CN107058231A - Carry recombined adhenovirus of antigen-4 fusion protein gene and its preparation method and application - Google Patents

Abstract

The present invention relates to recombined adhenovirus for carrying antigen-4 fusion protein gene and its preparation method and application.Specifically, the present invention relates to a kind of recombined adhenovirus of the coded sequence for the fusion protein for carrying heat shock protein and carcinomebryonic antigen, its preparation and application, the genomic deletion E1 areas and E3 areas of the adenovirus, and the expression cassette of the fusion protein coded sequence is inserted in E1 and/or E3 zone positions, the expression cassette includes successively：Promoter sequence, the coded sequence of the fusion protein and tailing signal sequence, wherein the heat shock protein is selected from：Hsp70 or Hsp70L1.The present invention recombined adhenovirus can efficiently mediated gene it is (especially antitumor) transfer and expression, be with a wide range of applications.

Description

Carry recombined adhenovirus of antigen-4 fusion protein gene and its preparation method and application

Technical field

The invention belongs to biological technical field, it is related to a kind of human cancer embryoantigen 576-669 (CEA_576-669) and heat shock protein Recombined adhenovirus of 70 sample molecule 1 (hsp70L1) fusions and its preparation method and application.

Background technology

Heat shock protein (Heat shock protein, HSP) is that a class is highly conserved in biological evolution, is widely present Class protein in protokaryon and eucaryote, the expression increase after cell is stressed.HSP is in the cell as companion point Son, participates in the processing of antigen and forms MHC- antigenic peptide complexes.

In recent years, the obvious adjuvant sample effect of HSP families causes the concern of domestic and international academia.Research shows that HSP exists After cell is stressed, extracellular, the important physiology mistake such as inducing antigen presenting cells and T cell maturation activation can be discharged into Journey, thus it is closely related with the physiology such as the antineoplastic immune, anti-infectious immunity, autoimmune disease of body and pathomechanism, It is the important immune molecule of a class (Srivastava PK, Annu Rev Immunol.2002；20:395–425).

Heat-shock protein family can be divided into four classes according to molecular weight：Hsp90 families, Hsp70 families, HSP60 families and Small molecule Hsp families, wherein Hsp70 families are a most conservative and topmost classes in HSP.Many studies have shown that, tumour cell Or Hsp70- peptide complexes, the Hsp70 molecules in virus infected cell source are combined polypeptide antigen or are crosslinked with polypeptide antigen Compound, can substantially induce produce antigentic specificity CD8⁺CTL, Hsp70 is used alone or polypeptide is used alone Antigen is then without this effect, therefore the immunogenicity of adjuvant sample effects and small-molecular peptides of the HSP in antigen presentation pathway is that HSP is situated between Immunization therapy institute indispensable two piths (Todryk SM etc., the Immunology.2003 led；110:1-9； Manjili MH etc., Expert Opin Biol Ther.2004；4:363-373；Blachere NE etc., J Immunother Emphasis Tumor Immunol.1993；14:352-356；Li Z etc., Behring Inst Mitt.1994；94:37- 47；Moroi Y etc., Proc Natl Acad Sci USA.2000；97:3485–90).

The specific CTL reaction of HSP inductions is due to the acceptor that there is HSP on professional antigen presenting cell (APC), can be with Make endocytosis (Arnold-Schild D etc., J Immunol.1999s of the APC specifically by hsp receptor mediate antigen peptide； 162:3757-3760)；The antigen that HSP is combined is to be relied on by TAP and TAP non-dependent approach in intracellular, by submission to MHC-I On quasi-molecule, and subsequent ctl response is induced, this acceptor is proved to be present on the APC such as DC, macrophage and B cell, and (Castellino F, J Exp Med.2000 are not present in T cell；191:1957-1964)；It is understood that HSP is logical It is an important mechanisms of the HSP as adjuvant that hsp receptor, which is crossed, with APC interactions.Research shows, this receptor-mediated antigen The efficiency of submission mode is higher 10000 times than swallowing.

HSP70 molecules can exercise the effect of similar cell factor sample extracellular.HSP70 can produce high affine with monocyte The combination of power, induces its to produce IL-6, IL-1 isoreactivity cell factor, it is possible to induce DC ripe, raise its MHC-II and CD86 developed by molecule, secretion IL-12 and TNF-α, so as to strengthen DC antigen submission ability (Svensson PA etc.； Atherosclerosis.2006；185:32-38).Therefore, can using HSP as immunologic adjuvant collaboration tumour antigen induction DC With the submission of targeting mediate antigen, the submission efficiency of enhancement antigen, so as to induce stronger ctl response.

On the basis of it specify that HSP unique adjuvant effect, HSP adjuvant effect inducing antitumor and anti-infective is utilized Immune therefore to turn into focus, the vaccine of new generation based on HSP enters clinical experimental study one after another.Vaccine based on HSP constitutes general Bracketing mainly has following several ways：1st, from tumour cell or virus infected cell purifying HSP- peptide complexes, with purifying HSP- peptide complexes vivo immunizations excite antitumor or anti-infectious immunity.HSP peptide complexes, which are used for oncotherapy, to be had significantly Advantage, because the HSP peptide complexes extracted are used directly for immunization therapy, it is not necessary to identify the tomour specific included in it Property epitope.Unfortunately, individualized treatment scheme is used this scheme, that is, HSP- peptide complexes used, which are immunized, to be needed to come more Come from autologous tumor cell.2nd, HSP is obtained by protokaryon and eukaryotic expression, the epitope peptide for making synthesis by external crosslinked is incorporated into HSP peptide binding pockets, then with crosslinking HSP- peptide complexes inducing antitumor or anti-infectious immunity.3rd, the fusion protein of HSP antigens It is immune.4th, the DNA vaccination or adenovirus vaccine of HSP antigen genes amalgamation and expression.

The gp96- peptide complexes (HSPPC-96) in autologous tumor source have been enter into a variety of pernicious by representative of clear-cell carcinoma The clinical test of tumour, have proven at present patient have to multiple different application dosage good tolerance (Caudill MM and Li Z.Expert Opin Biol Ther.2001；1(3):539-47.).Cohen etc. treats 36 IV phase kidneys with HSPPC-96 The clinical I phases of cell cancer and the II phase clinical effectivenesses of III-IV phase melanomas show that treatment group's life quality is significantly improved (Cohen L etc.；Melanoma Res.200212(5):505-11.).Belli treats metastasis melanin tumor with HSPPC-96, Have no in obvious toxic-side effects, 28 patients, 2 CR (complete response), 3 SD (stable disease), CR Duration has exceeded 559 and 703 days respectively；Learn from else's experience and control the PMNC of patient and make in ELISPOT detections, 23 11 melanoma-specific T cells dramatically increase (Belli F etc., J Clin Oncol.2002；20(20):4169-80.). The vaccine HspE7 (SGN-00101) that Stressgen companies are constituted with the fusion protein of HSP65 and HPV HPV-16 E7s is to HPV infection Treatment with related neoplasms has been found to safe and effective, now positive to carry out phase III clinical trial.It can be seen that, the epidemic disease of new generation based on HSP Seedling has extraordinary potential applicability in clinical practice (Maciag PC, Paterson Y.Curr Opin Mol Ther.2005；7(3): 256-263；Hunt S.Curr Opin Mol Ther.2001；3(4):413-417.).

Immunology teaching and research room of Second Military Medical University, PLA passes through to important antigen presenting cell --- tree A kind of prominent shape cell cdna library large scale sequencing, it was found that recruit of HSP 70 family, by name HSP70L1, and gram Grand its full-length cDNA (GenBank accession number is AF143723), its expression product has applied for national inventing patent (application number： 99124271.8)。

Because HSP70L1 has more than 90% sequence identity and similitude on amino acid sequence with mouse HSP70, Sequence label containing classical HSP70 families in sequence, existing experimental result has confirmed that DC-HSP can be lured by heat Lead, PMA/TNF α are stimulated and the induced expression such as bacterium, similar to the characteristic of classical heat shock protein.Biological function experiment prompting The albumen has certain immunologic adjuvant sample effect, can with inducing dendritic shape cell secretion of cytokines, by FITC marks and There is DC-HSP binding sites in the effect of known HSP family members, tentative confirmation DC surfaces, with non-with classical HSP70 families Often similar the characteristics of.Research shows, HSP70L1 for simulation tumour antigen have good adjuvant sample effect (Wan T etc., Blood, 2004；103:1747-1754)；HSP70L1 and specific tumor antigen CEA_576-669Fusion protokaryon albumen can also lure Obvious anti tumor immune response (Chinese invention patent application number is given birth in artificial delivery：200410053313.X；Wu Y etc., Cancer Res, 2005；65:4947-4954).

Still efficiently mediated gene it can shift and express and can be used as effective antitumor in the urgent need to developing in this area Gene transfer vector and products thereof.

The content of the invention

An object of the present invention is just being to provide a kind of recombined adhenovirus obtained by homologous recombination, uses the restructuring gland Effective carrying to Hsp70L1 and human cancer embryoantigen antigen-4 fusion protein gene, transfer and expression can be achieved in virus.The present invention's is another Purpose is to provide a kind of human dendritic cell transfected by the recombined adhenovirus, and it can be used for the oncotherapy of animal.The present invention A further object be to provide the recombined adhenovirus of the present invention or purposes of the human dendritic cell in treatment of cancer of its sensitization.

There is provided a kind of recombined adhenovirus obtained by homologous recombination in the first aspect of the present invention, it carries heat The coded sequence of the fusion protein of shock protein and carcinomebryonic antigen, the genomic deletion E1 areas and E3 areas of the adenovirus, and E1 and/or E3 zone positions insert the expression cassette of the fusion protein coded sequence, and the expression cassette includes successively：Promoter sequence Row, the coded sequence of the fusion protein and tailing signal sequence, wherein the heat shock protein is selected from：Hsp70 or Hsp70L1。

In a preference, the heat shock protein is Hsp70L1.

In an embodiment of the invention, the carcinomebryonic antigen is selected from：The full length sequence of human cancer embryoantigen or containing people The fragment of 576-669 of carcinomebryonic antigen.

In a preference, the carcinomebryonic antigen is the fragment of 576-669 of human cancer embryoantigen.

In yet another embodiment of the present invention, the coded sequence of the fusion protein has successively from 5' to 3'：Cancer Embryonal antigen coded sequence, optional connection peptide-coding sequence and heat shock protein coded sequence.

In a preference, the fusion protein has SEQ ID NO:Amino acid sequence shown in 2, or by SEQ ID NO:It is nucleotide sequence coded shown in 1.

In another preference, the length of the connection peptide is 0-10 amino acid, and preferably 1-5 amino acid is more excellent 2-4 amino acid is selected, the coded sequence of preferably described connection peptide is SalI restriction enzyme sites.

In yet another embodiment of the present invention, the recombined adhenovirus is obtained by the homologous recombination method being selected from the group ：Intracellular homologous recombination, endotoxin test method, COS/TPC cotransfections homologous recombination or location specific restructuring, preferably Plasmid DNA homologous recombination in intracellular virus DNA homologous recombination or bacterium.

In yet another embodiment of the present invention, the adenovirus is Ad5 type adenovirus.

In a preference, the two ends of the adenovirus genomic dna are combined with terminal peptide TP.

In yet another embodiment of the present invention, the recombined adhenovirus is carried based on the gland virus expression being selected from the group What body was built：PAdEasy-1, pShuttle-CMV or pShuttle-IRES.

In a preference, contained promoter sequence is giant cell in the expression cassette of the fusion protein coded sequence Viral promotors.In another preference, the tailing signal sequence is SV40PolyA.

There is provided a kind of BMDC of the recombined adhenovirus sensitization with the present invention in the second aspect of the present invention.

There is provided a kind of pharmaceutical composition in the third aspect of the present invention, it includes：

(a) recombined adhenovirus of the present invention of effective dose, BMDC of the present invention or with the present invention The T cell of Dendritic Cells Induced, and

(b) acceptable carrier, excipient or adjuvant in pharmacy or immunology.

There is provided a kind of method of Prepare restructuring adenovirus in the fourth aspect of the present invention, the recombined adhenovirus is carried The coded sequence of the fusion protein of heat shock protein and carcinomebryonic antigen, this method comprises the following steps：

(a) replication-defective adenoviral in missing E1 areas and E3 areas in genome is provided；

(b) carrier for the fusion protein coded sequence expression box for carrying heat shock protein and carcinomebryonic antigen is provided；

(c) adenovirus of step (a) is made to carry out homologous recombination with the carrier of step (b), to obtain recombined adhenovirus.

In a preference, methods described also comprises the following steps：

(d) with the recombined adhenovirus transfection host cell of gained in step (c), and

(e) recombined adhenovirus is obtained from the host cell being transfected.

In another preference, the host cell expression e1a gene, preferably described host cell is：HEK293 people Embryonic kidney cell line.

In another preference, the restructuring is carried out by the method for endotoxin test method.

In another preference, the replication-defective adenoviral is selected from：PAdEasy-1, pShuttle-CMV or pShuttle-IRES。

In another preference, the used method of the transfection is：Calcium phosphate DNA is co-precipitated infection protocol or liposome Infection protocol, preferred liposome infection protocol.

In another preference, step (d) is by cell lysis, discontinuous CsCL gradient centrifugations, continuous CsCl ladders What the four step rule purifying of degree centrifugation and dialysis was obtained.

There is provided a kind of method for the BMDC for preparing sensitization, methods described bag in the fifth aspect of the present invention Include：The recombined adhenovirus loaded dendritic cells prepared with the recombined adhenovirus of the present invention or with the above method of the present invention.

In a preference, the BMDC behaviour cells of monocytic origin BMDC.

There is provided the recombined adhenovirus described in the present invention 1, dendron shape of the present invention in the sixth aspect of the present invention Cell is preparing the medicine group for preventing or treating CEA positive tumors with the T cell of the Dendritic Cells Induced of the present invention Purposes in compound.

In a preference, described pharmaceutical composition is vaccine.

In another preference, the CEA positive tumors are selected from：Straight colon cancer, non-small cell lung cancer, stomach cancer, pancreas Cancer, liver cancer, kidney, breast cancer or oophoroma.

A kind of abductive approach of CEA positive tumors specific T-cells is additionally provided in another aspect of the present invention, including Following steps：Using the dendritic cells in vitro stimulation human peripheral blood lymphocyte of sensitization of the present invention.

In another preference, the stimulation step includes：

(i) BMDC of sensitization is collected, and radioactivity is inactivated；

(ii) by after sensitization and inactivation BMDC press BMDC：T cell is 1:1-1000, preferably 1：20 Ratio is incubated jointly, and 10-200U/ml IL-2 is added in complete medium, is cultivated 7 ± 2 days；

Repeat step (i) and (ii) 3-10 time, collect the T cell bred, as CEA positive tumors specific T-cells.

The other side of the present invention, due to this disclosure, is apparent to those skilled in the art 's.

Brief description of the drawings

Fig. 1：Prepared AdCEA of the invention_576-669The PCR qualification results of hsp70L1 virus liquids.

Fig. 2：Prepared AdCEA of the invention_576-669Hsp70L1 transfected with human MoDC RT-PCR and real-time PCR qualification results.

Fig. 3：Prepared recombined adhenovirus transfected with human cells of monocytic origin BMDC (MoDC) of the invention, it is slight to promote MoDC ripe activation.A：Through AdCEA_576-669Hsp70L1 transfection after DC expression costimulatory molecules --- CD40, CD54, CD80, CD86, CD83 or HLA-DR level；B：By AdCEA_576-669After hsp70L1 transfections, DC stimulates the water of T cell propagation of the same race Flat slight up-regulation.

Fig. 4：Prepared recombined adhenovirus transfected with human cells of monocytic origin BMDC (MoDC) of the invention, induces CEA Peptide-specific CTL is produced.A：AdCEA_576-669The people MoDC of hsp70L1 genetic modifications substantially can induce HLA-A2.1 to limit Property CAP-1 antigentic specificities CD8⁺The generation of T cell；B：AdCEA_576-669The people MoDC groups induction of hsp70L1 genetic modifications CD8⁺T cell is in CEA_576-669After antigenic stimulus, the quantity and spot size for IFN-γ spot occur substantially increase；C： AdCEA_576-669The CD8 of the people MoDC groups induction of hsp70L1 genetic modifications⁺T cell kills CEA⁺LS174T and SW480 tumours The ability of cell substantially increases.

Fig. 5：Recombinate AdCEA_576-669Hsp70L1 vivo immunizations experimental animal induces the generation of CEA Peptide-specific CTLs. A：AdCEA_576-669Hsp70L1 immune groups and AdCEA_576-669The CD8 of hsp70L1-DC immune groups induction⁺CAP-1-MHC tetra- gathers Body⁺T cell ratio substantially increases；B：AdCEA_576-669Hsp70L1 immune groups and AdCEA_576-669Hsp70L1-DC immune groups are induced CD8⁺T cell is in CEA_576-669After antigenic stimulus, the quantity and spot size for IFN-γ spot occur substantially increase；C： AdCEA_576-669Hsp70L1 immune groups and AdCEA_576-669The CD8 of hsp70L1-DC immune groups induction⁺T cell kills CEA⁺'s The ability of LS174T and SW480 tumour cells substantially increases.

Fig. 6：Recombinate AdCEA5_76-669Hsp70L1 treats CEA positive tumor diseases.A：AdCEA_576-669Hsp70L1 is immunized Mouse derived cell treatment group and AdCEA_576-669The animal tumor growth of mouse derived cell treatment group is immunized in hsp70L1-DC Substantially suppressed；B：AdCEA_576-669Mouse derived cell treatment group and AdCEA is immunized in hsp70L1_576-669Hsp70L1-DC exempts from The animal survival phase of epidemic disease mouse derived cell treatment group is obviously prolonged.

Fig. 7：With the XhoI of pAdeno-x system construction recombined adhenovirus restricted enzyme cutting analysis (Fig. 7 A) and XbaI/ Double restricted digestion (Fig. 7 B) results of KpnI.

Embodiment

The present inventor's in-depth study by long-term, carrying HSP70L1 is constructed and special by methods of homologous recombination Property tumour antigen CEA_576-669Adenovirus vector (the AdCEA of fusion_576-669Hsp70L1), and using this recombined adhenovirus carry Body transfection human dendritic cell (AdCEA_576-669hsp70L1-DC).Research has shown that：Apply AdCEA in inside and outside_576-669hsp70L1 Or AdCEA_576-669BMDC (the AdCEA of hsp70L1 modifications_576-669Hsp70L1-DC it) can effectively induce and be directed to CEA_576-669The killer T cell generation of antigentic specificity, kills CEA⁺Tumour cell；And adopt feedback AdCEA_{576- 669}Hsp70L1 or AdCEA_576-669The lymphocyte that animal is immunized in hsp70L1-DC can extend CEA⁺The life of tumour tumor animal Deposit the phase and suppress growth of tumour cell.

Therefore, AdCEA_576-669Hsp70L1 or AdCEA_576-669Hsp70L1-DC can be applied to as therapeutic vaccine CEA⁺The treatment of tumour.On this basis, the present inventor completes the present invention.

In the present invention, term " expressing fusion protein box " refers to the expression cassette containing elements below：Heat shock protein and cancer Component needed for the coded sequence of the fusion protein of embryonal antigen, and expression includes promoter and polyadenylation signal sequence. In addition, the expression cassette can also optionally contain other sequences, including but be not limited to：Enhancer, secretion signal peptide sequence Deng.

It can be any common promoter to go for the promoter in expression cassette of the present invention, and it can be group Constitutive promoter or inducible promoter.It is preferred that the promoter is the strong promoter of composing type, such as cytomegalovirus starts Other promoters suitable for eukaryotic expression such as son, bovine papilloma virus promoter.

As used herein, some parts for " being operably coupled to " refer to such a situation, i.e. linear DNA molecule being capable of shadow Ring the activity of same linear DNA molecule other parts.If for example, signal peptide DNA is as precursor expression and participates in dividing for polypeptide Secrete, then signal peptide (secretion targeting sequencing) DNA is exactly to be operably coupled to polypeptid DNA；If turn of promoter control sequence Record, then it is to be operably coupled to coded sequence.Typically, " be operably coupled to " mean adjoining, and for secretion before Leading sequence then means adjacent in reading frame.

Heat shock protein and carcinomebryonic antigen fusion protein

As used herein, term " heat shock protein " includes heat shock protein and heat shock protein sample albumen, representational Example includes (but being not limited to)：Hsp70, Hsp70L1 (Hsp70-like protein 1) and other heat shock proteins, it is excellent Elect Hsp70, Hsp70L1 as.The sequence of these heat shock proteins can be obtained from Genbank databases.

As used herein, term " fusion protein of carcinomebryonic antigen and heat shock protein ", " CEA-Hsp fusion proteins " etc. can Used interchangeably, all refers to by the amino acid of the amino acid sequence and heat shock protein (such as Hsp70 or Hsp70L1) of carcinomebryonic antigen element The albumen of sequence fusion, wherein therebetween can be with and without connection peptide sequence.In addition, the fusion protein can With the methionine or signal peptide with and without starting.

As used herein, " carcinomebryonic antigen element " refers to a part of amino in the fusion protein in term fusion protein Acid sequence, the sequence has substantially the same amino with natural or variation total length carcinomebryonic antigen or its immunogenic fragments Acid sequence, and with the bioactivity substantially the same with natural carcinomebryonic antigen.It is preferred that carcinomebryonic antigen element be people's cancer embryo Antigen or its immunogenic fragments are more preferably the human cancer embryoantigen or its immunogenic fragments of total length (as containing 576-669 The fragment of CEA amino acid sequences).

The sequence of carcinomebryonic antigen and heat shock protein can be derived from people, may originate from inhuman animal.However, it is preferred that It is the native sequences of people.It should be understood that the carcinomebryonic antigen of the present invention and the amino acid sequence or nucleotide sequence of heat shock protein are also wrapped Include its homologous sequence (such as homology is higher than 90%, 95%, 98% or 99% sequence) or by the sequence through conservative replacement, Delete or add the sequence with same or similar function obtained.

As used herein, term " connection peptide " or " amino acid linking arm " are used interchangeably, and are referred to positioned at carcinomebryonic antigen amino Small peptide between acid sequence and heat shock protein amino acid sequence, playing connection function.Connection peptide may be present or be not present, and it is grown Degree is usually 0-20 amino acid, and preferably 2-10 amino acid is most preferably 2-4 amino acid.Technical staff can be according to This area conventional method is (such as referring to PNAS 1998；95:5929-5934；Protein Eng, 2000；13(5):309-312 etc. Document) design connection peptide.Generally, connection peptide does not influence or had a strong impact on carcinomebryonic antigen to be formed with heat shock protein amino acid sequence It is correct to fold and space conformation.

It is preferred that connection peptide example include (but being not limited to)：In order to be conducive to protein folding into separate structure Domain, as linking arm is suitable with sequences such as SGGGGSGGGG；In order to be conducive to protease that CEA-Hsp70L1 is cut two Independent protein molecular, can make linking arm with the restriction enzyme site (IEGR) of active Ⅹ factor.Similar, chymotrypsin 1, pawpaw egg The restriction enzyme site of white enzyme, fibrinolysin, plasmase, trypsase etc. can also be configured as amino acid linking arm；For Be conducive to purifying, can be using 6His as linking arm, to purify CEA-Hsp70L1 fusion proteins with metal affinity chromatography；It is above-mentioned The combination of three kinds of schemes may be designed as new amino acid linking arm, and such as NVVVHQAHHHHHHEFTYK linking arms are exactly fusion Protease cutting site (NIa protease) and metal affinity chromatography site 6His.

In addition, another it is preferable that carcinomebryonic antigen element and heat shock protein element are directly connected to, and without appointing What connection peptide.

The DNA sequence dna of coding fusion protein of the present invention, can be all artificial synthesized.Also the side that can be expanded or be synthesized with PCR Method obtains the DNA sequences encoding of carcinomebryonic antigen and/or heat shock protein, is then stitched together, and forms the coding present invention and melts The DNA sequence dna of hop protein.

The structure of adenovirus vector

Current adenovirus vector is mostly from both serotypes of adenovirus Ad2 and Ad5.Should suitable for gene therapy Adenovirus vector belongs to replication-defective virus.Because the E1 protein functions encoded are that other all adenoviral genes are effective Necessary to expression, therefore most common strategy is exactly to substitute virus E1 region sequences with exogenous DNA, is carried by the way that incasing cells is trans The replication defective adenoviral carrier produced for E1 region sequences.It is preferred to use Δ E1 Δ E3 replication defect type adenopathies in the present invention Malicious expression vector, such as pAdEasy-1 (are purchased from Strategen companies).

Host packaging cell available for the present invention is can to express any host cell of adenovirus E 1 area gene, because The cell can provide the E1 areas gene expression product needed for replication-defective adenoviral is replicated.A kind of preferred conventional packaging is thin Born of the same parents are 293 cells, and the cell is transformed through Ad5 adenovirus DNA fragments by human embryonic kidney cells, is loaded with and is integrated into cellular genome The viral gene fragment such as adenovirus E 1 area, and continuous expression E1 areas albumen, Neng Wei E1 areas displaced type adenovirus vector provides anti- Formula is compensated.

Viral infection is not influenceed using E3 areas missing, the exogenous DNA capacity of carrier can be increased by cutting E3 areas.Cut E1 and E3 areas, foreign gene insertion capacity can be of about 8kb.And cloning bigger exogenous dna fragment then needs to cut other virus sequences, The function of missing is provided by the way that complementary cell system or helper virus are trans again.

The inventors discovered that being directly connected to method (for example with Adeno-X using the extracellular virus recommended in this area Expression system) it is difficult to efficiently obtain the recombined adhenovirus containing fusion protein of the invention, linear pAd skeletons are with containing the restructuring gland Joint efficiency is low in vitro for the linear fragment of the expressed intact box of virus, and the recombined adhenovirus of homologous recombination is obtained after conversion bacterium Probability it is extremely low.

However, the present inventor by long-term and experiment repeatedly surprisingly, it is found that, it is anti-using the method for homologous recombination And the recombined adhenovirus of the present invention can be efficiently obtained, and the recombined adhenovirus of gained has high infectious and low pathogeny to target cell Property, pole is significantly better than the result obtained using extracellular virus conjugation.

Therefore, recombined adhenovirus of the invention preferably uses the method for homologous recombination to obtain.Can be same using what is be selected from the group Source recombination method, for example：(1) intracellular homologous recombination, such as intracellular virus DNA homology are recombinated；(2) endotoxin test method, Such as plasmid DNA homologous recombination in bacterium；(3) COS/TPC cotransfections homologous recombination；Or the restructuring of (4) location specific.In the present invention It is preferred to use endotoxin test method method, for example with pAdEasy-1, pShuttle-CMV or pShuttle-IRES, and will contains There are Escherichia coli (such as E.coli BJ5183) of the linear fragment conversion containing adenoviral backbone of expressing fusion protein box, make They are in endotoxin test method.

The BMDC of recombined adhenovirus sensitization and the CEA T cells with antigenic specificity by the cell induction

BMDC (Dendritic Cells, DC) is a kind of the resisting with specific function existed in normal human Former presenting cells, are called " the natural adjuvant " of body, can directly absorb, process, presenting antigen, stimulating internal primary tape T cell activation, is immune response " initiator ".In addition, DC can also promote B cell by direct or indirect mode Propagation and activation, regulate and control humoral immune response；Stimulate memory T cell to activate and induce secondary immune response；With NK interaction shadows Ring nonspecific, innate immunity.DC is in the key link of tumour immunity, can absorb with working process tumour antigen simultaneously Transfer body active specificity antineoplastic immunity reaction killing tumor cell.

A kind of BMDC by recombined adhenovirus sensitization of the present invention is provided in the present invention, it can be effectively used for induction CEA T cells with antigenic specificity.The present invention can be caused using the common method as known in the art for primed dendritic shape cell BMDC sensitization, preferably with the present invention recombined adhenovirus virus transfection is carried out to BMDC.

Can be according to method commonly used in the art, the CEA antigens further induced with the BMDC of sensitization of the present invention Specific T-cells.In an embodiment of the invention, stimulated using the dendritic cells in vitro of sensitization of the present invention Human peripheral lymphocyte.Methods described preferably includes following steps：(i) the BMDC cell of sensitization is collected, and to it Inactivated (such as radioactivity inactivation)；(ii) BMDC of sensitization and inactivation is pressed into BMDC：T cell is 1:1- 1000th, preferably 1：20 ratio is incubated jointly, and 10-200U/ml IL-2, culture 7 ± 2 are added preferably in complete medium My god；(iii) repeat step (i) and (ii) 3-10 time, collect the T cell bred, as CEA positive tumors specific T-cells.

Composition

A kind of pharmaceutical composition is additionally provided in the present invention, it includes：(a) recombined adhenovirus of the invention of effective dose, Acceptable carrier on BMDC or the T cell with Dendritic Cells Induced of the present invention, and (b) pharmacy or immunology, Excipient or adjuvant.The composition can be used for preventing or treating CEA positive tumors.As used herein, " the CEA positives are swollen for term Knurl " or " CEA⁺Tumour " include refer on tumor cell membrane altimeter reach CEA antigens tumour, representational example include (but It is not limited to)：The malignant tumours such as straight colon cancer, non-small cell lung cancer, stomach cancer, cancer of pancreas.

As used herein, " active material " refers to the recombined adhenovirus of the present invention, the dendron shape of the recombined adhenovirus sensitization Cell, CEA T cells with antigenic specificity or combinations thereof.

Recombined adhenovirus, the BMDC of the recombined adhenovirus sensitization or the CEA antigen specific Ts of the present invention is being made After cell, can by the active material of required purity with it is optional physiologically, acceptable carrier in pharmacy or immunology, Excipient or stabilizer (Remington's Pharmaceutical Sciences, 16 editions, Osol, A., Ed., [1980]), Mixed with lyophilized form or aqueous solution form, so that the composition of the present invention is made.Acceptable carrier, excipient or steady It under dosage and concentration used should be nontoxic to determine agent for recipient, and can contain buffer solution such as phosphate, lemon Hydrochlorate or other organic acid buffer liquids；Antioxidant such as vitamin C；Low molecule amount (being less than about 10 residues) polypeptide；Protein Such as seralbumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyvinylpyrrolidone；Amino acid such as glycine, paddy Glutamine, asparagine, arginine or lysine；Monose, disaccharides and other carbohydrate such as glucose, mannose or paste Essence；Chelating agent such as EDTA；Sugar alcohol such as mannitol or D-sorbite；The gegenion of forming salt such as sodium；And/or non-ionic table Face activating agent such as Tween, Pluronics or polyethylene glycol (PEG).As most common example, water and physiological saline can be made For the carrier in invention formulation.

In yet another embodiment of the present invention, active material of the invention accounts for the 0.001- of the composition total weight 99.9wt%, preferably 1-95wt%, more preferably 5-90wt%, further preferably 10-80wt%.

Composition (or preparation) containing active material of the present invention can be applied to the individual for needing to treat with usual manner, this A little modes include (but being not limited to)：Intramuscular injection, hypodermic injection, intravenous injection etc..The amount for bestowing active material of the present invention can By professional (such as doctor) according to the concrete condition of object, such as illness, the course of disease, age, sex, other associated treatments are true It is fixed.Generally, the amount for giving active material of the present invention is：Recombined adhenovirus：10⁷-10¹¹PFU/ kg body weights, preferably 10⁸-5× 10¹⁰PFU/ kg body weights, more preferably preferably 10⁹-10¹⁰PFU/ kg body weights；Present invention restructuring gland expresses the DC cells of sensitization： 10³-10⁷Sensitization DC cells/kg body weight, preferably 10⁵-5×10⁶Sensitization DC cells/kg body weight, more preferably 10⁵-10⁶Cause Quick DC cells/kg body weight；CEA antigenspecific T lymphocytes of the present invention：10⁵-10⁹CEA antigenspecific T lymphocytes/ Kg body weight, preferably 10⁶-10⁸CEA antigenspecific T lymphocytes/kg body weight, more preferably 10⁶-10⁷CEA antigen-specifics Property T cell/kg body weight.

Treatment and/or prevention CEA can be also included in the composition of the present invention⁺Other active materials of tumour or with these its Its agents in combination is applied (as applied simultaneously or successively).

In a preference, treatment and/or the prevention CEA⁺Other active materials of tumour are selected from：Chemotherapy of tumors Medicine, such as oxaliplatin；Or the BMDC of tumour antigen impact, tumour antigen can be that known Antigenic Peptide can also be swollen Oncocyte lysate.

Main advantages of the present invention

1. the recombined adhenovirus of the present invention is expressed in intracellular by infection cell, " CEA-Hsp fusion proteins " is overcome former The following weak point of nuclear expression：1) expression technology has certain difficulty, it is difficult to obtains large-tonnage product and is controlled for clinic in future Treat purpose；2) hyperenergia of the fusion protein inducing dendritic shape cell maturation, is difficult to be used in control volume using prokaryotic expression method Amount, it is easy to the too strong side effect of immune response occur；3) hsp70L1 native form is intracellular protein, is perhaps played in intracellular Main function；4) fusion protein produced in prokaryotic expression form lacks correctly to fold waits eukaryotic protein with glycosylation modified The characteristics of (its native form)；5) fusion protein extracellular by with acceptor combination endocytosis form entering intracellular, according to immune Principle is learned, extracellular protein mainly passes through the CD4 of MHC-II classpath present antigens, that is, inducing antigen-specific⁺T cell； And intracellular protein mainly passes through the CD8 of MHC-I classpath present antigens, that is, inducing antigen-specific⁺T cell, the present invention Main purpose is exactly to realize the antigen submission of the MHC-I classpaths.

2. the recombined adhenovirus of the present invention has high infectious and low pathogenic to target cell, it can mediate safe efficiently Gene expression shift and express, have the advantages that it is carcinogenic, small to Mutation probability, for Antioncogene treatment provide preferable base Because of transfer vector；

3. the recombined adhenovirus of the present invention and its BMDC of sensitization can effectively induce the killing of CEA antigentic specificities Property T cell generation, and the latter has the strain of killing CEA positive tumor cells and suppresses CEA positive tumor growths, extends tumor animal The effect of life cycle.So as to prevent and treat the new strategy provided for CEA positive tumors.

Embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip Part is (for example, the equal reference of basic molecular biology manipulations《Molecular Cloning:A Laboratory guide》The second edition, Science Press, 1992；Substantially Cytology operates equal reference《Cell culture experiments guide》The first edition, Science Press, 2001) described in condition, or according to Condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number are calculated by weight.

Unless otherwise defined, all specialties used in text known to scientific words and one skilled in the art with anticipating Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the present invention.Described in text Preferable implementation only present a demonstration and be used with material.

Embodiment 1.AdCEA_576-669Hsp70L1 design and preparation

Restriction enzyme and Taq enzyme are purchased from NEB companies in this experiment；PQE 30 and Host Strains M15 is public purchased from Qiagen Department；Pre- transfection AdEasy-1 E.coli BJ5183, pShuttle-CMV is purchased from Strategen companies；LS174T is purchased from ATCC。

The induction of people mononuclear origin BMDC：Healthy volunteer anticoagulation 200ml is taken, it is close using Ficoll 1.077 Gradient centrifugation is spent, human peripheral blood single nucleus cell is obtained；Immunomagnetic beads person monocytic cell, in short, by 2 × 10⁸People PMNC and 200 μ lCD14 immunomagnetic beadses are incubated 15 minutes in 4 DEG C；The human peripheral of immunomagnetic beads will be combined Mononuclearcell is placed in magnetic field, washes away negative cells, and positive cell, as human peripheral CD14 are gone out from post⁺Monokaryon is thin Born of the same parents；By the people CD14 of fresh separated⁺Monocyte (Mo) is according to 10⁶Individual cell/ml cultures are containing 10%FBS, human GM-CSF In (500U/ml), people IL-4 (10ng/ml) culture mediums of RPMI 1640.After 6 days, suspension cell is collected, i.e., is come for people's monokaryon Source BMDC (MoDC).

Amplification obtains Hsp70L1 total length in the people's mononuclear origin BMDC (MoDC) stimulated from LPS (lipopolysaccharides) CDNA (according to bibliography 16).With RT-PCR method (sense primer 5 '- CTCGTCGACGCGGCCATCGGAGTTCACCTG-3’(SEQ ID NO:3) ,-GGGGTACCAG of anti-sense primer 5 ' ATGCTATCTCAATAGAGATTGCT-3’(SEQ ID NO:4)；Response parameter：95 DEG C 15 seconds, 53 DEG C 30 seconds, 72 DEG C 40 seconds, 72 DEG C extend 10 minutes after 29 circulations) cloned people Hsp70L1cDNA, SalI and kpnI restriction enzyme sites are introduced in upstream and downstream primer. Separate total serum IgE from LS174T cells, with RT-PCR method (sense primer 5 '- ATTGAGCTCGTGAGTGCAAACCGCAGTGACC-3’(SEQ ID NO:5), anti-sense primer is：5’- TATGTCGACGACTATGGAATTATTGCGGCC-3’(SEQ ID NO:6)；Response parameter：95 DEG C, 15 seconds, 56.5 DEG C, 30 Second, 72 DEG C, 45 seconds, 72 DEG C extend 10 minutes after 29 circulations) human cloning CEA_576-669In cDNA, upstream and downstream primer introduce sacI and SalI restriction enzyme sites.

By CEA_576-669CDNA segments are inserted into plasmid pQE30, form pQE30CEA_576-669；Hsp70L1cDNA is inserted Enter to pQE30CEA_576-669, form restructuring pQE30CEA_576-669hsp70L1.Thus obtained restructuring pQE30CEA_{576- 669}In hsp70L1, CEA_576-669CDNA be located at hsp70L1cDNA 5 ' end, centre connected by restriction enzyme site SalI, Its gene order such as SEQ ID NO:Shown in 1.CDNA genes are verified by nucleotide sequence analysis.To recombinate pQE30CEA_576-669Hsp70L1 is template, expands CEA_576-669Hsp70L1 fragment (response parameters：95 DEG C, 15 seconds, 57 DEG C, 30 Second, 72 DEG C, 90 seconds, 72 DEG C extend 10 minutes after 29 circulations), the primer sequence the following is upstream：5’- CCGGTACCATGGTGAGTGCAAACCGCAGTGACC-3’(SEQ ID NO:7)；Downstream：5’- CCCTCGAGTTAAGATGCTATCTCAATAGAGATTGC-3’(SEQ ID NO:8)。

The CEA of amplification_576-669Shuttle plasmid pShuttle- is inserted into after hsp70L1 piece cracked ends kpnI and xhoI digestions CMV, is built into restructuring pShuttle CEA_576-669hsp70L1；Through sequencing identification it is correct after, by PmeI linearization for enzyme restriction, turn Change pre- E.coli BJ5183 (the conventional CaCl for having transfected replication defect type pAdEasy-1₂, heat shock procedures)；Through card, that resists Property screening obtain recombinant adenovirus plasmid pAdCEA_576-669Hsp70L1, after being confirmed through restricted enzyme cutting analysis, by the sun of acquisition Property clone, further amplification, a large amount of extracting pAd CEA_576-669Hsp70L1, after PacI linearization for enzyme restriction, for transfecting HEK293 cells, carry out the packaging of adenovirus.

(1) Prepare restructuring adenovirus crude extract

DMEM nutrient solutions, the hyclone FBS of this experiment use are purchased from PAA companies；Transfection reagent is purchased from Polyplus- Transfection companies.HEK293 cells are purchased from Shanghai cell institute of the Chinese Academy of Sciences.

By HEK293 cells (cell of human embryonic kidney cell line 293 of expression adenovirus E 1 A, E1B albumen) 10%FBS's DMEM nutrient solutions, 37 DEG C, 5%CO₂Cultivated in environment to 50-70% fusions.It will be linearized using liposome method pAdCEA_576-669Hsp70L1 is transfected to HEK293 cells, and operation is carried out with reference to specification.What addition was isometric after 2h contains 10% The fresh DMEM nutrient solutions containing 10%FBS are changed after FBS DMEM nutrient solutions, 24h, continue to cultivate.Culture was to 10-14 days When, there is cytopathic effect, including cell adhesion is reduced, come off, being rounded, the change of beading sample is presented.There is lesion in collection Cell, multigelation 3 times is prepared into recombined adhenovirus crude extract, saved backup in -70 DEG C.

(2) single plaque purification virus

This experiment uses the agarose of Sigma companies.

By HEK293 cells (3 × 10⁵Individual cells/well) 6 orifice plates are inoculated in, to during 70% fusion, add recombined adhenovirus Crude extract, 37 DEG C of infection 2h, discards virus liquid, adds 1.25% agarose semisolid culturemedium, incubated at room temperature 30 minutes is waited to coagulate Gu after, place 37 DEG C, 5%CO₂Cultivated in environment to there is plaque.The single plaque of picking (chooses 3 clones), re-infects HEK293 cells, through repeated amplification, prepare virus liquid, enter performing PCR identification (response parameter：95 DEG C, 15 seconds, 62 DEG C, 30 seconds, 72 DEG C, 90 seconds, 35 circulation after 72 DEG C extend 10 minutes).As a result pAd CEA are shown_576-669Hsp70L1 contains 1812bp characteristic bars Band (Fig. 1).

The primer sequence is as follows：Upstream：5’-GGAAATGCGGCCGCATGGTGAGTGCAAAC-3’(SEQ ID NO: 9)；Downstream：5’-AATCGGCTCGAGTTAAGATGCTATCTCAAT-3’(SEQ ID NO:10).

(3)AdCEA_576-669Hsp70L1 amplification and purifying

pAdCEA_576-669Hsp70L1 a large amount of amplifications：HEK293 cells are laid on 60mm trainings with 50-70% rate of converging Support in ware, added with 30-50% volume ratio and contain vial supernatant.After the appearance of CPE phenomenons, there is 1/3 to 1/2 cell detachment During floating, cell is collected by centrifugation.Cell is resuspended in 2.0ml PBS.Freeze cell in liquid nitrogen, dissolved in 37 DEG C of water-baths, Acutely vibration.This step is carried out 3 times altogether.The viral supernatants of acquisition are further infected to the HEK293 cells of 100mm culture dishes, pressed Preceding method collects virus, and and then infected the HEK293 cells of multiple 100mm culture dishes, to obtain the disease of sufficient amount Poison.

The purifying of recombined adhenovirus：Carrying out discontinuous CsCl gradient centrifugations respectively, (density is respectively 1.4g/ml and 1.2g/ ) and continuous CsCl gradient centrifugations (1.3g/ml) ml.Dialysis removes CsCl, and recombined adhenovirus is stored in into buffer solution (10mM Tris, 4% sucrose, 2mM MgCl₂,pH 8.0)。

Also, many experiments, which demonstrate the recombined adhenovirus for obtaining the present invention using the method in above-mentioned (1)~(3), to be had Very high reappearance.

(4) AdCEA is determined_576-669Hsp70L1 titres

Routinely Plaque assays method determines pAdCEA_576-669Hsp70L1 titres are 1.2 х 10¹²PFU/ml。

Embodiment 2. recombinates AdCEA_576-669Hsp70L1 Infection in Vitro people's mononuclear origin BMDC and expression

RPMI1640 culture mediums and Ficoll1.077 are purchased from PAA companies in this experiment；Human GM-CSF and people IL-4 are purchased from Peprotech companies；CD14 immunomagnetic beadses, MS posts and separator frame are purchased from Miltenyl Biotec companies；AdLacZ is purchased from Strategen companies.

The abductive approach of people's mononuclear origin BMDC (MoDC) is as described in example 1 above.

The MoDC collected is resuspended with the culture mediums of RPMI 1640 containing 10%FBS, adjustment concentration is 10⁷Individual cell/ml. According to MOI=1000：1, with restructuring AdCEA_576-669Hsp70L1 (prepared in such as embodiment 1) infection MoDC.Collected after 24h Cell, extracts total serum IgE, carries out RT-PCR (CEA_576-669：Response parameter：94 DEG C, 30 seconds, 54 DEG C, 30 seconds, 72 DEG C, 30 seconds, 35 72 DEG C extend 10 minutes after circulation；hsp70L1：Response parameter：94 DEG C, 30 seconds, 60 DEG C, 30 seconds, 72 DEG C, 30 seconds, after 35 circulations 72 DEG C extend 10 minutes；CEA_576-669hsp70L1：Response parameter：94 DEG C, 30 seconds, 60 DEG C, 30 seconds, 72 DEG C, 30 seconds, 35 circulations Afterwards 72 DEG C extend 10 minutes) and real-time PCR (each same RT-PCR of response parameter) identification.Another setting is uninfected by, AdLacZ (is purchased from Strategen companies, voluntarily expand and purify method as described in Example 1；MOI=1000：1) infected group is compareed.As a result figure is seen 2。

The result shows：AdCEA_576-669After the people MoDC of hsp70L1 infection, CEA is expressed_576-669, Hsp70L1, and CEAhsp70L1mRNA level is significantly higher than untransfected group and Ad LacZ transfection groups.RT-PCR primer sequence is as follows：

CEA_576-669(279bp)：Upstream：5’-ATTGAGCTCGTGAGTGCAAACCGCAGTGACC-3’(SEQ ID NO: 11)；Downstream：5’-TATGTCGACGACTATGGAATTATTGCGGCC-3’(SEQ ID NO:12)

Hsp70L1(122bp)：Upstream：5’-CAGCATGTGAGGCCGTCTA-3’(SEQ ID NO:13)；Downstream：5’- TGCTGCCAATCCAACAAT-3’(SEQ ID NO:14)

CEA_576-669Hsp70L1(111bp)：Upstream：5’-AAATCACGCCAAATAATAACGG-3’(SEQ ID NO: 15)；Downstream：5’-TGCAGCCCAGGTGAACTCC-3’(SEQ ID NO:16).

Embodiment 3. recombinates AdCEA_576-669Hsp70L1 genetic modifications promote people MoDC ripe activation

Each antibody is purchased from B.D.Pharmingen companies in the present embodiment；CFSE is purchased from Calbiochem companies；CD3 is immunized Magnetic bead is purchased from Miltenyl Biotech companies；Protokaryon PROTEIN C EA_576-669Hsp70L1 is purified simultaneously by this room using conventional method (picking conversion has pQE30-CEA for preservation_576-669The M15 bacterium of hsp70L1 plasmids, after induced expression, it is expressed with inclusion body Form is present in intracellular；Inclusion body is by washing and dissolves, and upper metal ion-chelant post and DEAE column chromatographies purify CEA_{576- 669}PBS is dissolved in after hsp70L1 fusion proteins, dialysis, -80 DEG C are stored in).

By the human peripheral CD14 of fresh separated⁺Monocyte (Mo) is with 10⁶The culture of individual cell/ml containing 10%FBS, In human GM-CSF (500U/ml), people IL-4 (10ng/ml) RPMI1640 culture mediums.After 6 days, it is people to collect suspension cell Mononuclear origin BMDC (MoDC).

The MoDC collected is resuspended with the culture mediums of RPMI 1640 containing 10%FBS, adjustment concentration is 10⁷Individual cell/ml； According to MOI=1000：1, respectively with AdLacZ or Ad CEA_576-669Hsp70L1 washes away free virus after infecting MoDC, 2h, after Continuous culture is in above-mentioned culture medium；CEA_576-669Hsp70L1 albumen is incubated according to 100 μ g/ml and MoDC.MoDC is collected after 48h, Carry out tests below：

(1) Phenotypic examination

With the antibody labeling MoDC cells of anti-costimulatory molecules CD40, CD54, CD83, CD86 and MHCII molecule, and carry out Fluidic cell (FACSCalibur, BD company) is analyzed.As a result show, compared with being uninfected by group and AdLacZ groups, recombined adhenovirus Ad CEA_576-669The level of people MoDC expression costimulatory molecules CD54, CD86 and MHC-II molecules of hsp70L1 genetic modifications is light Degree up-regulation；Expression CD83 and CD40 level does not change (Fig. 3 A).

(2) the lymphopoietic abilities of T of the same race are stimulated

Separate CD3 of the same race⁺T lymphocytes (apply immunomagnetic beads method, such as embodiment 2).By MoDC and CD3 of the same race⁺T drenches Bar cell is according to MoDC：CD3⁺T lymphocyte=1:10 ratio is mixed 5 days, detects the proliferative conditions of T cell.T cell Marked in advance with CFSE, with the T lymphocytes % (T cell of reflection propagation) of flow cytomery CFSE low expressions.

As a result show, compared with being uninfected by group and AdLacZ groups, recombined adhenovirus Ad CEA_576-669Hsp70L1 genes are repaiied The people MoDC of decorations stimulates CD3 of the same race⁺The lymphopoietic levels of T are slightly raised (Fig. 3 B).

Above-mentioned result of the test shows, recombinates AdCEA_576-669Hsp70L1 genetic modifications promote people MoDC ripe activation.

Embodiment 4. recombinates AdCEA_576-669The people MoDC induction CEA Peptide-specific CTLs of hsp70L1 genetic modifications are produced

In the present embodiment, the CAP-1-MHC tetramers are purchased from ProImmune companies；CD8 immunomagnetic beadses are purchased from Miltenyl Biotech companies；ELISPOT culture plates are purchased from Millipore companies；Anti-human IFN-γ monoclonal antibody is public purchased from B.D.Pharmingen Department；7-AAD is purchased from Sigma companies；Recombinant C EA_576-669Antigen purifies (prokaryotic expression carrier pQE30-CEA by this room_576-669 In M15 bacterium after induced expression, the CEA of restructuring_576-669Albumen is mainly present in bacterium intracellular, inclusion body warp in the form of inclusion body Cross after washing and dissolving, cross metal ion-chelant column chromatography preliminary purification recombinant C EA_576-669Albumen, then by conventional method Renaturation, dialysis, filtration sterilization packing are stored in -80 DEG C).The anti-human HLA-A2.1 antibody of FITC- couplings is purchased from B.D.Pharmingen companies.

The anti-human HLA-A2 antibody labelings that human peripheral blood single nucleus cell is coupled through FITC- in advance, flow cytometry analysis HLA-A2 positive samples are chosen, immunomagnetic beads CD14 is carried out⁺Monocyte, and MoDC induction.Feel according to embodiment 3 Contaminate people HLA-A2⁺MoDC is collected after MoDC, 48h, 4 parts are distributed into；A and itself CD3⁺T lymphocytes are according to 1:20 ratio Mixed culture, remaining 3 parts are frozen in -80 DEG C with the frozen stock solution containing 5%DMSO, 95%FBS.After culture 7 days, a jelly of recovering The restructuring AdCEA deposited_576-669The people MoDC of hsp70L1 genetic modifications, co-culture system is added to by it according to same ratio In, after cultivating 3 days, human IL-2 (100U/ml) is added, continues to cultivate.The stimulation of the 3rd circulation is carried out with method.After 21 days, enter The following experiment of row：

(1) the restricted CEA of HLA-A2.1_605-613Antigenic Peptide (CAP-1) T lymphocyte specific ratio (CD8⁺CAP-1- The MHC tetramers⁺%)

The cell that culture is terminated is collected, Bicolor-code, flow cytometer point are carried out with the anti-CD8 and CAP-1-MHC tetramers Analyse CD8⁺The CAP-1-MHC tetramers⁺%.As a result as shown in Figure 4 A, the result shows：With being uninfected by, AdLacZ infected groups, CEA_576-669Hsp70L1 sensitization DC groups (Ctrl, AdLacZ and CEAhsp70L1 are shown respectively as in figure) compare, AdCEA_{576- 669}The people MoDC (AdCEAhsp70L1 is shown as in figure) of hsp70L1 genetic modifications can substantially induce CAP-1 antigentic specificities CD8⁺The generation of T cell.

(2) the restricted CEA of HLA-A2.1_576-669Antigentic specificity CD8⁺The level of T cell secretion of gamma-IFN

Separate the CD8 in co-culture system⁺T lymphocytes, itself MoDC with recovery is according to 5：1 ratio culture, system Middle addition people CEA_576-669Albumen (10 μ g/ml), culture to 96 well culture plates for being coated with anti-human IFN-γ, after cultivating 3 days eventually Only, the detection of IFN-γ secretion spot is carried out according to ELISPOT specifications.

As a result as shown in Figure 4 B, the result shows：With being uninfected by, AdLacZ infected groups and CEA_576-669Hsp70L1 sensitization DC groups (Ctrl, AdLacZ and CEAhsp70L1 are shown respectively as in figure) compare, AdCEA_576-669The people of hsp70L1 genetic modifications The CD8 of MoDC groups (AdCEAhsp70L1 is shown as in figure) induction⁺T cell is in CEA_576-669After antigenic stimulus, there is IFN-γ spot Quantity and spot size substantially increase, illustrate in the cultivating system contain more CEA specific Cs D8⁺T cell.

(3) to CEA⁺The lethal effect of tumour cell

Separate the CD8 in co-culture system⁺T lymphocytes, the CEA marked with CFSE⁺Tumor cell line (LS174T and SW480 is purchased from ATCC) according to 10：1 ratio is co-cultured, after 4h, adds 7-AAD, thin with flow cytomery tumour immediately Death (the CFSE of born of the same parents⁺7-AAD⁺%).

As a result show, with being uninfected by, AdLacZ infected groups and CEA_576-669Hsp70L1 sensitization DC groups compare, AdCEA_{576- 669}The CD8 of the people MoDC groups induction of hsp70L1 genetic modifications⁺T cell kills CEA⁺LS174T and SW480 tumour cells energy Power substantially increases (accompanying drawing 4C).

To sum up, AdCEA is recombinated_576-669The people MoDC of hsp70L1 genetic modifications can be stronger induction CEA_576-669Antigen is special Different in nature CTL is produced.Especially, Ad CEA_576-669The human dendritic cell of hsp70L1 modifications is than protokaryon PROTEIN C EA_{576- 669}Hsp70L1 impact human dendritic cell can be stronger induction CEA specific CTLs generation, show Ad CEA_576-669Hsp70L1 modification human dendritic cell induction to Cap-1 (a kind of HLA-A2 restricted peptides in CEA albumen) Specific CD8⁺T cell, killing CEA⁺The ability of tumour cell is stronger.

Embodiment 5. recombinates AdCEA_576-669Hsp70L1 vivo immunizations experimental animal induces CEA_576-669Peptide-specific CTL Generation

4 week old HLA-A2.1/Kb transgenic mices (being purchased from Jackson Laboratory) bone marrow cell is taken, according to 1 х 10⁷Individual cell/ml cultures are containing 10%FBS, and mouse GM-CSF (10ng/ml), mouse IL-4 (1ng/ml) RPMI 1640 is cultivated Inhaled in base, after three days and abandon suspension cell, attached cell continues to cultivate in above-mentioned culture medium, after 6 days, collects suspension cell, As derived from bone marrow BMDC.

Cell is resuspended with the culture mediums of RPMI 1640 containing 10%FBS, adjustment concentration is 1 х 10⁷Individual cell/ml, according to MOI=1000:1 adds recombined adhenovirus AdLacZ or Ad CEA_576-669After hsp70L1,2h, free virus is washed away, it is a part of For being immunized, remaining freezes, for follow-up immunization.

By 30 4 week old HLA-A2.1/Kb transgenic mice (female-male proportions 1:1, purchased from JacksonLaboratory) press It is divided into five groups according to randomly assigne；1. untreated fish group；2. AdLacZ immune groups, are injected intraperitoneally, 10⁹PFU/ is only；③AdCEA_{576- 669}Hsp70L1 immune groups, intraperitoneal injection, 10⁹PFU/ is only；4. the bone marrow derived BMDC of AdLacZ genetic modifications is exempted from Epidemic disease group (AdLacZ-DC), intraperitoneal injection, 10⁶AdLacZ-DC cells/only；⑦Ad CEA_576-669Hsp70L1 genetic modifications it is small Mouse derived from bone marrow dendritic cell vaccination group (Ad CEA_576-669Hsp70L1-DC), it is injected intraperitoneally, 10⁶Ad CEA_{576- 669}Hsp70L1-DC cells/only；Immunization protocol is to be immunized once every 7 days, is immunized three times altogether.After 21 days, tested as follows：

(1) the restricted CAP-1 Antigenic Peptides T lymphocyte specific ratio (CD8 of HLA-A2.1⁺CAP-1-MHC tetra- gathers Body⁺%) determine

Each group mouse boosting cell is separated, Bicolor-code is carried out to splenocyte with anti-CD8 antibody and the CAP-1-MHC tetramers, Flow cytometry analysis CD8⁺The CAP-1-MHC tetramers⁺T percentage of lymphocyte.

As a result show, compared with non-immune group and AdLacZ or AdLacZ-DC immune groups, AdCEAhsp70L1 immune groups and AdCEA_576-669The CD8 of hsp70L1-DC immune groups induction⁺CAP-1-TCR⁺T cell ratio substantially increases (Fig. 5 A).

(2) the restricted CEA of HLA-A2.1_576-669Antigentic specificity CD8⁺The level of T cell secretion of gamma-IFN

Separate the CD8 in each group mouse boosting cell⁺T lymphocytes, DC is originated according to 5 with autogenous bone marrow：1 ratio culture is extremely It is coated with 96 well culture plates of anti-mouse IFN-γ, system and adds mouse IL-2 (100U/ml), and CEA_576-669Antigen (10 μ g/ ml)；Culture is terminated after 3 days, and the detection of IFN-γ secretion spot is then carried out according to ELISPOT specifications.

As a result show, compared with non-immune group and AdLacZ or AdLacZ-DC immune groups, AdCEA_576-669Hsp70L1 exempts from Epidemic disease group and AdCEA_576-669The CD8 of hsp70L1-DC immune groups induction⁺T cell is in CEA_576-669After antigenic stimulus, there is IFN-γ The quantity and spot size of spot substantially increase, and illustrate AdCEA_576-669Hsp70L1 and AdCEA_576-669Hsp70L1-DC immunity energies More CEA are produced in enough inductors_576-669Antigentic specificity CD8⁺T cell (Fig. 5 B).

(3) to CEA⁺The lethal effect of tumour cell

Separate the CD8 in each group mouse boosting cell⁺T lymphocytes, the CEA marked with CFSE⁺Tumor cell line (LS174T And SW480) according to 10：1 ratio is co-cultured, after 4h, adds 7-AAD, dead with flow cytomery tumour cell immediately Die (CFSE⁺7-AAD⁺%).

As a result show, compared with non-immune group and AdLacZ or AdLacZ-DC immune groups, AdCEA_576-669Hsp70L1 exempts from Epidemic disease group and AdCEA_576-669The CD8 of hsp70L1-DC immune groups induction⁺T cell kills CEA⁺LS174T and SW480 tumour cells Ability substantially increase, show AdCEA_576-669Hsp70L1 or AdCEA_576-669Hsp70L1-DC is immune to be produced in inductor More CEA antigentic specificities CD8⁺CTL (Fig. 5 C).

To sum up, AdCEA_576-669Hsp70L1 immune groups and AdCEA_576-669Hsp70L1-DC immune groups can be induced substantially The generation of CEA Peptide-specific CTLs.

Embodiment 6. recombinates AdCEA_576-669Hsp70L1 treats CEA positive tumor diseases

By 50 4 week old HLA-A2.1/Kb transgenic mice (female-male proportions 1:1, purchased from Jackson Laboratory) It is divided into five groups according to randomly assigne；1. untreated fish group；2. AdLacZ immune groups, abdominal channels, 10⁹PFU/ is only；③AdCEA_{576- 669}Hsp70L1 immune groups, abdominal channels, 10⁹PFU/ is only；4. the bone marrow derived BMDC of AdLacZ genetic modifications is exempted from Epidemic disease group (AdLacZ-DC)；Abdominal channels, 10⁶AdLacZ-DC/ is only；⑤AdCEA_576-669The mouse bone marrow cells of hsp70L1 genetic modifications Dendritic cell group abdominal channels, 10⁶AdCEA_576-669Hsp70L-DC/ is only；Immunization protocol is every 7 days immune one It is secondary, it is immunized three times altogether.After 21 days, separating mouse splenocyte, armpit, abdomen bone ditch Mesenteric lymph node cell are removed after red blood cell, It is resuspended with PBS, according to 5 х 10⁷/ only, it is injected intravenously to CEA⁺Tumour tumor-bearing model.

Set up CEA⁺Tumour tumor-bearing model：By 1 х 10⁶LS174T cells, in subcutaneous vaccination in the nude mice of 4 week old； 50 nude mices are inoculated with altogether, are divided into 4 groups according to randomly assigne after three days, are received cell therapy：①Wei treatment groups；2. mouse is not immunized Derived cell treatment group；3. mouse derived cell treatment group is immunized in AdLacZ；Or the treatment of mouse derived cell is immunized in AdLacZ-DC Group；④AdCEA_576-669Mouse derived cell treatment group is immunized in hsp70L1；Or AdCEA_576-669Mouse source is immunized in hsp70-DC Cell therapy group.

Therapeutic scheme：Start to receive cell therapy within 3 days after inoculated tumour；It is repeated once every after 7 days.

It is to appear to survey after tumour, the measurement and the observation of life cycle of tumor size are every other day carried out, 3 are observed altogether Month.

As a result show, with non-treatment group, not to be immunized mouse derived cell treatment group and AdLacZ or AdLacZ-DC immune Mouse derived cell treatment group compares, AdCEA_576-669Mouse derived cell treatment group and AdCEA is immunized in hsp70L1_{576- 669}The animal tumor growth that mouse derived cell treatment group is immunized in hsp70L1-DC is suppressed (Fig. 6 A) by obvious；Life cycle is obvious Extend (Fig. 6 B).

The application pAdeno-x system construction recombined adhenovirus of embodiment 7. AdCEA_576-669hsp70L1

Such as embodiment 1, to recombinate pQE30CEA_576-669Hsp70L1 is template, expands CEA_576-669Hsp70L1 fragments are (anti- Answer parameter：95 DEG C, 15 seconds, 57 DEG C, 30 seconds, 72 DEG C, 90 seconds, 72 DEG C extend 10 minutes after 29 circulations), the primer sequence is as follows For upstream：Sense primer：5’-TTCC T CTAGA ATG GTG AGT GCA AAC CGC AGT GAC-3’(SEQ ID NO: 17)；Anti-sense primer：5’-AAGT GGTAC CAGA TGC TAT CTC AAT AGA GAT-3’(SEQ ID NO:18) expand CEA_576-669Shuttle plasmid pShuttle2 is inserted into after hsp70L1 piece cracked ends kpnI and xbaI digestions, CMV promoter is introduced With PolyA tails, restructuring pShuttle CEA are built into_576-669hsp70L1；Through sequencing identification it is correct after, by PI-SceI and I- CeuI double digestions, obtain the linear fragment of the expressed intact box containing CMV promoter, purpose fragment and PolyA tails, Ran Houyu PAdeno-X linear backbone is connected with ligase in vitro；Convert Escherichia coli (conventional CaCl₂, heat shock procedures)；Through ammonia Benzyl resistance screening can obtain 1-2 resistant strain, extract plasmid, enter the restricted enzyme cutting analysis (Fig. 7 A) and XbaI/ for the XhoI that passes through The double restricted digestions (Fig. 7 B) of KpnI find that due restriction fragment distribution and mesh does not occur in the plasmid in resistant strain Fragment.

The above results show：The linear fragment of linear pAd skeletons and the expressed intact box containing the recombined adhenovirus is in body Outer joint efficiency is very low, and the probability for the recombined adhenovirus for obtaining homologous recombination after conversion bacterium is very low.

All documents referred in the present invention are all incorporated as reference in this application, independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Bibliography

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2.Todryk SM,Gough MJ,Pockley AG.Facets of heat shock protein 70show immunotherapeutic potential.Immunology.2003；110:1-9.

3.Manjili MH,Wang XY,MacDonald IJ,Arnouk H,Yang GY,Pritchard MT, Subjeck JR.Cancer immunotherapy and heat-shock proteins:promises and challenges.Expert Opin Biol Ther.2004；4(3):363-73

4.Blachere NE,Udono H,Janetzki S,Li Z,Heike M,Srivastava PK.Heat shock protein vaccines against cancer.J Immunother Emphasis Tumor Immunol.1993；14:352-356.

5.Li Z,Srivastava PK.A critical contemplation on the role of heat shock proteins in transfer of antigenic peptides during antigen presentation.Behring Inst Mitt.1994；94:37-47.

6.Moroi Y,Mayhew M,Trcka J,et al.Induction of cellular immunity by immunization with novel hybrid peptides complexed to heat shock protein 70.Proc Natl Acad Sci U S A 2000；97:3485–90

7.Arnold-Schild D,Hanau D,Spehner D,Schmid C,Rammensee HG,de la Salle H,Schild H.Cutting edge:receptor-mediated endocytosis of heat shock proteins by professional antigen-presenting cells.J Immunol.1999；162(7):3757-60.

8.Castellino F,Boucher PE,Eichelberg K,Mayhew M,Rothman JE,Houghton AN,Germain RN。Receptor-mediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class I antigen presentation via two distinct processing pathways..J Exp Med.2000；191(11): 1957-64.

9.Svensson PA,Asea A,Englund MC,Bausero MA, M,Wiklund O,Ohlsson BG,Carlsson LM,Carlsson B.Major role of HSP70as a paracrine inducer of cytokine production in human oxidized LDL treated macrophages.Atherosclerosis.2006Mar；185(1):32-8.Epub 2005Jul 1.

10.Caudill MM,Li Z.HSPPC-96:a personalised cancer vaccine.Expert Opin Biol Ther.2001 May；1(3):539-47.

11.Cohen L,de Moor C,Parker PA,Amato RJ.Quality of life in patients with metastatic renal cell carcinoma participating in a phase I trial of an autologous tumor-derived vaccine.Urol Oncol.2002；7(3):119-24.

12.Cohen L,Parker PA,Sterner J,De Moor C.Quality of life in patients with malignant melanoma participating in a phase I trial of an autologous tumour-derived vaccine.Melanoma Res.2002 12(5):505-11.

13.Belli F,Testori A,Rivoltini L,Maio M,Andreola G,Sertoli MR,Gallino G,Piris A,Cattelan A,Lazzari I,Carrabba M,Scita G,Santantonio C,Pilla L, Tragni G,Lombardo C,Arienti F,MarchianòA,Queirolo P,Bertolini F,Cova A,Lamaj E,Ascani L,Camerini R,Corsi M,Cascinelli N,Lewis JJ,Srivastava P,Parmiani G.Vaccination of metastatic melanoma patients with autologous tumor-derived heat shock protein gp96-peptide complexes:clinical and immunologic findings.J Clin Oncol.2002；20(20):4169-80.

14.Maciag PC,Paterson Y.Technology evaluation:HspE7(Stressgen).Curr Opin Mol Ther.2005；7(3):256-63.

15.Hunt S.Technology evaluation:HspE7,StressGen Biotechnologies Corp.Curr Opin Mol Ther.2001；3(4):413-7.

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17.Wu Y,Wan T,Zhou X,Wang B,Yang F,Li N,Chen G,Dai S,Liu S,Zhang M, Cao X.Hsp70-like protein 1 fusion protein enhances induction of carcinoembryonic antigen-specific CD8⁺CTL response by dendritic cell vaccine.Cancer Res.2005；65(11):4947-4954.

Sequence table

<110>Second Military Medical University, PLA

<120>Carry recombined adhenovirus of antigen-4 fusion protein gene and its preparation method and application

<130> 095767

<160> 18

<170> PatentIn version 3.3

<210> 1

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<220>

<221> CDS

<222> (1)..(1812)

<400> 1

gtg agt gca aac cgc agt gac cca gtc acc ctg gat gtc ctc tat ggg 48

Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly

1 5 10 15

ccg gac acc ccc atc att tcc ccc cca gac tcg tct tac ctt tcg gga 96

Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly

20 25 30

gcg aac ctc aac ctc tcc tgc cac tcg gcc tct aac cca tcc ccg cag 144

Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln

35 40 45

tat tct tgg cgt atc aat ggg ata ccg cag caa cac aca caa gtt ctc 192

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

50 55 60

ttt atc gcc aaa atc acg cca aat aat aac ggg acc tat gcc tgt ttt 240

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

65 70 75 80

gtc tct aac ttg gct act ggc cgc aat aat tcc ata gtc gtc gac gcg 288

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Val Asp Ala

85 90 95

gcc atc gga gtt cac ctg ggc tgc acc tca gca tgt gag gcc gtc tat 336

Ala Ile Gly Val His Leu Gly Cys Thr Ser Ala Cys Glu Ala Val Tyr

100 105 110

aag gat ggc cgg gct ggt gtg gtt gca aat gat gcc ggt gac cga gtt 384

Lys Asp Gly Arg Ala Gly Val Val Ala Asn Asp Ala Gly Asp Arg Val

115 120 125

act cca gct gtt gtt gct tac tca gaa aat gaa gag att gtt gga ttg 432

Thr Pro Ala Val Val Ala Tyr Ser Glu Asn Glu Glu Ile Val Gly Leu

130 135 140

gca gca aaa caa agt aga ata aga aat att tca aat aca gta atg aaa 480

Ala Ala Lys Gln Ser Arg Ile Arg Asn Ile Ser Asn Thr Val Met Lys

145 150 155 160

gta aag cag atc ctg ggc aga agc tcc agt gat cca caa gct cag aaa 528

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

165 170 175

tac atc gcg gaa agt aaa tgt tta gtc att gaa aaa aat ggg aaa tta 576

Tyr Ile Ala Glu Ser Lys Cys Leu Val Ile Glu Lys Asn Gly Lys Leu

180 185 190

cga tat gaa ata gat act gga gaa gaa aca aaa ttt gtt aac cca gaa 624

Arg Tyr Glu Ile Asp Thr Gly Glu Glu Thr Lys Phe Val Asn Pro Glu

195 200 205

gat gtt gcc aga ctg ata ttt agt aaa atg aaa gaa acg gca cat tct 672

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

210 215 220

gta ttg ggc tca gat gca aat gat gta gtt att act gtc ccg ttt gat 720

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

225 230 235 240

ttt gga gaa aag caa aaa aat gct ctt gga gaa gca gct aga gct gct 768

Phe Gly Glu Lys Gln Lys Asn Ala Leu Gly Glu Ala Ala Arg Ala Ala

245 250 255

gga ttt aat gtt ttg cga tta att cac gaa ccg tct gca gct ctt ctt 816

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

260 265 270

gct tat gga att gga caa gac tcc cct act gga aaa agc aat att ttg 864

Ala Tyr Gly Ile Gly Gln Asp Ser Pro Thr Gly Lys Ser Asn Ile Leu

275 280 285

gtg ttt aag ctt gga gga aca tcc tta tct ctc agc gtc atg gaa gtt 912

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

290 295 300

aac agt gga ata tat cgg gtt ctt tca aca aac act gat gat aac atc 960

Asn Ser Gly Ile Tyr Arg Val Leu Ser Thr Asn Thr Asp Asp Asn Ile

305 310 315 320

ggt ggt gca cat ttc aca gaa acc tta gca cag tat cta gct tct gag 1008

Gly Gly Ala His Phe Thr Glu Thr Leu Ala Gln Tyr Leu Ala Ser Glu

325 330 335

ttc caa aga tcc ttc aaa cat gat gtg aga gga aat gcg cga gcc atg 1056

Phe Gln Arg Ser Phe Lys His Asp Val Arg Gly Asn Ala Arg Ala Met

340 345 350

atg aaa tta acg aac agt gct gaa gta gcg aaa cat tct ttg tca acc 1104

Met Lys Leu Thr Asn Ser Ala Glu Val Ala Lys His Ser Leu Ser Thr

355 360 365

ttg gga agt gcc aac tgt ttt ctt gac tca tta tat gaa ggt caa gat 1152

Leu Gly Ser Ala Asn Cys Phe Leu Asp Ser Leu Tyr Glu Gly Gln Asp

370 375 380

ttt gat tgc aat gtg tcc aga gca aga ttt gaa ctt ctt tgt tct cca 1200

Phe Asp Cys Asn Val Ser Arg Ala Arg Phe Glu Leu Leu Cys Ser Pro

385 390 395 400

ctt ttt aat aag tgt ata gaa gca atc aga gga ctc tta gat caa aat 1248

Leu Phe Asn Lys Cys Ile Glu Ala Ile Arg Gly Leu Leu Asp Gln Asn

405 410 415

gga ttt aca gca gat gat atc aac aag gtt gtc ctt tgt gga ggg tct 1296

Gly Phe Thr Ala Asp Asp Ile Asn Lys Val Val Leu Cys Gly Gly Ser

420 425 430

tct cga atc cca aag cta cag caa ctg att aaa gat ctt ttc cca gct 1344

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

435 440 445

gtt gag ctt ctc aat tct atc cct cct gat gaa gtg atc cct att ggt 1392

Val Glu Leu Leu Asn Ser Ile Pro Pro Asp Glu Val Ile Pro Ile Gly

450 455 460

gca gct ata gaa gca gga att ctt att ggg aaa gaa aac ctg ttg gtg 1440

Ala Ala Ile Glu Ala Gly Ile Leu Ile Gly Lys Glu Asn Leu Leu Val

465 470 475 480

gaa gac tct ctt atg ata gag tgt tca gcc aga gat att tta gtt aag 1488

Glu Asp Ser Leu Met Ile Glu Cys Ser Ala Arg Asp Ile Leu Val Lys

485 490 495

ggt gtg gac gaa tca gga gcc agt aga ttc aca gtg ctg ttt cca tca 1536

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

500 505 510

ggg act cct ttg cca gct cga aga caa cac aca ttg caa gcc cct gga 1584

Gly Thr Pro Leu Pro Ala Arg Arg Gln His Thr Leu Gln Ala Pro Gly

515 520 525

agc ata tct tca gtg tgc ctt gaa ctc tat gag tct gat ggg aag aac 1632

Ser Ile Ser Ser Val Cys Leu Glu Leu Tyr Glu Ser Asp Gly Lys Asn

530 535 540

tct gcc aaa gag gaa acc aag ttt gca cag gtt gta ctc cag gat tta 1680

Ser Ala Lys Glu Glu Thr Lys Phe Ala Gln Val Val Leu Gln Asp Leu

545 550 555 560

gat aaa aaa gaa aat gga tta cgt gat ata tta gct gtt ctt act atg 1728

Asp Lys Lys Glu Asn Gly Leu Arg Asp Ile Leu Ala Val Leu Thr Met

565 570 575

aaa agg gat gga tct tta cat gtg aca tgc aca gat caa gaa act gga 1776

Lys Arg Asp Gly Ser Leu His Val Thr Cys Thr Asp Gln Glu Thr Gly

580 585 590

aaa tgt gaa gca atc tct att gag ata gca tct taa 1812

Lys Cys Glu Ala Ile Ser Ile Glu Ile Ala Ser

595 600

<210> 2

<211> 603

<212> PRT

<213>Artificial sequence

<400> 2

Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly

1 5 10 15

Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly

20 25 30

Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln

35 40 45

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

50 55 60

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

65 70 75 80

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Val Asp Ala

85 90 95

Ala Ile Gly Val His Leu Gly Cys Thr Ser Ala Cys Glu Ala Val Tyr

100 105 110

Lys Asp Gly Arg Ala Gly Val Val Ala Asn Asp Ala Gly Asp Arg Val

115 120 125

Thr Pro Ala Val Val Ala Tyr Ser Glu Asn Glu Glu Ile Val Gly Leu

130 135 140

Ala Ala Lys Gln Ser Arg Ile Arg Asn Ile Ser Asn Thr Val Met Lys

145 150 155 160

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

165 170 175

Tyr Ile Ala Glu Ser Lys Cys Leu Val Ile Glu Lys Asn Gly Lys Leu

180 185 190

Arg Tyr Glu Ile Asp Thr Gly Glu Glu Thr Lys Phe Val Asn Pro Glu

195 200 205

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

210 215 220

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

225 230 235 240

Phe Gly Glu Lys Gln Lys Asn Ala Leu Gly Glu Ala Ala Arg Ala Ala

245 250 255

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

260 265 270

Ala Tyr Gly Ile Gly Gln Asp Ser Pro Thr Gly Lys Ser Asn Ile Leu

275 280 285

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

290 295 300

Asn Ser Gly Ile Tyr Arg Val Leu Ser Thr Asn Thr Asp Asp Asn Ile

305 310 315 320

Gly Gly Ala His Phe Thr Glu Thr Leu Ala Gln Tyr Leu Ala Ser Glu

325 330 335

Phe Gln Arg Ser Phe Lys His Asp Val Arg Gly Asn Ala Arg Ala Met

340 345 350

Met Lys Leu Thr Asn Ser Ala Glu Val Ala Lys His Ser Leu Ser Thr

355 360 365

Leu Gly Ser Ala Asn Cys Phe Leu Asp Ser Leu Tyr Glu Gly Gln Asp

370 375 380

Phe Asp Cys Asn Val Ser Arg Ala Arg Phe Glu Leu Leu Cys Ser Pro

385 390 395 400

Leu Phe Asn Lys Cys Ile Glu Ala Ile Arg Gly Leu Leu Asp Gln Asn

405 410 415

Gly Phe Thr Ala Asp Asp Ile Asn Lys Val Val Leu Cys Gly Gly Ser

420 425 430

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

435 440 445

Val Glu Leu Leu Asn Ser Ile Pro Pro Asp Glu Val Ile Pro Ile Gly

450 455 460

Ala Ala Ile Glu Ala Gly Ile Leu Ile Gly Lys Glu Asn Leu Leu Val

465 470 475 480

Glu Asp Ser Leu Met Ile Glu Cys Ser Ala Arg Asp Ile Leu Val Lys

485 490 495

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

500 505 510

Gly Thr Pro Leu Pro Ala Arg Arg Gln His Thr Leu Gln Ala Pro Gly

515 520 525

Ser Ile Ser Ser Val Cys Leu Glu Leu Tyr Glu Ser Asp Gly Lys Asn

530 535 540

Ser Ala Lys Glu Glu Thr Lys Phe Ala Gln Val Val Leu Gln Asp Leu

545 550 555 560

Asp Lys Lys Glu Asn Gly Leu Arg Asp Ile Leu Ala Val Leu Thr Met

565 570 575

Lys Arg Asp Gly Ser Leu His Val Thr Cys Thr Asp Gln Glu Thr Gly

580 585 590

Lys Cys Glu Ala Ile Ser Ile Glu Ile Ala Ser

595 600

<210> 3

<211> 30

<212> DNA

<213>Artificial sequence

<400> 3

ctcgtcgacg cggccatcgg agttcacctg 30

<210> 4

<211> 33

<212> DNA

<213>Artificial sequence

<400> 4

ggggtaccag atgctatctc aatagagatt gct 33

<210> 5

<211> 31

<212> DNA

<213>Artificial sequence

<400> 5

attgagctcg tgagtgcaaa ccgcagtgac c 31

<210> 6

<211> 30

<212> DNA

<213>Artificial sequence

<400> 6

tatgtcgacg actatggaat tattgcggcc 30

<210> 7

<211> 33

<212> DNA

<213>Artificial sequence

<400> 7

ccggtaccat ggtgagtgca aaccgcagtg acc 33

<210> 8

<211> 35

<212> DNA

<213>Artificial sequence

<400> 8

ccctcgagtt aagatgctat ctcaatagag attgc 35

<210> 9

<211> 29

<212> DNA

<213>Artificial sequence

<400> 9

ggaaatgcgg ccgcatggtg agtgcaaac 29

<210> 10

<211> 30

<212> DNA

<213>Artificial sequence

<400> 10

aatcggctcg agttaagatg ctatctcaat 30

<210> 11

<211> 31

<212> DNA

<213>Artificial sequence

<400> 11

attgagctcg tgagtgcaaa ccgcagtgac c 31

<210> 12

<211> 30

<212> DNA

<213>Artificial sequence

<400> 12

tatgtcgacg actatggaat tattgcggcc 30

<210> 13

<211> 19

<212> DNA

<213>Artificial sequence

<400> 13

cagcatgtga ggccgtcta 19

<210> 14

<211> 18

<212> DNA

<213>Artificial sequence

<400> 14

tgctgccaat ccaacaat 18

<210> 15

<211> 22

<212> DNA

<213>Artificial sequence

<400> 15

aaatcacgcc aaataataac gg 22

<210> 16

<211> 19

<212> DNA

<213>Artificial sequence

<400> 16

tgcagcccag gtgaactcc 19

<210> 17

<211> 34

<212> DNA

<213>Artificial sequence

<400> 17

ttcctctaga atggtgagtg caaaccgcag tgac 34

<210> 18

<211> 31

<212> DNA

<213>Artificial sequence

<400> 18

aagtggtacc agatgctatc tcaatagaga t 31

Claims (9)

1. the method that the recombined adhenovirus that a kind of use homologous recombination is obtained prepares the BMDC of sensitization, methods described includes The step of primed dendritic shape cell external with the recombined adhenovirus, wherein, the recombined adhenovirus carry heat shock protein with The coded sequence of the fusion protein of carcinomebryonic antigen, its genomic deletion E1 areas and E3 areas, and in the insertion of E1 and/or E3 zone positions The expression cassette of the fusion protein coded sequence, the expression cassette includes successively：The coding of promoter sequence, the fusion protein Sequence and tailing signal sequence, wherein the heat shock protein is Hsp70L1, and wherein described adenovirus is pAdEasy-1.

2. the method as described in claim 1, it is characterised in that the carcinomebryonic antigen is selected from：The full length sequence of human cancer embryoantigen Or the fragment of 576-669 containing human cancer embryoantigen.

3. the method as described in claim 1, it is characterised in that the coded sequence of the fusion protein has successively from 5' to 3' Have：Carcinomebryonic antigen coded sequence, optional connection peptide-coding sequence and heat shock protein coded sequence.

4. the method as described in claim 1, it is characterised in that the homologous recombination is selected from：Intracellular homologous recombination；In bacterium Homologous recombination；COS/TPC cotransfection homologous recombinations；Or location specific restructuring.

5. BMDC prepared by a kind of method described in use claim 1.

6. a kind of pharmaceutical composition, it is characterised in that it includes：

(a) BMDC described in the claim 5 of effective dose or the T with the Dendritic Cells Induced described in claim 5 Cell, and

(b) acceptable carrier, excipient or adjuvant in pharmacy or immunology.

7. a kind of method prepared for the recombined adhenovirus in claim 1 methods described, the recombined adhenovirus carries heat The coded sequence of the fusion protein of shock protein and carcinomebryonic antigen, it is characterised in that this method comprises the following steps：

(a) replication-defective adenoviral in missing E1 areas and E3 areas in genome is provided；

(b) carrier for the fusion protein coded sequence expression box for carrying heat shock protein and carcinomebryonic antigen is provided；

(c) adenovirus of step (a) is made to carry out homologous recombination with the carrier of step (b), to obtain recombined adhenovirus.

8. a kind of method for the BMDC for preparing sensitization, methods described includes：Prepared with the method described in claim 7 Recombined adhenovirus in-vitro transfection BMDC.

9. BMDC described in claim 5 is being prepared with the T cell of the Dendritic Cells Induced described in claim 5 The purposes in pharmaceutical composition for preventing or treating CEA positive tumors.