places, C-terminal constant region (preceding 7 aminoacid of representing with runic and underscore) begins at amino acid residue 679 places of example A (SEQ ID NO:1), amino acid residue 348 places of example B (SEQ ID NO:2) begin, and begin at amino acid residue 524 places of example C (SEQ ID NO:3).

Embodiment 5

Contain flagellin and Yersinia pestis

The biological activity of F1 and the antigenic fusogenic peptide of V

In order to prepare coding as single flagellin and Yersinia pestis F1 and antigenic expression plasmid of V of planting protein, remove the major part of nucleotide sequence of the hypervariable region of coding Salmonella enteritidis flagellin, and with placed in-line F1 and V sequence replacing, described F1 and V sequence are separated (seeing top example A, SEQ ID NO:1) by 6 amino acid whose 8 nucleotide bridges of coding.In the BL21 cell, produce recombinant protein, and by affinitive layer purification on the affine resin of metal.Use Acrodisc chromatography filter to remove endotoxin and contaminative nucleic acid.In order to determine whether gained protein keeps the flagellin activity, negative and positive RAW264.7 cell of TLR5-and three fusion rotein incubations with TLR5-, and the degree of definite tumor necrosis factor generation.TLR5-negative RA W cell is used for controlling and may has any contaminative factor of influence in this mensuration.As shown in Figure 8, the fusion rotein that contains flagellin and Yersinia pestis F1 and V protein has kept the flagellin biological activity in the TLR5-positive cell.This protein does not transmit signal in TLR5-negative RA W264.7 cell.

With the fusion rotein incubation of the positive RAW264.7 cell of Toll-sample receptor 5 (TLR5) negative RA W264.7 cell or TLR5-(cell line that produces by construct stable transfection RAW264.7 cell) and the coding flagellin that increases concentration and Yersinia pestis F1 and V protein 4 hours, measure the TNF-alpha content of culture medium then by ELISA with the enhanced yellow fluorescence protein of coding TLR5-.

For whether single protein of planting of determining flagellin+F1+V or containing all three kinds of components can provide protection at the deadly attack of Yersinia pestis CO92, only use phosphate-buffered saline (PBS) or contain the vaccine of three kinds of protein-every kind of F1 of 1mg flagellin+5mg and V or contain a kind of protein (flagellin/F1/V that comprises flagellin, F1 and V; Vaccine immunity 10mg) and reinforcement C3H/HeJ mouse.After 4 weeks, strengthen mice, use about 150 LD then with identical scheme ₅₀Yersinia pestis CO92 attack.Before attack, mice is got blood and determines that by ELISA anti--F1 IgG tires.As shown in table 2, F1 of flagellin+Yersinia pestis and V antigen or contain flagellin and the F1 of Yersinia pestis and the antigenic fusion rotein of V provide the lethal of Yersinia pestis is breathed the protection fully of attacking.

Immunity	Anti-F1 IgG tires	Anti-VIgG tires	Survival number percent
					PBS	6.6×10 2	1.2×10 3	0/10	0
Flagellin+F1+V	4.5×10 6	4.2×10 6	10/10	100
					Flagellin/F1/V	5×10 6	6.3×10 6	4/4	100

Table 2. is with F1 and the V antigen of flagellin+Yersinia pestis or contain flagellin and the mice of F1 of Yersinia pestis and the antigenic fusion protein immunization of V breathes the protection result of study of attacking to the lethal of Yersinia pestis

Above illustrated the present invention, and not as restriction of the present invention.The present invention limits by following claim, comprising the equivalent of claim.

Claims (22)

1. fusion rotein, it comprises:

(a) flagellin adjuvant, this flagellin adjuvant comprises:

(i) the terminal constant region of flagellin N-; With

The (ii) terminal constant region of flagellin C-; With

(b) tumor antigen between terminal constant region of N-and the terminal constant region of C-.

2. the fusion rotein of claim 1, wherein said flagellin adjuvant comprises the flagellin hypervariable region of disappearance, does not have the flagellin hypervariable region in the perhaps described flagellin adjuvant.

3. the fusion rotein of claim 1, wherein said tumor antigen is inserted in (i) hypervariable region, (ii) between terminal constant region of flagellin N-and the hypervariable region, perhaps (iii) between terminal constant region of flagellin C-and the hypervariable region.

4. the fusion rotein of claim 1, wherein tumor antigen is a breast tumor antigen.

5. the fusion rotein of claim 1, wherein tumor antigen is cancer/testis antigen.

6. the nucleic acid of the fusion rotein of the claim 1 of encoding.

7. the carrier that comprises the nucleic acid of claim 6.

8. the host cell that comprises the carrier of the nucleic acid of claim 6 or claim 7.

9. prepare the method for the fusion rotein of claim 1, this method comprises: enough producing under the condition of described fusion rotein, cultivate the host cell of claim 8 in culture medium.

10. the method for claim 9 is wherein collected fusion rotein from host cell or culture medium.

11. immunogenic composition, it comprises and is in fusion rotein in pharmaceutically suitable carrier, claim 1.

12. produce the method at the immunne response of tumor antigen in mammalian subject, this method comprises with effective generation in this mammalian subject uses the fusion rotein of claim 1 or the immunogenic composition of claim 11 at the amount of the immunne response of described tumor antigen to described mammalian subject.

13. the method for tumor in the treatment mammalian subject, this method comprise with the treatment effective dose this mammalian subject is used the fusion rotein of claim 1 or the immunogenic composition of claim 11.

14. the method for claim 13, wherein said tumor is a breast tumor.

15. immunogenic composition, it comprises in pharmaceutically suitable carrier:

(a) flagellin adjuvant; With

(b) tumor antigen.

16. the immunogenic composition of claim 15, wherein said tumor antigen are breast tumor antigen.

17. the immunogenic composition of claim 15, wherein said tumor antigen is cancer/testis antigen.

18. the immunogenic composition of claim 17, wherein said immunogenic composition comprises two or more cancer/testis antigens.

19. the immunogenic composition of claim 15, wherein said tumor antigen and the coupling of flagellin adjuvant.

20. produce the method at the immunne response of tumor antigen in mammalian subject, this method comprises to produce in this mammalian subject at the immunne response of tumor antigen effectively measures the immunogenic composition of described mammalian subject being used claim 15.

21. the method for tumor in the treatment mammalian subject, this method comprise the immunogenic composition of this mammalian subject being used claim 15 with the treatment effective dose.

22. the method for claim 21, wherein said tumor is a breast tumor.

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