CN101134102B - 5T4 antigen-expressed vector - Google Patents

Abstract

The present invention provides 5T4 tumour-associated antigen (TAA) for use in a method of immunotherapy of tumours. The invention also relates to a recombinant poxvirus vector from which at least one immune evasion gene has been deleted, which comprises a nucleic acid sequence encoding a 5T4 TAA and the use thereof in vaccinating against and in treating tumours.

Description

Express the carrier of 5T4 antigen

The application is dividing an application of following application: the applying date: on November 18th, 1999; Application number: 99815632.9 (PCT/GB99/03859); Denomination of invention: " polypeptide ".

The invention provides the 5T4 tumor associated antigen (TAA) for tumour immunotherapy.The invention still further relates to the recombinant poxvirus carrier and the purposes in anti-tumor vaccine inoculation and oncotherapy thereof that wherein lack at least one immune evasion gene, described recombinant poxvirus carrier comprises the nucleotide sequence of the 5T4TAA that encodes.

Invention field

The present invention relates to can be used for exciting the tumor associated antigen (TAA) of receiver's anti-tumor immune response.Specifically, the present invention relates to 5T4 antigen and the purposes in immunization therapy thereof.

Background of invention

Multiple carcinoembryonic antigen or tumor associated antigen (TAA) in human and animal's tumor have been identified and have characterized.In general, TAA is the antigen of expressing in the fetal development process, regulating downwards in adult's cell, and therefore the adult lacks TAA or it exists level very low usually.Observe tumor cell and recover to express TAA, so propose TAA is used for diagnosing tumor, targeting and immunization therapy.

Specifically, the tumor antigen of clone's T cell recognition causes great interest in exploitation antigenic specificity cancer vaccine recently.Yet many tumor associated antigens are the not mutated low histo-differentiation antigen of immunogenicity.The weak possible cause of its immunogenicity is self tolerance.Therefore, they almost can not become the antigenic peptides that suitable enhance immunity is replied.

However, find that the part tumor associated antigen is regular relevant with the tumor of most of individualities.This class antigen is noticeable especially vaccine candidate object.The albumen that comprises melanoma differentiation antigen (MDA), melanoma antigen and several MAGE families of the identification of T lymphocyte.But the result that clinical trial so far obtains confirms, is extremely difficult to these antigen induction therapeutic T cell.The human immune system to a reason of the tangible hypoergia of many tumor antigens may be: they are normal, nonmutationed autoantigens.Therefore, not mutated self cellular antigens are used for the major obstacle of tumour immunotherapy in the tolerance of breaking this antigen.For example the muroid zona pellucida antigen of muroid poxvirus recombinant expression can make mice forfeiture reproductive performance.Although these data show the recombinant poxvirus of available expression autoantigen and break self tolerance, still need to optimize its effectiveness, so that making a definite diagnosis tumor, effective treatment becomes possibility.

Fully characterized TAA5T4 (referring to WO89/07947).It is that wide expression is in the 72kDa of various cancers glycoprotein, and the expression and distribution of normal mature tissue very limited (seeing Table 1).As if it and colon cancer and stomach cancer metastasis are closely related.The complete nucleotide sequence of known person 5T4 (Myers etc., 1994J Biol Chem169:9319-24).

Table 1

The distribution of people 5T4

Tumor type 5T4 frequency (%)

Breast carcinoma 84

Ovarian cancer 71

Gastric cancer 74

Colon cancer 85

(Starzynska etc., Eur J Gastroenterol Hepatol 1998Jun; 10 (6): 479-84; Starzynska etc., BrJCancer1994May; 69 (5): 899-902; Starzynska etc., Br J Cancer1992Nov; 66 (5): 867-9)

Although because the mechanism that may relate to has proposed 5T4 as labelling (Carsberg etc., (1996) Int JCancer 1996 Sep 27 of tumour progression and metastatic potential; 68 (1): 84-92), but do not propose 5T4 as immunotherapy medicaments.Breaking itself also is not confirmed with the immunologic tolerance of restricted manner at the 5T4 of mature tissue's expression.Therefore, can not predict that can 5T4 become effective antigen of anticancer immunotherapy.

Summary of the invention

If the therapeutic outcome of succeeing, the immunotherapy for the treatment of of cancer depends on multiple factor.Comprise the ability that activated cell toxic T lymphocyte (CTL) replys, the ability that excites antibody response, and importantly break the ability of receiver's immunologic tolerance.Verified at present, react with the immunization therapy of as above judging that 5T4 immunity inoculation receiver succeeds.Specifically, confirmed to excite antibody response with the 5T4 immunity inoculation.

Therefore, the invention provides the viral vector of the nucleic acid of expressing coding 5T4 antigen.

5T4 antigen can effectively excite the immunotherapeutical antitumor to reply in receiver's expression.Described viral vector preferably helps the CTL of antigen expressed is replied, and preferably poxvirus vector such as vaccinia virus vector.Other virus and the non-virus carrier that is fit to transmit 5T4 antigen below described.

" carrier " used herein can be anyly can transmit or nucleic acid is remained on the factor in the host cell, comprises viral vector, plasmid, naked nucleic acid, with the compound nucleic acid of polypeptide or other molecule and be fixed on nucleic acid on the solid phase particles.Below describe this class carrier in detail.Certainly the present invention is at its broadest any concrete carrier that is not limited to transmit the 5T4 code nucleic acid.

" nucleic acid " as referred to herein can be natural or synthetic DNA or RNA or its any compositions.Only limit to the effect that they have coding 5T4 antigen according to nucleic acid of the present invention, the organelle of host organisms can be translated described nucleic acid like this.Therefore, for example can modify natural acid to increase its stability.But modifying DNA and/or RNA, especially RNA are to improve its composition to the resistance of nuclease.For example known modification to ribonucleotide comprise 2 '-O-methyl, 2 '-fluorine-based, 2 '-amino and '-the O-pi-allyl.The nucleic acid of modifying according to the present invention can comprise chemical modification, carries out chemical modification with the body internal stability, the enhancing that increase described nucleic acid or coordinates its transmission or reduce the clearance rate of body.The example that this class is modified comprises that the locational chemistry of each base of ribose and/or phosphoric acid and/or specific RNA sequence replaces.Referring to for example WO92/03568; U.S.5,118,672; Hobbs etc., (1973) Biochemistry12:5138; Guschlbauer etc., (1977) Nucleic Acids Res.4:1933; Schibaharu etc., (1987) Nucleic AcidsRes.15:4403; Pieken etc., (1991) Science253:314, wherein each document all specifically is attached to herein by reference.

Refer to that according to the present invention's " expression " 5T4 antigen the host living beings somatic cell produces 5T4 antigen by the nucleic acid of translation (optional transcribing) coding 5T4.Therefore, 5T4 original position in described cell produces.Because 5T4 is transmembrane protein, so the outer part of its born of the same parents is presented on its celliferous surface.Thereby in case of necessity, term " expression " comprises the signal that provides necessary to guarantee correctly to process 5T4, makes it be presented on cell surface and can interact with host immune system.

Term used herein " polypeptide " refers to that monomer wherein is aminoacid and the polymer that links together by peptide bond or disulfide bond." polypeptide " refers to that total length natural amino acid chain or its fragment are as desired polypeptides district, synthesizing amino acid chain or its compositions in the selected combination.Therefore " its fragment " refers to about 500 amino acid lengths of about 8-of the part in the full-length polypeptide, more preferably from about about 200 aminoacid of 8-even the aminoacid sequence of about 50 or 100 amino acid lengths of 10-more preferably from about.In addition, can comprise alpha-non-natural amino acid, for example Beta-alanine, phenylglycine and homoarginine.Also use the common amino acid of non-genomic coding in the present invention.

5T4 antigen is the polypeptide that is called 5T4, and for example in WO89/07947 it is characterized.One preferred aspect, 5T4 is the people 5T4 that characterizes as (the same) such as Myers, its sequence is seen GenBank accession number Z29083, this paper is called SEQ.ID.No.1.Yet, the present invention includes various 5T4 and 5T4 allelic variation body, comprise that the mice 5T4 (GenBank accession number AJ012160) shown in the dog 5T4 shown in this paper SEQ ID NO 3 and this paper SEQ ID NO 2 and fragment (preferred visibly different epi-position) and its keep the antigenic variant that comprises aminoacid insertion, disappearance or replacement of 5T4.Such fragment is described in more detail hereinafter.

Second aspect, the 5T4 antigen that the present invention relates to modify." antigen of modification " used herein is truncate, the prolongation that is different from natural 5T4 or the 5T4 polypeptide that suddenlys change with other method.Find that the fragments of peptides that 5T4 produces can be used as 5T4 specific antigen determinant.Such peptide can be in receiver's body in conjunction with the HLA molecule and induce the CTL at wild type 5T4 to reply, more effective than total length 5T4 usually.In addition, can make the 5T4 polypeptide mutant by aminoacid insertion, disappearance or replacement; Mutant peptide is preferably in receiver's body more effectively in conjunction with HLA, induces stronger CTL to reply.Peptide can be any length, but best 5-25 aminoacid, preferred 6-15 aminoacid, preferred about 9 amino acid lengths.

Modified peptides is the HLA CTL epi-position of 5T4 preferably.What more effective CTL that " peptide is in conjunction with prediction " obtains according to the program of utilizing K.Parker (NIH) design replied predicts the outcome, and can modify this class epi-position, and described program can network address below find: Http:// www- Bimas.dcrt.nih.gov/c gi-bin/molbio/ken parker comboform(also can consult 1994.J.Immunol.152:163 such as Parker, K.C).

One preferred aspect, " modification " 5T4 comprises that the peptide of being combined with transit peptides or adjuvant or otherwise connecting is in order to strengthen the ability that it excites immunne response.For example peptide and the transit peptides that does not rely on TAP can be merged effectively to be transitted to HLA and (summary is consulted Yewdell etc., 1998J Immunother21:127-31 with enhanced CT L epi-position with the HLA interaction of molecules; Fu etc., (1998) J Virol72:1469-81).

The third aspect the invention provides the method that excites immunne response the receiver, comprises with the nucleic acid immunization receiver of coding 5T4 antigen and expresses the step of 5T4 antigen the receiver.

Excite immunne response with the nucleic acid immunization inoculation of expressing 5T4 as mentioned above.By comprising " causing (priming) " agent of antigen, after effectively causing with initiator, give described immune system then and comprise second dose or " reinforcement " agent of appending antigen, thereby can excite immunity.

The carrier that is used for immunity inoculation can be any virus or non-virus carrier.No matter used 5T4 antigen is total length 5T4 or its peptide, all can be the 5T4 antigen of modifying, and 5T4 antigen can be homology (namely deriving from the species identical with the receiver) or allos in the source.

The immunne response that preferably excites is that the CTL of participating in activation 5T4 specificity cell toxicity T lymphocyte replys.

Preferably described replying is that effective inhibition, prevention or the anti-tumor immunotherapy that reverses receiver's tumor development are replied.

Fourth aspect the invention provides and uses the compositions that the 5T4 antigen preparation is used for immunization therapy receiver tumor.

Utilize the immunity inoculation of 5T4 antigen preferably can in receiver's body, break immunologic tolerance to 5T4.

According to the 5th aspect, the invention provides the vaccine combination that comprises 5T4 antigen.Vaccine combination can comprise homology 5T4 antigen, allos 5T4 antigen or sudden change 5T4 antigen.

The mode that the vaccine that contains 5T4 antigen can be similar to the application 5T4 code nucleic acid of identical purpose is used for tumour immunity or oncotherapy.Described vaccine combination preferably comprises one or more adjuvants.

The 6th aspect the present invention includes the expression vector of coding 5T4 antigen, and this carrier can be used for expressing 5T4 and produces the 5T4 antigen that is applicable to vaccine combination.Described carrier can be prokaryotic vector or eukaryotic vector, and preferably can express the carrier of 5T4 in mammalian cell.

5T4 antigen can be the 5T4 antigen in any source, and can be the 5T4 antigen of modifying, for example 5T4 antigen as herein described.

The 7th aspect the invention provides and uses the compositions that the 5T4 antigen preparation is used for immune receiver.Utilize the immunity inoculation of 5T4 to comprise the vaccine combination according to fifth aspect present invention that gives described receiver's immunity effective dose.

Eight aspect the invention provides and uses the compositions that the 5T4 antigen preparation is used for making receiver's sterillization (sterilisation).Give 5T4 antigen and can make receiver's sterillization effectively.Described receiver is preferably women receiver (female subject).

The invention still further relates to and use 5T4 targeting molecule, as anti-5T4 antibody such as anti-5T4scFvs.These antibody can be used for the natural or external source 5T4 of (i) original position targeting and/or (ii) original position with the immunostimulant molecule such as B7.1 is passed to natural or external source 5T4 (Carroll etc. (1998) J Natl CancerInst90 (24): 1881-7).This can strengthen the immunogenicity of 5T4 in described receiver's body.The invention still further relates to the carrier of sequential use coding 5T4 antigen and anti-5T4 antibody such as anti-5T4svFvs.Anti-5T4svFvs antibody can be used as the naked DNA (for example in the plasmid of short promoter region that comprises coding DNA and its generation of control) of encoding said antibody, expression vector (can be virus or non-virus carrier) or the albumen form that comprises described coded sequence gives.Therefore, what the invention provides that the carrier of coding 5T4 antigen and optional and molecules of immunization stimulus merge can be in conjunction with the factor of 5T4, and they independently are used for the treatment of tumor, for example sequential use.

In an embodiment again, the present invention includes the therapeutic alliance that comprises enzyme treatment/prodrugs therapy and utilize the immunization therapy of 5T4.For example enzyme treatment/prodrugs therapy can comprise in the tumor or systems communicate P450 (optional with retroviral vector or slow virus carrier transmission) and cyclophosphamide (CPA), carries out the systemic immunity therapeutic with 5T4 then and induces.

Therefore, the invention still further relates to the carrier of coding 5T4 antigen, the use in conjunction of prodrug/enzyme, they respectively, simultaneously respectively or unite and be used for the treatment of tumor.

In an embodiment again, 5T4 or 5T4 peptide and hepatitis B core antigen can be merged to strengthen t helper cell and antibody response (Schodel etc., 1996Intervirology39:104-10).

So, according to a ninth aspect of the present invention, providing the recombinant poxvirus that wherein lacks an immune evasion gene at least carrier, it comprises the nucleotide sequence of codes for tumor related antigen (TAA).

A little less than the TAA antigenicity, immune system is identified as " self " component with it, so its tolerance degree is very big.Although use poxvirus vector described antigen is offered, make at least part of this tolerance of obeying, the immunogenicity effect that observes with most poxvirus vectors is limited.It is believed that the natural immune evasion gene that is present in poxvirus of disappearance may be to having useful effect with the TAA immunity inoculation.The poxvirus vector of disappearance immune evasion gene may be able to be broken the immunologic tolerance that the autoantigen of coding is comprised TAA, therefore can make the host strengthen immunne response to weak immunogenicity autoantigen or other autoantigen.

The tenth aspect the invention provides the method that excites immunne response mammal, comprises giving the recombinant poxvirus carrier of described mammal according to ninth aspect present invention, excites described mammal to the immunne response of TAA thus.

Known antigens such as TAA depend on generation CTL and reply, and depend on by MHCI approach process antigen to provide protectiveness effect or therapeutical effect, CTL to reply in receiver's body.It is believed that long-term antigen presentation prolongs the retention time of high-level CTL.The poxvirus that a eleventh aspect of the present invention provides lytic activity to reduce is to strengthen the receiver to the ctl response of antigen.

The 12 aspect the invention provides application and excites immunne response at TAA according to the present invention the 9th, the tenth recombinant poxvirus carrier on the one hand mammal.

The 13 aspect the invention provides the 5T4 antigen that is used as tumor associated target in immunization therapy.

The 14 aspect, the invention provides and use the recombinant poxvirus carrier and break mammal to weak immunogenic immunologic tolerance and excite immunne response to it, at least one immune evasion gene delection or sudden change and comprise the weak immunogenic nucleotide sequence of coding in the described recombinant poxvirus carrier.

Provide other side of the present invention in appended claims and following description with in discussing.These aspects provide under independent sub-section titles.Yet, should be noted that the content under each sub-section titles not necessarily is limited to its concrete sub-section titles.

Detailed Description Of The Invention

Transmit or express the carrier of 5T4 antigen

Available virus or non-virus technology transmission are according to 5T4 polypeptide of the present invention.

Non-viral system includes but not limited to the DNA transfection method.Comprise in this transfection and to utilize non-virus carrier that the 5T4 gene is passed to the mammiferous method of target.

Conventional transfection method comprises transfection, cation face or amphiphile (CFA) (the Nature Biiotechnology199614 that the transfection of electroporation, biological nucleic acid emission (nucleic acidbiolistics), lipid mediation, acid mediated transfection, liposome, immunoliposome, lipofection (lipofectin), the cationics of tight tuberculosis mediate; 556), polyvalent cation such as spermine, cation lipid or polylysine, 1,2-two (oily acyloxy)-3-(trimethyl amino (ammonio)) propane (DOTAP)-cholesterol complex (Wolff and Trubetskoy 1998 Nature Biotechnology16:421) and its compositions.

The viral system includes but not limited to adenovirus vector, adeno associated virus (AAV) carrier, herpesvirus vector, retroviral vector, slow virus carrier or baculovirus vector, Venezuelan equine encephalitis virus (VEE), poxvirus is as canary pox virus (1995Vaccine13:539-549 such as Taylor), entomopox virus (the 144th page of the international poxvirus special meeting in LiY etc. 1998 the 13 boundary, summary), penguin poxvirus (penguine pox) (J GenVirol.199879:1637-46 such as Standerd) alphavirus and based on the dna vector of alphavirus.

The retrovirus example includes but not limited to: muroid leucovirus (MLV), HIV (human immunodeficiency virus) (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney muroid leucovirus (Mo-MLV), FBR muroid osteosarcoma virus (FBRMSV), Moloney muroid sarcoma virus (Mo-MSV), Abelson muroid leucovirus (A-MLV), avian myelocytomatosis virus-29 (MC29) and birds protoerythrocyte hypertrophy virus (AEV).

The retrovirus list of details is seen (" Retroviruses " 1997ColdSpring Harbour Laboratory Press Eds:JM Coffin, SM Hughes, HE Varmus 758-763 pages or leaves) such as Coffin.

Slow virus is divided into primates and non-human primate group.Primates slow virus example includes but not limited to: HIV (human immunodeficiency virus) (HIV), namely mankind itself's immunity deficit syndrome (AIDS) is pathogenic former, and simian immunodeficiency virus (SIV).Non-human primate slow virus group comprises prototype " slow virus " Wei Sina/chronic progressive pneumonia virus of sheep (VMV) and relevant caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV) and the bovine immunodeficiency virus of describing recently (BIV).

One of lentiviridae and other type retrovirus are tangible differently to be that slow virus can infect somatoblast and can infect Unseparated Cell (1992EMBO.J 11:3053-3058 such as Lewis again; Lewis and Emerman1994J.Virol.68:510-516).On the contrary, other retrovirus such as MLV can not infect Unseparated Cell, as the cell of configuration example such as muscle, brain, lung and liver organization.

Carrier of the present invention can be assembled into the carrier that intron is interrupted (split-intron).PCT patent application WO99/15683 and WO99/15684 have described the carrier that intron is interrupted.

If the feature of adenovirus is combined with the hereditary stability of retrovirus/slow virus, then described adenovirus can be used for the target cell of transduceing basically, it is become can stablize the instantaneous retrovirus generation cell that infects adjacent cells.The retrovirus that such engineering can be expressed 5T4 antigen produces cell implantable bioartificial body such as animal or human's class, is used for the treatment of angiogenesis and/or cancer.

Poxvirus vector

Preferably use poxvirus vector in the present invention.Engineered poxvirus is used for recombinant gene expression and is used as live recombined vaccines.Need to use in the nucleic acid importing poxvirus genome group of recombinant technique with encoding exogenous antigen.If described nucleic acid is incorporated into the nonessential viral DNA of described viral biocycle site, then the new recombinant poxvirus that produces may be communicable, that is to say, infects foreign cell, expresses the DNA sequence of integrating thus.Zhi Bei recombinant poxvirus can be used as live vaccine by this way, is used for preventing and/or treating disease and infectious disease.

5T4 expresses in recombinant poxvirus such as vaccinia virus and the nucleic acid of vaccine promoter with coding 5T4 need be connected.Make up plasmid vector (be also referred to as and insert carrier), be used for the virus sequence of the described nucleic acid flank by donor plasmid and the homologous recombination that is present between the homologous sequence of parental virus inserts vaccinia virus 1982PNAS79:7415-7419 such as () Mackett with nucleic acid.One type insertion carrier is composed as follows: the vaccinia virus promoter that (a) comprises transcriptional start site; (b) be positioned at the restriction endonuclease cloning site that the transcriptional start site downstream is used for several uniquenesses of insertion nucleic acid; (c) cloning site of the nonessential homology region of nucleic acid insertion viral genome as described in the nonessential vaccinia virus sequence of promoter flank (as thymidine kinase (TK) gene) and the guidance; (d) antibacterial origin of replication and the antibiotic resistance labelling that in escherichia coli, copies and select.Mackett has described this class carrier example, and (Mackett etc. 1984, J.Virol.49:857-864).

The separation quality grain of the nucleic acid that comprises the needs insertion is transfected in cell culture such as the chick embryo fibroblast with parental virus such as poxvirus.Homology variola dna in the plasmid and the reorganization between the viral genome correspondingly produce the recombinant poxvirus of modifying because of having promoter-gene constructs at its genomic locus that does not influence viral vigor.

As mentioned above, insert described nucleic acid in the virus district of the viral viability of the recombinant virus that does not influence generation (inserting the district).For example allow to form reorganization and the not serious viral DNA section that influences the district of recombinant virus viability, the easily such district of evaluation in a kind of virus by random test.A district that is easy to use and is present in many viruses is thymidine kinase (TK) gene.For example the TK gene sees all the poxvirus genome groups [hare poxvirus: J.Virology60:920 such as Upton (1986) (rabbit fibroma virus) that detects; Goat capripoxvirus: J.Gen.Virol.70:525 such as Gershon (1989) (Kenya sheep-1); Vaccinia subgroup virus: J.Virol46:530 such as Weir (1983) (vaccinia virus); Virology135:561 such as Esposito (1984) (monkey pox virus and smallpox virus); PNAS such as Hruby, 80:3411 (1983) (vaccinia virus); Virology143:399 such as Kilpatrick (1985) (yaba monkey tumour virus); Fowlpox virus: J.Gen.Virol69:1275 such as Binns (1988) (bird pox virus); Virology156:355 such as Boyle (1987) (bird pox virus); J.Virological Method20:341 (1988) such as Schnitzlein (bird pox virus, quail poxvirus); Entomopox virus (J.Gen.Virol73:3235-3240 (1992) such as Lytvyn].

In vaccinia virus, except the TK district, other inserts the district and comprises for example HindIIIM.

In bird pox virus, except the TK district, other inserts the district and comprises that for example EPO applies for AIDS Research and HumanRetroviruses7:991-998 (1991) such as the described BamHI J[Jenkins of 0308220A1], EcoRI-HindIII fragment, BamHI fragment, EcoRV-HindIII fragment, BamHI fragment and HindIII fragment.[J.of Virol67:3069-3076 (1993) such as Calvert; Vaccine6:497-503 such as Taylor (1988); J.ofGen.Virol71:621-628 (1990) such as Spehner etc. (1990) and Boursnell].

In pig pox virus, preferably insert the site and comprise the thymidine kinase gene district.

Can easily select promoter according to host and target cell type.For example when poxvirus, should use the poxvirus promoter, for example vaccinia virus 7.5K or 40K or bird pox virus C1.Also can use the artificial constructed thing that contains suitable poxvirus sequence.Also can unite and use enhancer element to improve expression.In addition, the preferred inducible promoter that uses in the part embodiment, inducible promoter also is that this area is known.

Can detect exogenous gene expression with enzyme assay or immunoassay (for example immunoprecipitation, radioimmunoassay or immunoblotting assay).The naturally occurring membrane glycoprotein that the recombinant vaccinia virus infection cell produces is by glycosylation, and the transporte to cells surface.Can obtain high expression level by using strong promoter.

Other requirement that is used for the viral vector of vaccine comprises good immunogenicity and safety.MVA is the replication defect type vaccinia virus strain, has good safety records.MVA can not copy in most cell types and normal human tissue.Transformant type such as BHK21 cell observation in minority copy to MVA.Carroll etc. (1997) confirm that recombinant MVA is good equally with conventional vaccinia virus recombinant carrier aspect generation protectiveness CD8+T cell response, is the effective substitute that copies the competence vaccinia virus of more common use.The vaccinia virus strain that is produced by MVA or stand-alone development, have and make MVA be specially adapted to the Strain of the MVA feature of vaccine, also be applicable to the present invention.

Described carrier is vaccinia virus vector such as MVA or NYVAC preferably.The viral ankara (MVA) of vaccinia virus strain improvement or by the Strain of its generation most preferably.The alternative carrier of vaccinia virus vector comprises fowlpox virus carrier such as bird pox virus or is called the canary pox virus of ALVAC and by the Strain of its generation, they can infect and in the human cell express recombinant protein, but reproducible not.

In one aspect of the invention, described poxvirus vector lacks at least one immune evasion gene.

Virus especially has the unitary Item capacity, and big virus such as the poxvirus of the several genes of therefore can encoding, and has developed the technology of multiple its host immune system of escape.For example they can escape nonspecific defense, for example complement, interferon and inflammatory reaction, and also they can disturb or block effect of cytokines.Lack multiple such immune evasion polypeptide from MVA, only stay the albumen that has interferon-resistant in the left distal end district.

Poxvirus is generally big DNA viruses, causes actute infection rather than latent infection.Their many antigenic proteins of encoding make to be difficult to change antigenicity, therefore depend on active immunity and escape, and escape immune system to protect himself.They have the several genes of coded polypeptide, described polypeptide produces immune many aspects and disturbs: they destroy effect, interference complement, cytokine activity, inflammatory reaction and the CTL recognition reaction (summary of interferon, Smith etc., (1997) Immunol Rev159:137-154).Remove the ability that weak immunogen that these albumen help to strengthen the poxvirus vector coding excites receiver's immunne response.

Immune evasion gene or polypeptide are to help described virus to escape gene or its product of immune system.The effect of described gene or the best interference immuning system of gene outcome is the effect of interference immuning system on a level at least.Many methods can reach this purpose, and for example analogies by soluble cytokine receptor is provided etc. provide the competition thing interfering signal approach of signaling molecule.

The immune evasion gene includes but not limited to following gene:

Interferon is escaped the gene vaccinia virus and is had at least 3 genes that disturb the IFN effect.25Kd polypeptide of E3L gene expression, this polypeptide and the competition of P1 protein kinase are in conjunction with dsRNA, and this process makes P1 activation, eIF2 α phosphorylation, and causes assembling translation initiation complex.This approach generally activates IFN and works, but stop E3L to express therefore translation initiation is not obstructed, and can hinder this approach.

The 10.5Kd polypeptide of K3L gene expression also disturbs the P1 activity, because it is effective eIF2 α analogies, and plays a role as P1 protein kinase competition thing.So its binding mode is similar to E3L.

Prediction A18R gene code unwindase, as if it disturb 2 ', 5 '-oligoadenylate approach, and that this approach is IFN is reactive.2 ', 5 '-A activator RNA se L, its effect is to stop the virus translation.It seems that the A18R expression has reduced by 2 ', 5 '-A level in infection cell.

The B5R gene outcome of the known vaccinia virus of complement and factor H height correlation, factor H is the instrumentality that substitutes complement pathway.Different with classical pathway, antigen just can activate this approach separately.Therefore the B5R gene outcome can be disturbed alternative complement pathway.

The C21L gene is relevant in conjunction with albumen with human C4b again, and acts on the cell that its surface has C4b, is combined with the CR1 complement receptors to stop.

Soluble cytokine receptor vaccinia virus WR B15R gene (at the B16R of vaccinia virus Copenhagen Strain) product is relevant with IL-1 beta receptor IL-1R.

The A53R of vaccinia virus Copenhagen Strain, i.e. WR gene ORF SalF19R coding TNF receptor.Yet these two genes that it is believed that wild-type virus are the inactivation because the ORF fracture all.

It is believed that B8R gene code solubility IFN-γ receptor, provide another IFN escape mechanism for described virus again.

Inflammation it is believed that many genes participate in stoping the inflammatory reaction to viral infection.These genes comprise A44L, K2L, B13R and B22R.

One aspect of the present invention, recombinant poxvirus carrier lack most of immune evasion genes.Preferably lack all immune evasion genes.Therefore, one aspect of the present invention, described poxvirus vector be wherein K3L interferon-resistant protein gene destroyed or the disappearance the recombinant MVA carrier.

The preferred poxvirus that the intended recipient is safe from danger.Therefore for example being preferred for human virus is: perhaps limited poxvirus such as the fowlpox virus of host range, the perhaps attenuated strain of the poxvirus of attenuation such as vaccinia virus (comprising NYVAC and MVA).Though it is effective that non-vaccinia virus strain is used for the receiver of preexist variola immunity, most preferably the vaccinia virus attenuated strain.

A kind of construction is imported in the cell that infects MVA, make its homologous recombination, described construction comprises the nucleic acid of a kind of 5T4 of coding at least, and this nucleic acid flank be that a natural disappearance in the MVA genome is as lacking the MVA DNA sequence of II.

After in case described construction imports described eukaryotic cell and 5T4DNA and viral DNA reorganization, preferably by means of the separable needed vaccinia virus recombinant of labelling (Proc.Natl.Acad.Sci.USA79 such as Nakano, 1593-1596[1982], Mol.Cell.Biol.1918-1924[1985 such as Franke], Mol.Cell.Biol.3403-3409[1985 such as Chakrabarti], Virology97-105[1986 such as Fathi].

The construction that needs to insert can be linearity or annular.Preferred annular DNA, especially plasmid.Described construction be included in the natural disappearance of MVA genome for example lack the left side of II and the sequence of right side flank (Altenburger, W., Suter, C.P. and Altenburger J. (1989) Arch.Virol.105,15-27).Between the flanking sequence of natural disappearance, insert exogenous DNA array.

In order to express at least a nucleic acid, be necessary to make the required adjusting sequence of described transcribed nucleic acid to be present in described nucleic acid upstream.Those skilled in the art understand this class and regulate sequence, comprise for example EP-A-198, the adjusting sequence of the 328 vaccinia virus 11kDa genes of describing and the adjusting sequence (EP-A-110,385) of 7.5kDa gene.

Described construction can import the MVA infection cell by transfection, and described transfection method for example comprises: calcium phosphate precipitation (Virol.52 such as Graham, 456-467[1973; Cell777-785[1979 such as Wigler] electroporation (EMBOJ.1 such as Neumann, 841-845[1982]), micro-injection (Meth.Enzymology101 such as Graessmann, 482-492 (1983)), liposome (Methods in Enzymology101 such as Straubinger, 512-527 (1983)) spheroplast (Schaffner, Proc.Natl.Acad.Sci.USA77,2163-2167 (1980)) or other method well known by persons skilled in the art.The preferred liposome transfection.

The present invention recombinates and causes carrier and strengthen carrier and can have tropism to the mammal particular cell types.For example, can engineered recombinant vector of the present invention to infect sole duty (professional) APC such as dendritic cell and macrophage.Known dendritic cell are successfully the optimal coordination person (orchestrator) of immunne response, especially the cell-mediated optimal coordination person who replys. confirmed can induce when the input homologous gene animal or human body efficient immune (referring to Nestle FO etc. with antigen or the viral vector extracorporeal treatment dendritic cell that comprise this target antigen, with peptide-or the dendritic cell immunity inoculation melanoma patient of tumor lysate-burst process, Nat Med.1998Mar; 4 (3): 328-32 and Kim CJDeng, the external sensitized T lymphocyte of dendritic cell of the poxvirus infection of usefulness coding MART-1/Melan A, J Immunother.1997Jul; 20 (4): 276-86.Described recombinant vector also can infected tumor's cell.Perhaps, described recombinant vector can infect any mammalian cell.

Other example of carrier comprises external transmission system, and external transmission system includes but not limited to the transfection of DNA transfection method such as electroporation, the biological emission of DNA, lipid mediation and tightly ties the transfection of DNA mediation.

Described carrier can be a kind of plasmid DNA carrier." plasmid " used herein refer to for the allogeneic dna sequence DNA transfered cell to express or to copy the independent component of allogeneic dna sequence DNA.Select and use this class component fully in technical staff's technical merit. can utilize many plasmids, and select suitable plasmid to depend on the desired use of plasmid, namely be for DNA amplification or expressible dna, need to insert plasmid the DNA size and will be with described plasmid transformed host cells.Every kind of plasmid according to its effect (DNA amplification or expressible dna) with contain different components with the host cell of its coupling.The plasmid component generally comprises but is not limited to one or more following components: origin of replication, one or more marker gene, enhancer element, promoter, transcription terminator and signal sequence.

Expression plasmid and cloned plasmids generally all comprise the nucleotide sequence that described plasmid can be copied in one or more selected host cells.Usually this sequence is to make described plasmid be independent of the sequence that host chromosome DNA copies in cloned plasmids, comprises origin of replication or autonomous replication sequence.This sequence to various antibacterials, yeast and virus is known.The origin of replication of plasmid pBR322 is applicable to most of gram negative bacteria, and 2m plasmid replication starting point is applicable to yeast, and various virus replication starting point (for example SV40, polyoma virus, adenovirus) can be used for the cloned plasmids of mammalian cell.In general, the mammal expression plasmid does not need the origin of replication component, unless these expression plasmids are used for high-level dna replication dna competence mammalian cell such as COS cell.

Most of expression plasmids are shuttle plasmids, and namely they can copy at least one class organism, but can be transfected into another kind of organism expressing.For example a kind of plasmid of clone in escherichia coli is gone into yeast or mammalian cell with identical plasmid transfection then, copies although it can not be independent of host cell chromosome.

Preferably expression and cloned plasmids can contain the selection gene, are also referred to as selected marker.This gene code grows in the transformed host cell survival of selecting culture medium or the necessary albumen of growing.The plasmid transformed host cells of not involved selection gene can not be survived in culture medium.Usually select the albumen of gene code to give resistance, the complementary auxotrophy of antibiotic and other toxin (for example ampicillin, neomycin, methotrexate or tetracycline) or the important nutrient that can not obtain from complex medium is provided.

About being fit to the selection genetic marker of yeast, can use any marker gene that helps transformant to select because of the marker gene phenotypic expression.The labelling that is fit to yeast is the gene of giving the resistance of antibiotic G418, hygromycin or bleomycin for example, or the former foster gene of auxotrophy yeast mutants such as URA3, LEU2, LYS2, TRP1 or HIS3 gene are provided.

Because can in escherichia coli, carry out plasmid replication easily, therefore preferably include escherichia coli genetic marker and escherichia coli origin of replication.These components can from escherichia coli plasmid pBR322,

Plasmid or pUC plasmid such as pUC18 or pUC19 obtain, and they not only comprise the escherichia coli origin of replication but also comprise the escherichia coli genetic marker of giving antibiotic such as amicillin resistance.

The selected marker that is fit to mammalian cell is the selected marker that makes it possible to identify the cell of taking in 5T4 nucleic acid, for example dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase or give G418 or the gene of hygromycin resistance.Described mammalian cell transformant is under the selection pressure, have only the transformant of taking in and expressing described labelling to survive. if DHFR or glutamine synthase (GS) labelling, applying of selection pressure can be: cultivate described transformant under the condition that described pressure constantly increases progressively, make the DNA that selects gene and the coding 5T4 that is connected all increase (at its chromosomal integration site) thus.Amplification is to repeat to produce the genes of a large amount of needs of the important albumen of preparation growth and the process that codified close-connected with it needs the gene of albumen with the series connection form in the reconstitution cell chromosome.Usually the DNA that increases thus is for the synthesis of the albumen that needs of requirement increase.

Express the promoter that contains host organisms identification usually and effectively be connected with 5T4 nucleic acid with cloned plasmids.This promoter can be induction type or composing type.Digest the promoter of removing source DNA by Restriction Enzyme, and the promoter sequence that separates is inserted described plasmid, thereby described promoter effectively is connected with the DNA of coding 5T4.Natural 5T4 promoter sequence and many allogeneic promoters all can be used for controlling amplification and/or the expression of 5T4DNA.Term " effectively connect " refers to the connection that the position relation of wherein said component makes that they can work with its predetermined way.Make the described coded sequence of expression under the condition that is fit to described control sequence with connected mode that coded sequence " effectively is connected " control sequence.

The promoter that is applicable to prokaryotic hosts comprises for example beta-lactamase and lactose promoter systems, alkali phosphatase, tryptophan (trp) promoter systems and hybrid promoter such as tac promoter.Disclose its nucleotide sequence, made those of skill in the art utilize joint or junctional complex effectively to be connected to provide the restriction site of any needs with the DNA of coding 5T4 with it thus.The promoter that is used for bacterial system generally also comprises the Shine-Delgarno sequence that effectively is connected with the DNA of coding 5T4.

Preferred expression plasmid is the bacterial expression plasmid that comprises phage (as phagex or the T7) promoter that can work in antibacterial.In an expression system the most widely used, can transcribe nucleic acid (Studier etc., the Methods in Enzymol.185 of encoding said fusion protein by t7 rna polymerase from described plasmid; 60-89,1990).In the e. coli bl21 that is used in combination the pET plasmid (DE3) host strain, in described host bacteria, produce t7 rna polymerase by λ lysogen DE3, and its expression is under the control of IPTG induction type lac UV5 promoter.This system's excess of successful Application produces many albumen.Perhaps can (Novagen, Madison USA) import described pol gene by bacteriophage lambda as the CE6 phage that is commercially available by infecting the int-phage.Other plasmid comprises plasmid such as the PLEX (Invitrogen that comprises λ PL promoter, NL), the plasmid such as pTrcHisXpressTm (Invitrogen) or pTrc99 (the Pharmacia Biotech that comprise the trc promoter, SE) or comprise the plasmid of tac promoter such as pKK223-3 (Pharmacia Biotech) or PMAL (new England Biolabs.MA, USA).

In addition, preferably include secretion sequence according to 5T4 gene of the present invention, secrete from bacterial host to help described polypeptide, it is produced with solubility native peptides rather than inclusion body.Can or suitably reclaim described peptide from culture medium from described antibacterial periplasmic space.

The suitable initiating sequence that is used for yeast host can be adjustment type or composing type, and is preferably obtained by high expressed yeast genes, especially genes of brewing yeast.Therefore can use following promoter: the promoter of TRP1 gene, ADHI or ADHII gene, acid phosphatase (PH05) gene; The promoter that the gene of the promoter of the coding a-that yeast engages or the pheromone gene of α-factor or coding glycolytic ferment is derived, for example promoter of following glycolytic ferment gene: Enolase, glyceraldehyde-3-phosphate dehydrogenase (GAP), glycerol 3-phosphate acid kinase (PGK), hexokinase, pyruvic carboxylase, phosphofructokinase, G-6-P isomerase, 3-phoshoglyceric acid mutase, pyruvate kinase, triose-phosphate isomerase, glucose phosphate isomerase or glucokinase gene, saccharomyces cerevisiae GAL4 gene, S.pombe nmt1 gene; Or TATA is in conjunction with the promoter of albumen (TBP) gene.In addition, can use hybrid promoter, hybrid promoter comprises the upstream activating sequence (UAS) of a yeast genes and comprises the downstream promoter element of the function TATA box of another yeast genes, for example comprises the UAS of yeast PH05 gene and the hybrid promoter (PH05-GAP hybrid promoter) of the downstream promoter element of the function TATA box that contains yeast GAP gene.Suitable composing type PH05 promoter for example is the acid phosphatase PH05 promoter of the shortening of removing upstream regulation element (UAS), as originates in nucleotide-173 and end at PH05 (173) promoter element of the nucleotide-9 of PH05 gene.

Plasmid in mammalian host cell, transcribe the 5T4 gene can be controlled by derived from virus genomic promoter, allos mammalian promoter (actin promoter or very strong promoter such as ribosomal protein promoter) with the natural promoter that is connected of 5T4 sequence, as long as this class promoter and described host cell systems coupling, described virus for example is polyoma virus, adenovirus, bird pox virus, bovine papilloma virus, avian sarcomata virus, cytomegalovirus (CMV), retrovirus and simian virus 40 (SV40).

In described plasmid, insert enhancer sequence and can strengthen the DNA that higher eucaryote is transcribed coding 5T4.Enhancer is relatively independent of orientation and position.Many enhancer sequence of known mammalian genes (for example elastoser and globulin).Yet people are ready to use the eukaryotic cell virus enhancer usually.Example comprises SV40 enhancer and the CMV early promoter enhancer of origin of replication rear side (bp100-270).Can enhancer be sheared in the described plasmid in 5 ' or the 3 ' position of 5T4DNA, still be preferably placed at 5 ' position of described promoter.

The eukaryon expression plasmid of coding 5T4 preferably comprises locus control region (LCR).LCR can instruct and be integrated into the expression that the chromatinic genetically modified high level of host cell does not rely on integration site; when being designed for gene therapy purposes plasmid or transgenic animal, when needs have been integrated in the chromosomal permanent transfecting eukaryotic cells based environment LCR particular importance when expressing the 5T4 gene at described plasmid.

Eukaryon expression plasmid also will contain termination transcribes the sequence essential with stable mRNA.This class sequence can obtain from 5 ' and the 3 ' untranslated region of eukaryotic cells or viral DNA or cDNA usually.These districts comprise the nucleotide section of the untranslated polyadenylation fragment partly of the mRNA that is transcribed into coding 5T4.

Expression plasmid comprises any can expression and the plasmid of regulating the effective 5T4 nucleic acid that is connected of sequence such as promoter region, and described adjusting sequence can be expressed this class DNA.Therefore, expression plasmid refers to express recombinant DNA or the RNA construction of described cloned DNA, for example plasmid, phage, recombinant virus or other plasmid when importing the suitable host cell.Suitable expression vector is that persons skilled in the art are known, comprises the plasmid that can copy and keep free plasmid or be integrated into the plasmid of host cell gene group in eucaryon and/or prokaryotic cell.The DNA of 5T4 of for example encoding can insert and be adapted at expressing in the mammalian cell in the plasmid of cDNA, for example cmv enhancer substrate grain such as pEVRF (Matthias etc., (1989) NAR17,6418).

Implementing the present invention useful especially is the expression plasmid that the DNA of transient expression coding 5T4 is provided in mammalian cell.Transient expression is usually directed to use the expression plasmid that can effectively copy in host cell, makes described host cell accumulate the expression plasmid of many copies, the high-caliber 5T4 of resynthesis.For purpose of the present invention, transient expression system can be used for for example identifying the 5T4 mutant, to identify the functional domain of potential phosphorylation site or the described albumen of characterized.

Structure uses conventional interconnection technique according to plasmid of the present invention.Make the cutting of the plasmid of separation or dna fragmentation, tailing, and need the mode that needs of plasmid to connect with generation again.Need, analyze to confirm the accurate sequence in the constructed plasmid in a known way.Construction expression plasmid, external preparation transcript, DNA import host cell and analyze to estimate the expression of 5T4 and the appropriate method of function is well known by persons skilled in the art.For example utilize based on the appropriate flags probe of sequence provided herein directly the rna blot analysis by conventional southern blotting technique analysis, the mRNA that quantitatively transcribes, dot blotting (DNA or RNA analyze) but or the gene in the in situ hybridization working sample exists, amplification and/or express.If necessary, those skilled in the art can easily design these methods that how to improve.

5T4 antigen, fragment and variant

5T4 antigen as referred to herein comprises that at least one common 5T4 antigen of reservation determines 5T4 peptide and other fragment of 5T4 of family.

" public antigen determines family " means that described derivant has at least one 5T4 antigen function.Antigen function comprise have one can with epi-position or the antigen site of the antibody cross reaction that produces at natural 5T4 polypeptide or degeneration 5T4 polypeptide or its fragment, or can and induce the ability of 5T4 specific immune response in conjunction with the HLA molecule.Therefore, 5T4 provided by the invention comprises shearing variant, amino acid variation body, the glycosylation variant that the mRNA of variable shearing primary transcription deposits yields encodes and other covalency 5T4 derivant that keeps 5T4 physiology and/or physical characteristic.Typical case's derivant comprises wherein by replacement, chemical method, enzyme method or utilizes the molecule of other appropriate method covalent modification albumen of the present invention of a part of alpha-non-natural amino acid. a such part can be a detectable part, for example enzyme or radiosiotope.In addition, comprise the natural 5T4 variant that sees specific species, preferred mammal.By the related gene of homologous genes family, this variant of allelic variation body codified of specific gene, or this variant is the variable shearing variant of 5T4 gene.

Keep public antigen to determine that the derivant of family can be the 5T4 fragment.The 5T4 fragment comprises its each domain and derived from the smaller polypeptides of described domain.Derived from the single 5T4 feature epi-position of little polypeptide best definition according to 5T4 of the present invention.Fragment almost can be any size in theory, and prerequisite is that they keep a 5T4 feature.Fragment is 5-400 amino acid length preferably.Think that long fragment is the total length 5T4 of truncate, and generally comprise in term " 5T4 " definition.Fragment is the less peptide of about 5-25 amino acid length preferably.The peptide of preferred about 9 amino acid lengths.

The 5T4 derivant comprises that also it can contain the mutant of aminoacid deletion, interpolation or replacement, and precondition is to possess at least one 5T4 features characteristic.Therefore, the conserved amino acid that can not change 5T4 character basically replaces, equally can be from 5 ' or 3 ' terminal truncate 5T4.Can the 5T4 fragment that the present invention includes be lacked and replace in addition.Make the DNA of coding 5T4 carry out in vitro mutagenesis, cause for example adding, exchange and/or lack one or more aminoacid, thereby prepare the 5T4 mutant by the DNA of coding 5T4.Replacement, disappearance that for example can be by recombinant methods 5T4 or insert variant, and screen according to the immune cross-reactivity of itself and native form 5T4.

In addition, utilize the program " peptide is in conjunction with prediction " of the K.Parker of NIH design can screen that (referring to Parker, K.C etc. 1994, J.Immunol.152:163) according to the good HLA binding ability of variant peptide.

The fragment of 5T4, mutant and other derivant preferably and 5T4 keep obvious homology." homology " used herein refers to that the total those skilled in the art of described two entities determine their source and intimate enough features.Homology is preferably used in and refers to sequence homogeneity.So, the 5T4 derivant preferably and SEQ ID NO:2 sequence have obvious homogeneity.

When homology referred to sequence homogeneity, " significantly homology " meaned that sequence homogeneity is higher than 40% according to direct sequence arrangement and contrast judgement, and preferred sequence homogeneity is higher than 45%, most preferably sequence homogeneity 50% or higher.

In addition available suitable homology algorithm, for example can determine sequence homology (or homogeneity) with default parameters.Preferably operation parameter is set at the BLAST algorithm of default parameters.The BLAST algorithm has a detailed description in http://www.ncbi.nih.gov/BLAST/blast_help.html network address, and it is attached to herein by reference.Search argument is defined as follows, and is preferably set to the default parameters of definition.

When BLAST estimated, " significantly homology " preferably was exactly to mate the EXPECT value at least about 7, preferably at least about 9, most preferably 10 or higher sequence.The EXPECT default threshold of BLAST retrieval is generally 10.

BLAST (Basic LocalAlignment Search Tool) is the searching algorithm of heuristicing of blastp, blastn, blastx, tblastn and the use of tblastx program; These programs utilize the Karlin that strengthens aspect several and the statistical method (referring to http://www.ncbi.nih.gov/BLAST/blast_help.html) of Altschul to give its discovery with significance.Blast program is applicable to the sequence similarity retrieval, for example identifies the homologue of search sequence.This program generally is not used in the retrieval of motif type.About the discussion to the basic problem of sequence library similarity retrieval, referring to (1994) Nature Genetics6:119-129 such as Altschul.

Can finish following work at obtainable 5 blast programs of http://www.ncbi.nlm.nih.gov:

Blastp comparing amino acid search sequence and protein sequence data base;

Blastn is nucleotide query sequence and nucleotide sequence database relatively;

Blastx is translation product and the protein sequence data base of 6 frame concepts of nucleotide query sequence (two strands) relatively;

Tblastn is albumen search sequence and the dynamic nucleotide sequence database of translating (two strands) of whole 6 frames relatively;

The tblastx relatively 6 frames translation of nucleotide query sequence translates with 6 frames of nucleotide sequence database.

BLAST uses following search argument:

HISTOGRAM shows the block diagram of keeping the score of each retrieval; Acquiescence yes.(referring to the Parameter H of BLAST handbook).

DESCRIPTIONS is defined as specified number or amount with the short number of describing of the matching sequence of report; Default limit is 100 descriptions.(referring to the V parameter of described handbook).Consult EXPECT and CUTOFF in addition.

ALIGNMENTS is defined as the report height with database sequence and keeps the score section to (HSP) specified number or amount; The acquiescence limit is 50.If the database sequence that is higher than this number satisfies the significance,statistical threshold value (referring to following EXPECT and CUTOFF) of report, then only report the coupling of maximum significance,statistical.(referring to the B parameter of BLAST handbook).

The significance,statistical threshold value of EXPECT report and database sequence coupling; Default value 10, according to the stochastic model (1990) of Karlin and Altschul, expection only can chance on 10 coupling things like this.If the significance,statistical of a coupling greater than the EXPECT threshold value, then can not be reported this coupling.Lower EXPECT threshold value is more strict, makes that the chance of report coupling is lower.Fractional value is acceptable.(referring to the parameter E of BLAST handbook).

Ending that the high section of keeping the score of CUTOFF report is right kept the score.Calculate default value according to EXPECT value (seeing above).Have only when the significance,statistical of the section that is classified as database sequence at least when being classified as the significance,statistical of the single HSP that equals the CUTOFF value of keeping the score, HSP that could the report database sequence.Higher CUTOFF value is more strict, makes that the chance of report coupling is littler.(referring to the parameter S of BLAST handbook).Usually can handle the significance threshold value more intuitively with EXPECT.

The matrix of alternately keeping the score of MATRIX regulation BLASTP, BLASTX, TBLASTN and TBLASTX.Default matrix is BLOSUM62 (Henikoff﹠amp; Henikoff, 1992).Effectively replace and select to comprise: PAM40, PAM120, PAM250 and IDENTITY.BLASTN does not have the available replacement matrix of keeping the score; When inquiring about, BLASTN specify the MATRIX indication can return the answer that makes mistakes.

STRAND just is limited to the TBLASTN retrieval described data base's cochain or following chain; Perhaps BLATN, BLASTX or TBLASTX retrieval are limited to the cochain of search sequence or the frame of following chain.

The FILTER shielding is according to Wootton ﹠amp; The low search sequence section of composition complexity that the SEG program of Federhen ((1993) Computers andChemistry17:149-163) is determined is perhaps according to Claverie ﹠amp; States ((1993) Computers and Chemistry17:191-201) if the XNU program determine or be BLASN then according to the definite section of being formed by repetitive sequence in the short period property of the DUST program (referring to http://www.ncbi.nlm.nih.gov) of Tatusov and Lipman.Filtration can be eliminated the report (for example hitting the abundant district of common acidic amino acid, the abundant district of basic amino acid or the abundant district of proline) that has statistical significance and do not have biological significance from blast output, stays the district that biological significance is more arranged of the search sequence of available and database sequence specificity coupling.

The low sequence of complexity of in nucleotide sequence, using letter " X " (for example " XXXXXXXXX ") to replace filter to find with letter " N " (for example " NNNNNNNNNNNNN ") and in protein sequence.

Filtration is only applicable to search sequence (or its translation product), is not suitable for database sequence.It is DUST that acquiescence is filtered for BLASTN, and other program is SEG.

When being used for the sequence of SWISS-PROT, not shielding any section with SEQ, XNU or the two is not abnormal conditions, therefore not expectability filter can the generation effect.In addition, in some cases, whole sequence is shielded, and illustrates the significance,statistical of any coupling of not filtering search sequence report doubtful.

NCBI-gi makes and shows NCBIgi identifier, login name and/or location name (locus name) in output.

Most preferably the simple BLAST searching algorithm that provides with http://www.ncbi.nlm.nih.gov/BLAST is carried out sequence relatively.

On the other hand, be used in the obtainable algorithm of http://biology.ncsa.uiuc.edu/BW30/BW.cgi such as FastA and can determine sequence homology.It is believed that FastA is better than BLAST for arranging the short sequence of contrast.The default parameters of the most handy http://biology.ncsa.uiuc.edu/BW30/BW.cgi uses the FastA algorithm.

Preferably provide albumen or derivatives thereof of the present invention with unpack format." separation " refers to identify described albumen or derivant, and do not contain one or more components of its natural surroundings.The 5T4 that separates comprises the 5T4 of reconstitution cell culture.The 5T4 that is present in the organism of express recombinant 5T4 gene, the 5T4 albumen that no matter whether separates includes in category of the present invention.

In human antineoplastic immune inoculation, preferably use non-human TAA.Therefore the invention provides dog 5T4.Preferably provide the dog 5T4 as human vaccine component, to excite the immunne response at people 5T4 human receiver.

The sequence of dog 5T4 is seen SEQ IDNO.3.Those skilled in the art for example utilize derived from the nucleic probe of SEQ IDNO.3 hybridizes the sequence that can obtain other dog source.

Therefore, exemplify the nucleotide sequence that nucleic acid can be characterized by encoding canine 5T4 albumen and hybridize with the selected fragment of DNA sequence shown in the SEQ IDNO.3 or described DNA sequence.Preferably under high stringent condition with this class sequence of the encoding canine 5T4 of SEQ IDNO:3 sequence hybridization.

Strict hybridization refers to that the Polynucleotide hybrid molecule is stable hybridization conditions.This class condition will be apparent to those skilled in the art.It is known to those skilled in the art that the stability of melting temperature (Tm) the reflection hybrid molecule of hybrid molecule, every reduction by 1% melting temperature of sequence homology just reduces about 1-1.5 ℃.In general, the stability of hybrid molecule is the function of Na ion concentration and temperature.Usually under high stringent condition, carry out hybridization, carry out the washing of different stringency then.

High stringency used herein refers to that only permission forms the condition of the nucleic acid array hybridizing of stablizing hybrid molecule in 65-68 ℃ 1M Na+.For example by in containing 6 * SSC, 5 * Denhardt, 1%SDS (sodium lauryl sulphate), 0.1Na+ pyrophosphate and the aqueous solution of 0.1mg/ml degeneration salmon sperm DNA as non-specific competition thing, hybridizing, can provide high stringent condition.After the hybridization, can carry out high stringency washing by several steps, (about 30 minutes) are carried out in last washing under hybridization temperature in 0.2-0.1 * SSC, 0.1%SDS.

Medium stringency refers to be equivalent in above-mentioned solution still in the condition of about 60-62 ℃ hybridization.In the case, the last washing carried out under hybridization temperature in 1 * SSC, 0.1%SDS.

Low stringency refers to be equivalent in above-mentioned solution in the condition of about 50-52 ℃ hybridization.In the case, the last washing carried out under hybridization temperature in 2 * SSC, 0.1%SDS.

Certainly can revise and with multiple buffer for example Methanamide be the basis buffer and temperature repeat these conditions.Denhardt solution and SSC are that those skilled in the art are known, other suitable hybridization buffer is also so (referring to editor (1989) Molecular Cloning:A Laboratory Manual such as for example Sambrook, Cold Spring Harbor LaboratoryPress, editor such as New York or Ausubel (1990) Current Protocols in MolecularBiology, John Wiley ﹠amp; Sons, Inc.).Because the length of probe and GC content also have influence, so have to rule of thumb determine best hybridization conditions.

In addition, the present invention preferably provides the nucleotide sequence that can hybridize with SEQ IDNO:3 fragment under stringent condition.Described fragment is preferably 15-50 base length.Preferred about 25 base length.

According to guidance provided herein, the method known according to this area can obtain nucleic acid of the present invention.For example utilize polymerase chain reaction (PCR) but by chemosynthesis or screening by thinking genomic library or the suitable cDNA library of the source preparation that has dog 5T4 and express with detection level, can obtain DNA of the present invention.

The chemical method of synthetic purpose nucleic acid is known in the art, comprises that oligonucleotide is synthetic on triester method, phosphite method, phosphoramidite method and H-phosphate ester method, PCR and other automatic primer (autoprimer) method and the solid support.If the complete nucleotide sequence of known described nucleic acid maybe can obtain the nucleotide sequence with described coding strand complementation, can use these methods.On the other hand, if known described target amino acid sequence, people can utilize the known of each amino acid residue and optimized encoding residue to infer possible nucleotide sequence.

An alternative method of separating the gene of encoding canine 5T4 is the described use round pcr of the 14th trifle according to Sambrook etc. 1989.The method need be used the oligonucleotide probe with dog 5T4 nucleic acid hybridization.The strategy of selecting oligonucleotide is below described.

With probe or the analytical tool screening library of design in order to identify genes of interest or its encoded protein.For the cDNA expression library, proper tools comprises that identification and specificity are in conjunction with monoclonal antibody or the polyclonal antibody of dog 5T4; The oligonucleotide of about 20-80 the base length of the known or uncertain dog 5T4cDNA of coding same species or different plant species; And/or complementation or homology cDNA or its fragment of coding homologous genes or hybrid gene.The proper probes in screening-gene group DNA library include but not limited to encode oligonucleotide, cDNA or its fragment of identical or hybrid dna; And/or homologous genes group DNA or its fragment.

Namely comprise nucleic acid disclosed herein derived from the oligonucleotide of sequence shown in the SEQ ID NO:3 with probe, the suitable cDNA of screening or genomic library under suitable hybridization conditions, the nucleic acid of separable acquisition encoding canine 5T4.Suitable library is commercially available or can be made by for example cell line, tissue sample etc.

Probe used herein for example comprises 10-50, preferred 15-30 most preferably at least about single stranded DNA or the RNA of the nucleotide sequence of 20 continuous bases for having, described continuous base and similar number shown in the SEQ ID NO:3 or the continuous base identical (or its complement) of more number more.The nucleotide sequence that probe is made in selection should have sufficient length and enough clear and definite, and it is minimum that false positive results is reduced to.Described nucleotide sequence is usually based on nucleotide sequence or the district of the conservative of dog 5T4 or height homology.Nucleic acid as probe can be degeneracies in one or more positions.When the preferred codon from species uses unknown species selection library, use degenerate oligonucleotide possibility particular importance.

Comprised the sequence of 5 ' and/or 3 ' coded sequence, expection coding ligand-binding site point etc. by its dominant area that makes up probe.For example full length cDNA clone disclosed herein or its fragment can be used as probe.The most handy suitable labeling method labelling nucleic probe of the present invention is in order to detect at any time after the hybridization.For example, a kind of suitable labeling method is radioactive label.The method for optimizing of labeled dna fragment is to utilize the Klenow fragment of the archaeal dna polymerase in the initiation reaction at random to mix α 32P dATP, and this is that this area is known.Usually ATP and the polynucleotide kinase with γ 32P-labelling carries out end labelling to oligonucleotide.Yet other method (for example on-radiation method) also can be used for the described fragment of labelling or oligonucleotide, comprises for example enzyme labelling, the fluorescent labeling of utilizing suitable fluorogen and biotinylation.

For example with after comprising described complete dog 5T4 coded sequence basically or screening described library based on the suitable oligonucleotide of the described DNA of a part, identify positive colony by detecting hybridization signal; Characterize the clone who is identified with Restriction Enzyme mapping and/or dna sequence analysis, for example provide sequence with this paper then and relatively detect, whether comprise the DNA (being that they comprise translation initiation codon and termination codon) of the complete dog 5T4 that encodes to determine them.If selected clone is not complete, they can be used for screening identical library or different library to obtain overlapping clone again.If described library is genomic library, then described overlapping clone can comprise exon and intron.If described library is the cDNA library, then described overlapping clone will comprise an open reading-frame.In both cases, by comparing with described DNA and putative amino acid sequence provided herein, can identify complete clone.

Imagination inserts by nucleotide replacement, nucleotide deletion, nucleotide or nucleic acid of the present invention can be easily modified in inversion and their combination of nucleotide section.This class mutant can be used for for example producing the dog 5T4 mutant with aminoacid sequence different with naturally occurring dog 5T4 sequence.Mutation can be (direct mutagenesis) or at random of expection.The sudden change of non-silent mutation can not place sequence outside the frame and preferably not produce to hybridize and produce the mRNA secondary structure as the complementation district of ring or hair clip.

Above-mentioned consideration also is applicable to and separates muroid (SEQ.ID.NO.2) or people (SEQ.ID.NO.1) the 5T4 antigen that substitutes.

Giving of the carrier of coding 5T4

Be to comprise that the carrier of described coding 5T4 antigen is as the composition of matter of active component according to Pharmaceutical composition of the present invention.Imagination comprises according to the active component of the Pharmaceutical composition of described active component of the present invention when when giving according to the definite dosage of case-specific, for example has the good activity that treats and/or prevents in treating and/or preventing tumor or other and cell proliferation diseases associated, infection and inflammatory conditions.Can adjust dosage to obtain optimum therapeutic response.But for example give every day several divided doses or according to the treatment situation urgency can reduce described dosage in proportion.

Described reactive compound can give in a usual manner, for example oral, intravenous (when under the water miscible situation), intramuscular, subcutaneous, intranasal, Intradermal or suppository approach or implantation (for example using the slow release molecule).According to route of administration, may need to avoid enzyme, acid and may make the material of other natural endowment of described composition inactivation that described active component is carried out coating with the described composition of protection.

In order to give described reactive compound by non-parenteral, will carry out coating with the material that prevents its inactivation, or utilization prevents that the material of its inactivation from giving described reactive compound.For example the available secondary auxiliary agent gives described reactive compound, or gives jointly with enzyme inhibitor, or gives described reactive compound with liposome.At its broadest application adjuvant, comprise any immune-stimulating compound such as interferon.The adjuvant of this paper design comprises resorcinol, non-ionic surface active agent such as polyethylene glycol oxide oil ether and n-hexadecyl polyvinylether.Enzyme inhibitor comprises the pancreas trypsin.

Liposome comprises water-Bao-oil-Bao-water CGF Emulsion and conventional liposome.

Described reactive compound also can parenteral or intraperitoneal give.Also available glycerol, liquid macrogol and composition thereof prepare dispersant with oil.Under routine storage and service condition, these preparations contain antiseptic to prevent growth of microorganism.

The medicament forms that is fit to injection comprises aseptic aqueous solution (when for water solublity) or dispersant and is used for the aseptic injectable solution of interim preparation or the aseptic powder of dispersant.In all cases, described medicament forms must be aseptic and also the fluid degree must be easy to the injection.Produce and holding conditions under must be stable, and must be antiseptical to microbial contamination such as antibacterial and fungus.Described carrier can be solvent or disperse medium, comprises for example water, ethanol, polyhydric alcohol (for example glycerol, propylene glycol and liquid macrogol etc.), their suitable mixture and vegetable oil.For example use coating such as lecithin, when dispersant, can keep proper flow by the grain graininess of maintenance needs and by the use surfactant.

For example parabens, methaform, phenol, sorbic acid, thimerosal (thirmerosal) etc. can prevent action of microorganisms can to use various antibacterial and antifungal.As a rule, preferably comprise isotonic agent, for example sugar or sodium chloride.In described compositions, use to postpone for example aluminum monostearate and the gelatin absorption that can prolong injectable composition of absorbent.

Being prepared as of aseptic injectable solution: in the suitable solvent that contains the various compositions of enumerating more than other (as required), add the described reactive compound of the amount of needs, filtration sterilization then.In general the preparation of dispersant is: described sterile active composition is added in the aseptic solvent, described solvent contain basic disperse medium and needed more than other composition of enumerating.If when being the aseptic powder for the preparation of aseptic injectable solution, preferred manufacturing procedure is vacuum drying and freeze drying technology, described active component and any other that this technology can obtain its above-mentioned aseptic filtration solution need the powder of composition.

When suitably protecting described reactive compound as mentioned above; for example available inert diluent or the edible carrier orally give that assimilates; perhaps it can be encapsulated in hard gelatin capsule or the Perle, perhaps its tablet forming perhaps can directly can be added it in dietary food product.For the therapeutic oral administration, described active component can with mixed with excipients, and use with forms such as the tablet of can ingesting, mouthful cheek sheet, lozenge, capsule, elixir, suspending agent, syrup, wafers.Reactive compound amount in the compositions that this class therapeutic is used makes can obtain suitable dosage.

Described tablet, lozenge, pill, capsule etc. can also contain following composition: adhesive such as tragakanta, acacin, corn starch or gelatin; Excipient such as dicalcium phosphate; Disintegrating agent such as corn starch, potato starch, alginic acid etc.; Lubricant such as magnesium stearate; And can add sweeting agent such as sucrose, lactose or glucide or flavoring agent such as Herba Menthae, wintergreen oil or Fructus Pruni pseudocerasi flavoring agent.When described dosage form was capsule, except the above-mentioned type material, it also can contain liquid-carrier.

Various other materials can or improve the physical form of described dosage form for coating.For example available Lac, sugar or the two are carried out coating to tablet, pill or capsule.Syrup or elixir can contain described active component, as the sucrose of sweeting agent, as methyl parahydroxybenzoate and propyl p-hydroxybenzoate, pigment and flavoring agent such as Fructus Pruni pseudocerasi or the orange flavoring agent of antiseptic.Certainly, any material for the preparation of any unit dosage forms should be pharmaceutical purity and nontoxic basically in described use amount.In addition, described reactive compound can add in slow releasing preparation and the prescription.

" pharmaceutically acceptable carrier and/or diluent " used herein comprises any solvent and all solvents, disperse medium, coating, antibacterial agent and antifungal, isotonic agent and delay absorbent etc.Use is applicable to that this class medium of pharmaceutically active substance and material are that this area is known.Unless any conventional media or material and described active component do not match, otherwise design its use in described therapeutic composition.Other active component also can add in the described compositions.

Preferred especially preparation is easy to the parenteral compositions of the unit dosage forms of administration and dosage homogeneous.Unit dosage forms used herein refers to be suitable for the physical property individual of the unit dose of making mammalian subject to be treated; Each unit contains the active substance of the scheduled volume that can produce needs treatment effect as calculated with the pharmaceutical carrier of needs.The specification table of the novel unit dosage forms of the present invention is shown and directly depends on the specific characteristic of (a) described active substance and the concrete treatment effect that need reach, and the intrinsic restriction of (b) adjustment technology, for example be used for the treatment of the inherent limitations of the active substance of curee's disease alive, its morbid state of described curee makes body health impaired.

For suitable effective administration, the main active of effective dose is adjusted into unit dosage forms with suitable pharmaceutically acceptable carrier.If compositions contains other active component, then determine its dosage with reference to routine dose and the administering mode of described composition.

Utilize conventional effectiveness testing experiment decision according to the dosage regimen of 5T4 expression vector of the present invention.Yet preferably include the continuous scheme that excites and strengthen step especially.People observe, and this class scheme can successfully better be eliminated immunologic tolerance and induce CTL to reply.In a preferred embodiment, use non-virus carrier and carry out exciting step as the plasmid of coding 5T4, use the viral vector of coding 5T4 such as poxvirus vector simultaneously and carry out booster immunization (referring to Schneider etc., 1998Nat Med4:397-402).

Give the method for 5T4 antigen

In general, available above general introduction about the method afford that gives the 5T4 code nucleic acid 5T4 antigen as the conventional vaccine preparation, in order to treat and/or prevent tumor.

The vaccine that can prepare 5T4 antigen usually.The known preparation that comprises the vaccine of 5T4 active component of those skilled in the art.This class vaccine is generally injectable liquid solution or suspension; Also can prepare the preceding available liquid dosage of injection is the solid form of solution or suspension.Also can prepare emulsifying agent, or with described albumen encapsulate capsule in liposome.Usually with described active immne ultimate constituent and mixed with excipients pharmaceutically acceptable and that mate with described active component.Suitable excipient for example is water, saline, glucose, glycerol, ethanol etc. and compositions thereof.

In addition, if necessary, described vaccine can comprise the auxiliary substance that trace strengthens described vaccine effect, for example wetting agent or emulsifying agent, pH buffer agent and/or adjuvant.Effectively the adjuvant example includes but not limited to aluminium hydroxide; N-acetyl group-muramyl-L-Threonyl-D-isoglutamine (thr-MDP); N-acetyl group-nor-muramyl-L-alanyl-D-isoglutamine (CGP11637; be called nor-MDP); N-acetyl group muramyl-L-alanyl-D-isoglutamine base-L-alanine-2-(1 '-2 '-two palmityls-sn-glyceryl-3-hydroxyl phosphorus acyloxy)-ethamine (CGP19835A; be called MTP-PE) and RIBI, it (is monophosphoryl lipid A that RIBI contains 3 components that useful 2% Squalene/Tween 80 emulsion extracts from antibacterial; trehalose dimycolate and cell wall skeleton (MPC+TDM+CWS)).

Other example of adjuvant and other material comprises aluminium hydroxide, aluminum phosphate, aluminium potassium sulfate (Alumen), beryllium sulfate, silicon dioxide, Kaolin, carbon, water-in-oil emulsion, O/w emulsion, muramyldipeptide, bacterial endotoxin, lipid X, spillikin bacillus (Corynebacterium parvum) (Propionobacterium acnes), bordetella pertussis (Bordetella pertussis), polyribonucleotide, sodium alginate, lanoline, LYSOLECITHIN SUNLECITHIN A, vitamin A, Saponin, liposome, levamisole, the DEAE-glucosan, block copolymer or other synthetic adjuvant.This class adjuvant can be from the commercially available acquisition in various sources, for example the Merck adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or incomplete Freund and Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan).

Normally used adjuvant for example has the mixture of Amphigen (oil-in-water), Alhydrogel (aluminium hydroxide) or Amphigen and Alhydrogel.It is human to have only the aluminium hydroxide approval to be used for.

The proportion of immunogen and adjuvant can alter a great deal, and prerequisite is that both content is effective dose.For example the content of aluminium hydroxide can be described vaccine mixture (Al ₂O ₃Be main) about 0.5%.Usually the immunogen final concentration of preparation vaccine is 0.2-200 μ g/ml, preferred 5-50 μ g/ml, 15 μ g/ml most preferably.

After the preparation, with the vaccine sterile chamber of packing into, sealed container is preserved in low temperature then, and for example 4 ℃, perhaps can be with its lyophilizing.Lyophilizing makes can the long-term preservation of stable form.

Usually for example inject (for example subcutaneous or intramuscular injection) by parenteral and give vaccine.Suitable other other preparation that gives pattern comprises the oral formulations under suppository and some situation.For suppository, traditional adhesive and carrier comprise for example poly alkylene glycol or triglyceride; Be that the mixture of 0.5%-10%, preferred 1%-2% can be made into such suppository with described active component content.Oral formulations comprises the conventional excipient that uses, for example pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc.The form of these compositionss can be solution, suspending agent, tablet, pill, capsule, slow releasing preparation or powder, and the content of active component is 10%-95%, preferred 25%-70%.When the described vaccine combination of lyophilizing, can copy the material of institute's lyophilizing before giving, for example copy as suspension.Copy preferably and carry out with buffer.

Utilize enteric coating can prepare orally give patient's capsule, tablet and pill, described enteric coating comprises for example Eudragit " S ", Eudragit " L ", cellulose acetate, cellulose acetate phthalate or hydroxypropyl emthylcellulose.

5T4 can be formulated as the vaccine of neutrality or salt form.Pharmaceutically acceptable salt comprises acid-addition salts (forming with the free amine group of described peptide), forms acid-addition salts with mineral acid (example hydrochloric acid or phosphoric acid) or organic acid (for example acetic acid, oxalic acid, tartaric acid and maleic acid).The salt that forms with free carboxy also can be derived from inorganic base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or hydrated ferric oxide. and such as the inorganic base of 2-aminopropane., trimethylamine, 2-ethyl amido alcohol, histidine and procaine.

Measurement to the effectiveness that gives 5T4

Can be according to the activity of any technology assessment 5T4 known in the art as the immunotherapeutical molecule, comprise that antagonist produces, induces CTL to reply mensuration with the tumor suppression pattern.Typical technology is described in following examples.

Further describe the present invention with following embodiment, its purpose only is for the present invention is described, wherein with reference to the following drawings.

Fig. 1 a illustrates a gene constructs;

Fig. 1 b illustrates a gene constructs;

Fig. 2 a is a photo figure;

Fig. 2 b is a photo figure;

Fig. 3 a is a coordinate diagram;

Fig. 3 b is a coordinate diagram;

Fig. 4 a is a coordinate diagram;

Fig. 4 b is a coordinate diagram;

Fig. 4 c is a coordinate diagram;

Fig. 5 is a coordinate diagram;

Fig. 6 is a coordinate diagram;

Fig. 7 is a coordinate diagram;

Fig. 8 is a coordinate diagram; With

Fig. 9 is a coordinate diagram.

Below slightly describe above-mentioned figure in detail:

Fig. 1 a is the collection of illustrative plates of vaccinia virus recombinant MVA.Under early stage/late promoter control that transgenic is in synthetic vaccinia virus.Under early stage/late promoter control that Lac Z gene is in vaccinia virus 7.5k.Therefore the DNA district of these gene flanks makes homologous recombination arrive this site derived from the disappearance district 2 of MVA.

Fig. 1 b is the collection of illustrative plates of vaccinia virus recombinant WR.Under early stage/late promoter control that transgenic is in synthetic vaccinia virus.Under early stage/late promoter control that Lac Z gene is in vaccinia virus 7.5k.Therefore the DNA district of described gene flank makes homologous recombination arrive this site derived from thymidine kinase (tk) gene of Wyeth Strain VV.

Fig. 2 illustrates the western blot analysis of the vaccinia virus recombinant of expressing human 5T4.Make sample walk electrophoresis at 12%SDS PAGE, and it is transported on the nitrocellulose filter.

Fig. 2 a: survey trace with the dilution MAb5T4 of 1:500 (anti-people 5T4).Put together antibody and ECL demonstration binding antibody with anti-mice HRP.The reorganization WR clone 1 of swimming lane 1: expressing human 5T4; The reorganization WR clone 2 of swimming lane 2: expressing human 5T4; Swimming lane 3: with WR infected B S-C-1 cell; Swimming lane 4: infected B S-C-1 cell not; Swimming lane 5:B16 melanoma cells; The B16 cell line of swimming lane 6: expressing human 5T4.

Fig. 2 b: anti-mice 5T4 surveys trace with the dilution rabbit of 1:500.Swimming lane 1: the reorganization WR that expresses LacZ; Swimming lane 2: the reorganization WR that expresses mice 5T4; The reorganization WR of swimming lane 3: expressing human 5T4; Swimming lane 4: the recombinant MVA of expressing mice 5T4.

Fig. 3 a and 3b are for showing with two width of cloth figures of MVA-h5T4 inoculation mice to the protective effect of attacking with the CT26 that expresses h5T4.

Fig. 4 a is for showing the figure to the B16 antitumor activity against various tumors of expressing h5T4 that induces with the MVA-h5T4 inoculation.

Fig. 4 b and 4c are for showing two width of cloth figure to the B16 antitumor activity against various tumors of expressing m5T4 that induce with the MVA-m5T4 inoculation.

Fig. 5 is for showing the figure of the oncotherapy effect that MVA-h5T4 induces in the mice of having set up lung tumor in advance.

Fig. 6 is for showing the figure that is lower than the mice of accepting control vaccine with the tumor developmental rate of MVA-m5T4 immunoprophylaxis mice.

Fig. 7 is for showing the figure that is lower than the mice of accepting the MVA-LacZ processing with the tumor load of MVA-m5T4 immunized mice.

Embodiment

Embodiment 1

The structure of vaccinia virus recombinant carrier

The breeding of vaccinia virus

Highly attenuated Strain MVA is derived from duplicating virus strain Ankara, and gone down to posterity more than 570 generations in former generation chick embryo fibroblast.Originally it is believed that MVA copies only limits to the CEF cell, because it is reported replication capacity minimum in mammalian cell.Yet, further analytical proof, baby hamster kidney cell (BHK-21) can support to produce the MVA of high titre.Therefore also can cultivate MVA (Carroll ﹠amp at BHK-21 or former generation CEF cell; Moss (1997) Virology238:198-211).

In order to prepare the CEF cell, 10 days instar chicken embryos are removed internal organs, and remove limbs and head, shred then, in 0.25% trypsin solution, carry out trypsinization and in 37 ℃ of incubations.Make cell suspending liquid pass through filter hole, washed cell uses Sorvall RC-3B with 2000rpm centrifugal concentrated 5 minutes (concentrated by centrifugation at2000rpm in a SorvallRC-3B at 1500rpm for 5mins).Cell suspension in containing the MEM of 10%FCS, is cultivated its equal portions at the 175cm culture bottle and in 37 ℃ CO2 gas incubator.When cell monolayer merges 95%, use trypsinization, it is inoculated into other culture bottle or 6 well culture plates.Perhaps transfer to 31 ℃ of incubators standby (Sutter and Moss (1992) Proc Natl Acad Sci USA89:10847-10851) with regard to primary culture.

Prepare rough, half purification and purified virus mother solution

The preparation virus stocks is used for preliminary recombinant virus analysis or is used as the virus stocks of infectious titer preparation subsequently.Centrifugal removal cell membrane and nucleus or or by comprising other centrifugal step of sucrose preventing that prefiguration from reaching recombiant protein product and organelle and polluting, thereby can half purification vaccinia virus preparation.Used method is the modification method of following literature method: Earl etc., be stated from: Ausubel etc. (editor), (1991) Current Protocols in MolecularBiology, the 16.16.1-16.16.17 page or leaf, New York: Greene Publishing Associates andWiley Interscience; Earl and Moss, the same, the 16.17.1-16.17.16 page or leaf; Earl and Moss, the same, the 16.18.1-16.18.10 page or leaf; Bronte etc., (1997) Proc Natl AcadSci USA94 (7): 3183-3188.

Rough virus

In 175 square centimeters of tissue culture flasks, cultivate MVA with CEF or BHK-21 (deriving from ATCC), cultivate WR with HeLa or BS-C-1 (ATCC).Briefly, infect confluent monolayer cell with MVA or WR with the about 1pfu of infection multiplicity (moi).It is viral to suspend with the 10mlMEM that contains 2%FCS, and adding comprises in 175 square centimeters of culture bottles of confluent monolayer cell.After 1 hour, add the MEM that 20ml contains 2%FCS in 37 ℃ of incubations again.After 48-72 hour, infection cell is scraped in the culture medium, with 1500g centrifugation 5 minutes.For rough virus formulation, each 175 square centimeters of culture bottles 2mlMEM (2%) suspension cell.Frozen-thawed cell 3 times, supersound process and equal portions are packed in the 1ml cold storage pipe.One of freeze thawing represents equal portions, and virus concentration is determined in titration.Virus stocks is preserved in below-20 ℃.

Half purification preparation

Press described (Earl etc. in the past; Earl and Moss; 1991) results infection cell.After centrifugal, with PBS (2ml/175cm ²Culture bottle) resuspension cell is on ice, with tight glass stopper homogenizer (tight fitting glass dounce homogeniser) homogenate 30-40 time back and forth.Test under microscope cell breakage situation.With centrifugal 5 minutes of 300g (4 ℃), to remove nucleus, organelle and cell membrane, keep supernatant.With 1ml/175cm ²Culture bottle resuspension cell precipitation thing, and repeated centrifugation.Merge supernatant, be divided into equal portions and preservation.

The purification preparation

Press described (Earl etc. in the past; Earl and Moss; 1991) results infection cell, and use 10mM Tris.CI, pH9.0 (2ml/ culture bottle) resuspension cell, after this this program remains on sample on ice.With 10mM Tris homogenate as mentioned above.(Misonics is USA) with peak power output (500W) supersound process cell pyrolysis liquid 1 minute with the ultrasonic cup of XL2016.Sample was placed 1 minute on ice, repeat ultrasonic nearly 3 times.Ultrasonic maximum 5ml once, and in ultrasonic procedure, constantly replenish ice.Cell pyrolysis liquid is added to 17ml36% sucrose in the SW-27 centrifuge tube lightly, and (10mM Tris.Cl, layer pH9.0) go up and form layering.(32,900xg) the centrifuge cell lysate is 80 minutes with 13500rpm under 4 ℃ with the SW-27 rotor.Abandon supernatant, viral precipitate is resuspended among the aseptic PBS, and with cup type Ultrasound Instrument supersound process 1 minute (on ice).The equal portions concentrating virus is preserved in below-20 ℃.

Embodiment 2

Structure and characterized are expressed the recombinant viral vector of 5T4

Mus 5T4 gene and people 5T4 gene clone are gone in WR (pSC65) (Chakrabarti etc., (1997) Biotechniques23:1094-7) and MVA (pLW22) transferring plasmid, go into corresponding virus genomic target area to allow homologous recombination.

The recombinant MVA of expressing human 5T4 and Mus 5T4 and WR

Excise 1.4kb Mus 5T4 and 1.4kb people 5T4 (P.Stern PatersonInstitute Manchester provides) gene from pBSII-m5T4 (pBluescript (Stratagene) that contains 5T4cDNA) and pBSII-h5T4 (Myers etc., (1994) JBC269:9319-9324) respectively by EcoRI and BamHI restrictive diges-tion." mend and put down " the terminal flush endization that makes fragment with dNTP and archaeal dna polymerase.(the MVA transferring plasmid is made up of the promoter in late period morning (Chakrabarti etc., 1997) of MCS upstream, and vicinity is the VV7.5Kb LacZ box for detection of recombinant virus to make the flush end terminal fragment be cloned into pLW22; In Fig. 1 PmeI site and the Sma I site (b) (Chakrabarti etc., 1997) of pSC65 a).PLW22 and pSC65 control homologous recombination respectively and enter disappearance district II and tk gene.

When being intended for the human receiver's of immunity inoculation construction, remove the LacZ gene that is under the control of 7.5k promoter.As (Wyatt etc., (1996) Vaccine14:1451-1458) as described in the former document, utilize anti-5T4 monoclonal antibody to identify the reorganization plaque through the live body immunostaining.

CEF cell culture B.Moss (NIH with plaque purification clone, Bethesda, USA) the wild type MVA that provides, and it is identical separation strain (Sutter and Moss (1992) the Proc NatlAcad Sci USA89:10847-10851 for the preparation of the recombinant virus of describing in the past, Sutter etc. (1994) Vaccine12:1032-1040, Hirsch etc. (1996) J Virol70:3741-3752, Carroll and Moss (1995) Biotechniques19:352-355, Wyatt etc. (1995) Virology210:202-205, Sutter etc., (1995) FEBS Lett.371:9-12, Wyatt etc. (1996) Vaccine14,1451-1458, Carroll etc. (1997) Vaccine, 15:387-394, Carroll and Moss (1997) Virology238:198-211).B.Moss (NIH) provide WR mother solution, derives from ATCC separated strain (referring to for example Earl and Moss, 1991).With HeLa S3 cell (ATCC) preparation WR mother solution.

Method for the preparation of recombinant MVA virus is similar to described method (Carroll﹠amp in the past; Moss (1997) Virology238:198-211).Briefly: infect BHK-21 or CEF cell with the MVA mother solution with 0.1 infection multiplicity.With 100 μ ld.H ₂O is 2 μ g with plasmid DNA dilution, with its with use aseptic d.H ₂The O dilution is that the 30 μ g lipofection reagent (BRL) of 100 μ l mix.At room temperature incubation added lipofection reagent/dna solution at the infection cell that is covered on the Opti MEM after 10 minutes.Behind 37 ℃ of incubations 5 hours, the sucking-off cell culture medium added the MEM that contains 2%FCS again.Incubation is harvesting after 36 hours, in the expression of measuring β-gal on CEF or the BH k-21 cell in the presence of 5-bromo-3-indyl-D-tilactase.The plaque that separates be the plaque of other at least purification 3 times.Behind the plaque purification with CEF or a small amount of virus stocks of BHK-21 cell preparation.

The scheme that makes up reorganization WR is similar to scheme (Carroll and Moss (1995) Biotechniques19:352-355 of document description in the past; Earl and Moss, 1991).Briefly: recombinate the same with MVA.But be to use the BS-C-1 cell, and use contains the top-layer agar of the substrate of LacZ gene 5-bromo-3-indyl-D-tilactase, exist under the BrdU, with 143Btk-cell analysis reorganization plaque. use dimethyl diaminophenazine chloride to detect the negative spontaneous tk-virus of LAC Z to estimate viral homogeneity.

Method (the Carroll ﹠amp of document description before use is similar to; Moss (1997) Virology238:198-211), adopt h5T4 and m5T4 specific antibody by direct plaque immunostaining preliminary analysis Recombinant Protein Expression.Briefly: recombinant virus forms plaque at monolayer BS-C-1 cell, fixes with acetone/methanol, handles (HoleN and Stern PL, (1990) Int J Cancer45 (1): 179-184) with MAb5T4.Put together antibody and dianisidine substrate demonstration reorganization 5T4 protein expression with anti-mice HRP.Because MAb identifies comformational epitope, so under non-reduced condition, pass through the further characterized 5T4 expressing protein of western blot analysis.As seen from Figure 2, the 72kDa albumen of the suitable size of recombinant virus high level expression.Press Carroll ﹠amp; The dual immunostaining of the described usefulness of Moss (1997) Virology238:198-211 detects the homogeneity of mother solution.

Embodiment 3

Illustrate the animal model of the immune cross protection of people 5T4 and mice 5T4

For whether the 5T4 gene outcome of determining a kind of species can induce immunity to 5T4 in another species body, with Mus tumor model test recombinant poxvirus.Mouse model is B16 based on the chemical induction adenocarcinoma CT26 (Brittain etc., (1980) Cancer Res.40:179-184) in BALB/c source with derived from the melanoma of C57B6 mice.CT26 system and B16 equal stable conversion expressing human 5T4 and Mus 5T4.Mouse mainline (produce the lung tuberosity, CT26) or subcutaneous injection (CT26 and B16) to produce subcutaneous single tumor mass.

At the 0th, 21 and 42 day, to each group (7 every group) BALB/c mouse with 1 * 10 ⁷PfuIV or IM inoculation MVA-h5T4 (being called OBA1 at this 5T4 antigen) construction.Use 5 * 10 then ⁵The tumor cell intravenous of people 5T4 stable transfection is attacked mice.Attacked back 14 days, and took out the mice lungs, and counting lung tuberosity.

As a result 3

Result shown in Fig. 3 a and the 3b confirms, presents anti-tumor activity with the MVA-h5T4 mice immunized when being the CT26 attack with the homogenic tumor of expressing h5T4 albumen.The Mann-Whitney statistical analysis shows, compares with the mice of inoculation MVA-LacZ or PBS, and the protection behind the inoculation MVA-h5T4 is significant (p＜0.05).

Embodiment 4

With 10 ⁷The C57BL6 mice is respectively organized in pfuMVA-h5T4 (IV or IM) or MVA-Lac Z (IV) inoculation, and every group of 5 mices at interval 3 weeks, inoculate 2 times.With 5 * 10 ⁵The B16-h5T4 cell is attacked mice.The 2 days records in every interval tumor size behind the tumor challenge.Calculate each tumor area.

With 10 ⁷The C57BL6 mice is respectively organized in pfuMVA-m5T4 (IV or IM) or MVA-Lac Z (IV) inoculation, and 5 every group and 7 mices at interval 3 weeks, inoculate 2 times.With 5 * 10 ⁵The B16-m5T4 cell is attacked mice.The 2 days records in every interval tumor size behind the tumor challenge.Calculate each tumor area.

As a result 4

Fig. 4 a shows that the mice of inoculation MVA-h5T4 has tangible Graft Versus Tumor when B16-h5T4 attacks mice.The Mann-Whitney statistical analysis of Fig. 4 a data confirms that MVA-LacZ compares with inoculation, and the tumor suppression behind the inoculation MVA-h5T4 is tangible (p＜0.05).

Fig. 4 b and 4c show that when attacking mice with B16-m5T4, inoculation MVA-m5T4 has tangible Graft Versus Tumor.The Mann-Whitney statistical analysis of Fig. 4 c data confirms that MVA-LacZ compares with inoculation, and the tumor retardance behind the inoculation MVA-m5T4 is significant (p＜0.05).

Embodiment 5

With 5 * 10 ⁵CT26-h5T4 cell IV injects female BALB/c mouse.Set up huge lung tumor after 3 days.Behind inoculated tumour the 3rd and 10 day with 10 ⁷PfuMVA-Lac Z, MVA-h5T4 (every group of 10 mices) or PBS (one group of 5 mice) handle mice.Behind inoculated tumour 14 days with lungs dyeing and counting tumor.

As a result 5

As shown in Figure 5, the MVA-h5T4 processing has remarkable therapeutical effect to the CT26 lung tuberosity of the expression h5T4 of foundation.Statistical analysis (Mann-Whitney) shows, compares with MVZ-Lac Z or PBS, and the MVA-h5T4 treatment has significance (p＜0.05).

Embodiment 6

CT26-m5T4 and B16-m5T4 autoantigen model

With 1 * 10 ⁷PfuMVA-LacZ (n=3) or MVA-m5T4 (n=6) IV inoculation C57BL6 mice, 3 week inoculations are 2 times at interval.Last immunity 3 weeks of back are with 5 * 10 ⁵Express the subcutaneous attack of the B16 mice of m5T4.A situation arises to monitor subcutaneous (S.C.) tumor.

Fig. 6 shows that the tumor development speed of inoculation MVA-m5T4 mice is lower than the mice of accepting control vaccine.In addition, compare the gross tumor volume of m5T4 immune mouse average little 5 times (the 13rd day time little 10 times) with accepting the mice that MVA-LacZ handles.This protection feature is parallel with the m5T4 antibody response that these mices produce.

Embodiment 7

With 1 * 10 ⁷PfuMVA-LacZ (n=5) or MVA-m5T4 (n=6) IV inoculation BALB/c mouse, 3 week inoculations are 2 times at interval.Last immunity 3 weeks of back are with 5 * 10 ⁵The CT26 that expresses m5T4 attacks mice.Take out the mice lungs in 12 days behind the attack mice, count tumor nodule in blind method mode.

As a result 7

Fig. 7 shows that the tumor load of MVA-m5T4 immune mouse is lower than the mice of accepting the MVA-LacZ processing.This protection feature is parallel with the m5T4 antibody response that these mices produce.

Embodiment 8

The 5T4 antibody response of inducing in C57 and BALB/c mouse

As mentioned above, with MVA-m5T4 and MVA-h5T4 immune mouse, got blood examination in 10 days after each immunity inoculation and survey.

As a result 8

After twice inoculation, MVA-m5T4 can all overcome immunologic tolerance at BALB/c and C57B16 mice.In addition, with DNA, then excite mice not at the performance of the antibody response of m5T4 with MVA.

Therefore, the tumor model two kinds of Mus confirms that the MVA that immunity inoculation is expressed Mus 5T4 has protection feature to the homogenic tumor cell of expressing m5T4.These antitumor characteristics are parallel with anti-m5T4 immunne response (measuring with ELISA).In addition, the generation that confirms this immunne response can not cause autoimmunity toxicity these animals.

Embodiment 9

The immunne response at 5T4 of utilizing activator (primer) enhance immunity to obtain

Induce effectiveness to the immunity of 5T4 in order to estimate MVA and naked DNA carrier BALB/c and C57BL6 mice, not only with the naked DNA of encoding murine 5T4 and people 5T4 but also use the continuous agitation of MVA carrier and the booster shot mice.More particularly, at the 0th day with 1 * 10 ⁷PfuMVA-5T4 intravenous, 50g pCl-h5T4 (25g/ hind leg) or 25g pCl-m5T4 (12.5g/ hind leg) i inoculate mice, and (reinforcement) inoculated MVA-5T4 for the second time at the 21st day.Get blood at the 29th day from mice, measure antibody titer with ELISA.

As a result 9

The titre that observes (be defined as OD and be higher than MVA-LacZ background OD2 dilution factor doubly) sees the following form 2 and 3.

Table 2: the Mus 5T4 antibody titer of the MVA of inoculation expressing human 5T4 and Mus 5T4 and the mice of dna vector

Table 3: the people 5T4 antibody titer of the MVA of inoculation expressing human 5T4 and Mus 5T4 and the mice of dna vector

The result shows that using DNA:MVA excites: allos 5T4 antigen (h5T4) is strengthened or MVA:MVA excites: allos or homology 5T4 (H5T4 or m5T4) strengthen effectively producing high titre antibody.

Embodiment 10

The modification type 5T4 that is used for cancer immunotherapy

People 5T4 can be modified strengthening its immunogenicity, thereby more effective immunization therapy reaction can be induced.For this reason, use program " peptide is in conjunction with prediction " (the http://www-bimas.dcrt.nih.gov/cgi-of the K.Parker design of NIH

Bin/molbio/ken_parder_comboform) (referring to Parker, 1994J.Immunol.152:163 such as K.C.) identifies HLA CTL epi-position and modifies this class epi-position, strengthening the combination with the HLA molecule, thereby produces more effective CTL and induces.Following result derives from people 5T49mer (table 4) and Mus (table 5) 5T49mer:

Table 4: in conjunction with the people 5T49mer of HLA A0201

Sequence number	Initial	Sequence	Dissociate the half-life
				1	97	FLTGNQLAV	319.939
2	364	ALIGAIFLL	284.974
				3	351	SLQTSYVFL	176.240
4	368	AIFLLVLYL	137.482
				5	283	GLPHIRVFL	117.493
6	358	FLGIVLALI	110.379
				7	81	NLTEVPTDL	87.586
8	95	NLFLTGNQL	79.041
				9	222	FLYLPRDVL	63.174
10	373	VLYLNRKGI	56.754
				11	365	LIGAIFLLV	30.890
12	290	FLDNNPWVC	28.109
				13	301	HMADMVTWL	27.207

Table 5: in conjunction with the Mus 5T4 of people HLA A0201

Sequence number	Initial	Sequence	Dissociate
				1	307	YMADMVAWL	3680.892
2	81	NLLEVPADL	324.068
				3	97	FLTGNQMTV	319.939
4	370	ALIGAIFLL	284.974
				5	228	FLFLPRDLL	178.158
6	357	SLQTSYVFL	176.240
				7	374	AIFLLVLYL	137.482
8	289	GLAHVKVFL	117.493
				9	364	FLGIVLALI	110.379
10	379	VLYLNRKG	56.754

Embodiment 11

Mutation H5T4 is to strengthen in conjunction with HLA A0201

The peptide that derives from Parker shows in conjunction with the above-mentioned data of predictor, and when people AA sequence when 301 beginnings sport HMADMVTWL by YMADMVAWL from the position, producing the half-life of dissociating with HLA A0201 increases by 10 times 9mer.This binding affinity increases the CTL inducing properties (consulting Overwijk etc. in addition, 1998J ExpMed188:277-86) that obviously strengthens the 5T4 polypeptide.

As a result 11

In addition, h5T49mer sports 4 times of the half-life risings of dissociating that NLLEVPADL causes h5T49mer and HLAA0201 since 81 by NLTEVPTDL.

Embodiment 12

Toxicity research

There is not autoimmune toxicity

The purpose of this research is that research is induced possible effect at the autoimmune toxicity of 5T4 at the muroid model.

Experimental design

With 10 ⁷The vaccinia virus recombinant MVA intravenous inoculation of pfu expressing human 5T4 (MVA-h5T4) and Mus 5T4 (MVA-m5T4) is respectively organized BALB/c and C57 BL6 mice (every group of 5 mices).As negative control, with the MVA that expresses escherichia coli LacZ (MVA-LacZ) or PBS inoculation mice.

In 14 months, female BALB/c mouse inoculation is amounted to 4 times.In 14 months to C57BL6 mouse inoculation 3 times.Inoculation back blood sampling product are estimated the 5T4 specific antibody with ELISA.

12a as a result

Antibody response: after inoculating 2 times, inoculation MVA-m5T4 and MVA-h5T4 mice serum contain high-caliber anti-m5T4 and anti-h5T4 (see Table 2 and table 3) respectively.

Toxicity: the healthy sign of disease of observing mice every day.14 months any time in the past, the physical appearance of inoculation MVA-m5T4 or MVA-h5T4 animal does not have different with the animal of inoculation MVA-Lac Z or PBS really.Prepared two reports about animal health by qualified veterinary's preparation, these two reports show that all animals all shows health.

Sum up

The MVA (MVA-h5T4) that in 14 months each is organized the MVA (MVA-m5T4) of BALB/c and C57BL6 mouse inoculation expression m5T4 antigen or expresses h5T4 antigen is up to 4 times, and detection toxicity sign.Have the antibody of the anti-m5T4 of high titre although show mice, in 14 months, do not have ill sign.Qualified veterinary has identified these animals, finds that they do not have the disease sign, and illustrating does not have autoimmune toxicity.

Therefore, can induce antibody response at h5T4 and m5T4 respectively with MVA-h5T4 or MVA-m5T4 inoculation BALB/c or C57BL6 mice.This replying do not have deleterious effects to mice health.

Embodiment 12b

The 5T4 autoimmune is to the influence of reproductive performance

Placenta Hominis human body and Mus has been found 5T4.Therefore, the immunne response of anti-mice 5T4 may hinder the mice pregnancy or influence foetus health.We have carried out deep research to illustrate these problems.The purpose of this research is to estimate at the influence to gestation of the immunne response of m5T4 at BALB/c and C57BL6 female mice simultaneously.

Experimental design

With 10 ⁷PfuMVA-m5T4, MVA-LacZ or PBS organize female BALB/c and C57BL6 mice (5 every group) intravenous inoculation 3 times to each.Postvaccinal special time (the 10th, 30 and 60 day) makes the mice copulation the last time, and the ability of estimating its gestation and producing healthy young Mus.

12b as a result

(i) research of C57 BL6 mice

Show 6:10 days research

	MVA-m5T4X3	MVA-LacZ?X3	PBS
				The gestation number	4/5＝80％	4/5＝80％	3/5＝60％
Survival rates	24	30	18
				The young size of average tire	6	7.5	6

Show 7:30 days research

	MVA-m5T4X3
		The gestation number	4/5＝80％
Survival rates	22
		The young size of average tire	5.5

Show 8:60 days research

	MVA-m5T4X3
		The gestation number	5/5＝100％
Survival rates	33
		The young size of average tire	6.6

The (ii) research of BALB/c mouse

Show 9:10 days research

	MVA-m5T4X3	MVA-LacZ?X3	PBS
				The gestation number	5/5＝100％	4/5＝80％	4/5＝80％
Survival rates
		30	23	22
The young size of average tire					6	5.75	5.5
	Survival is to the number of wean	24＝80.0％	19＝82.6％	22＝100％
The F:M ratio					13:11	14:5	11:11
	Average weight	10.4g	11.2g	10.1g

Show 7:45 days research

	MVA-m5T4X3
		The gestation number	4/4＝100％
Survival rates	24
		The young size of average tire	6
Survival is to the number of wean	24＝100.0％
		The F:M ratio	15：9
Average weight	11.4g

Sum up

With MVA recombinant virus injection BALB/c and the C57 BL6 mice of expressing m5T4, and induce anti-m5T4 antibody response.Allow these mice copulation, the pregnancy rate that confirms these mices and farrowing rate and mice identical (seeing Table 6-10) with the contrast virus immunity.In addition, the health to young Mus before weaning does not influence.Therefore, inoculation MVA-m5T4 and induce the m5T4 antibody response really can be in the following areas mice to be produced adverse effect: (i) reproductive performance of BALB/c and C57 BL6 mice; The viviparity of (ii) living is produced number and body weight and the survival rate of the preceding young Mus of (iii) weaning.

Embodiment 12c

The distribution of 5T4 in health adult tissue

The purpose of this research is that independent assessment is carried out in the distribution in normal human tissue to 5T4.Studies show that of part past is lower than Placenta Hominis related organization more than 1000 times even find the 5T4 expression in the relevant normal structure of little blood vessel, and the normal structure of still expressing 5T4 also may become the target of the immunne response of inducing at this tumor antigen.Do not know that described dyeing is specific or the part cross reactivity of the described MAb of reflection.

Experimental design

Under the GLP condition, estimate tissue slice from 32 histological types of 3 different donors by qualified pathologist.With the dye frozen section of each tissue specimen of 3 variable concentrations 5T4 specificity Mab.

12c as a result

Showing of conclusive table 11, comprise that the 5T4 of tissue slice of all vitals of brain, CNS, liver and kidney is expressed as feminine gender.Dyeing sees several non-vital tissues a little less than some of one or more donor tissue.It should be noted that and in the tissue of dying from the cancer individuality, observe the part positive staining.

Although adopt studies show that of immunohistochemical analysis in the past, in " the little blood vessel of part " of some non-cancer organ, observe certain 5T4 dyeing, described non-cancer organ " shows as weak dyeing " and comprises: kidney (glomerule), bladder (epithelium), small intestinal (chorioepithelium), uterus (endometrial gland), cervix uteri (cervical gland) and skin (substrate epithelium), this independent studies shows, there is not 5T4 to express on the vitals cell, and the expression of some normal structure is low and dispersion, expresses because almost find 5T4 in the specimen of all 3 donors.Therefore, these data show, compare with other tumor antigen of part that uses in the immunotherapy of tumors test, and the tissue expression of 5T4 is strict more limited.So these discoveries have reconfirmed that such viewpoint: 5T4 is the good candidate antigen of cancer immunotherapy.

The summary of table 11:5T4 tissue distribution

¹By determining the 5T4 staining power with respect to the naked eyes analysis of placental trophoblast (+++) and negative control dyeing (-).

²Tissue available from the cancer patient.

Embodiment 13

The anti-tumor in vivo efficacy data of ScFv fusion rotein

The purpose of this research is the effectiveness of a series of scfv fusion proteins of test.

Experimental design

CT26 cell (CT26-h5T4) and the CT26-neo of expressing human 5T4

With PBS, LscFv-1, LscFv-2, B7-scFv, ScFv-Ig precincubation cell.

In bhk cell system, express LscFv-1 and 2.By its histidine mark purification LscFv-1, adopt filtration system purification scFv-2 at the nickel post.Be purification B7-scFv by the His labelling from bhk cell, by Filter column purification scFv-Ig.Be defined in the concentration of the every kind of scFv that uses in the experiment with saturated protein content in conjunction with CT26-h5T4 cell needs in FACS measures.

Make every kind of scFv precincubation of CT26-h5T4 and CT26-neo cell and saturation capacity, and incubation 1 hour.Behind the washed cell, at flank subcutaneous injection 5 * 105 cells of homogenic BALB/c mouse.

Measure a tumor size every 1 day and calculate gross tumor volume.

As a result 13

Fig. 8: CT26-neo

If be LscFv-1, there is not significant difference between each acceptance group, described acceptance group behind tumor inoculation 36 days is compared with the PBS contrast, and its tumor size reduces by 3 times.

Fig. 9: CT26-h5T4

Handle tumor with all scFv constructions tumor growth is had significant effect.Disappear in the 36th day tumor with 4 in 5 mices of scFv-1 processing.At the 36th day, it is littler more than 60 times than the tumor of handling with PBS that scFv-1 handles tumor.

When the mouse black-in tumor cell system (B16) of expressing h5T4 with engineering carries out similar experiment, there is not antitumor action.

Sum up

As if in a word, B7 or IgG and 5T4 specificity scFv merge does not have favourable effect in CT26 and B16 muroid model.Because its binding affinity height, independent scFv may more effective (shown in BIACORE, comparing with B7-scFV).So these data show that independent scFv has significant effect to the retardance tumor, and the immunostimulant molecule that does not need to merge with scFv in the 5T4 pattern is realized blocking the effect of tumor.

Claims (9)

1. the viral vector of the non-virus carrier of coding 5T4 antigen and coding 5T4 antigen is exciting and is strengthening application in the medicine of scheme of step for the preparation of causing in receiver's body comprising of immunne response, and wherein said non-virus carrier is used for exciting step and described viral vector is used for strengthening step.

2. the application of claim 1, wherein said viral vector is poxvirus vector.

3. the application of claim 2, described poxvirus vector are selected from viral ankara, vaccinia subgroup virus, fowlpox virus, penguin poxvirus or the entomopox virus carrier of improvement.

4. the application of claim 1, wherein said viral vector is selected from Venezuelan equine encephalitis virus, Alphavirus, herpesvirus vector and adenovirus vector.

5. vaccine combination, it comprises the non-virus carrier of coding 5T4 antigen and the viral vector of coding 5T4 antigen, they are used for comprising that the scheme that excites and strengthen step causes immunne response in receiver's body, and wherein said non-virus carrier is used for exciting step and described viral vector is used for strengthening step.

6. the vaccine combination of claim 5, wherein said non-virus carrier is the DNA plasmid.

7. the vaccine combination of claim 5, wherein said viral vector is poxvirus vector.

8. the vaccine combination of claim 7, wherein said poxvirus vector is selected from viral ankara, vaccinia subgroup virus, fowlpox virus, penguin poxvirus or the entomopox virus carrier of improvement.

9. the vaccine combination of claim 5, wherein said viral vector is selected from Venezuelan equine encephalitis virus, Alphavirus, herpesvirus vector and adenovirus vector.

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