CN1782072A - Vector - Google Patents

Abstract

A vector comprising a nucleotide sequence of interest (''NOI'') encoding a product of interest (''POI'') is described. The NOI and/or the POI is capable of recognizing a tumor, such that in use the vector is capable of delivering the NOI and/or the POI to the tumor.

Description

Carrier

The application is to be on June 4th, 1998 applying date, and application number is 98805529.5, and denomination of invention is divided an application for the application for a patent for invention of " carrier ".

The present invention relates to a kind of carrier that is preferred in the medical science.

As well-known in the art, carrier is the instrument that allows or promote a kind of entity is passed to from a kind of environment another kind of environment.As an example, some carrier that is used for recombinant DNA technology allows to be transferred to target cell such as the entity of DNA section (such as the allogeneic dna sequence DNA section, such as allos cDNA section).Optional is that in case in described target cell, this carrier then can be used for keeping described allogeneic dna sequence DNA in this cell, maybe can be as the unit of dna replication dna.The example that is used for the carrier of recombinant DNA technology comprises plasmid, karyomit(e), artificial chromosome or virus.

Therefore, carrier can be used for protein and/or nucleotide sequence are passed to target cell, such as tumour cell.

Yet well-known, nucleotide sequence and protein are complicated molecules, and they can be from biogenetic derivation production, and are the most common from genetic engineering biology or cell culture production.In addition, produce nucleotide sequence and method of protein be complicated, workload big and expend cost.And, pharmacological property and other function aspects and the nucleotide sequence of some protein (such as the immunoglobulin (Ig) that derives from non-human organism source) can usually be different from the activity of the corresponding natural human immunoglobulin (Ig) that produces in the human cell with important mode.Information as a setting, immunoglobulin (Ig) are members of relevant multimeric protein family, and they are under normal circumstances by the emiocytosis of vertebrate bone-marrow-derived lymphocyte pedigree, and its exemplary functions is to combine for nonself macromole district specificity with differentiating.On behalf of this biological immune, immunoglobulin (Ig) reply an important component of repertoire, with " antibody " synonym.

A major cause of this class activity difference is that the proteinic glycosylation pattern difference that derives from different plant species (is summarized in Bebbington 1995; Be stated from MonoclonalAntibodies:the second generation H.Zola and edit the 165-181 page or leaf).In addition, system gives protein (those protein that especially contain the toxin territory) and nucleotide sequence may be induced generation other pharmacokinetics and toxicological issues (summarize, be stated from the 2.1st chapter of Monoclonal Antibodies (Birch and Lennox edit)) in Scheinberg and Chapman1995.

Therefore, the present invention manages to provide improved carrier system, to be used to transmit purpose nucleotide sequence and/or described nucleotide sequence expressed products.

According to first aspect of the present invention, a kind of carrier is provided, it comprises the nucleotide sequence (" NS ") of one section codes for tumor interact protein (" TIP ") and can randomly comprise one section purpose nucleotide sequence (" NOI "), described NOI coding purpose product (" POI "); Wherein said TIP can discern tumour, makes that in use this carrier can be passed to tumour with described NOI and/or POI.

According to second aspect of the present invention, the method that the purpose product (" POI ") of purpose nucleotide sequence (" NOI ") and/or described NOI coding is passed to tumour is provided, wherein utilizes the carrier that comprises described NOI and/or express described POI that described NOI and/or POI are passed to described tumour; Wherein said NOI and/or described POI can discern tumour; Wherein described NOI and/or described POI are passed to described tumour; And wherein this carrier is according to carrier of the present invention.

According to the 3rd aspect of the present invention, provide carrier that the purpose product (" POI ") of purpose nucleotide sequence (" NOI ") and/or described NOI coding is passed to the purposes of tumour, wherein utilize the carrier that comprises described NOI and/or express described POI that described NOI and/or POI are passed to described tumour; Wherein when described NOI and/or described POI were passed to tumour, described NOI and/or described POI can discern described tumour; And wherein this carrier is according to carrier of the present invention.

According to the 4th aspect of the present invention, provide treatment to need the curee's of described treatment method, this method comprises that the purpose product (" POI ") with purpose nucleotide sequence (" NOI ") and/or described NOI coding is passed to the method for tumour, wherein utilizes the carrier that comprises described NOI and/or express described POI that described NOI and/or POI are passed to described tumour; Wherein said NOI and/or described POI can discern tumour; Wherein described NOI and/or described POI are passed to described tumour; And wherein this carrier is according to carrier of the present invention.

According to the 5th aspect of the present invention, provide the purposes that is passed to tumor mass inside of the therapeutic gene that genophore will encode TIP, the preferred tumour of described TIP conjugated protein (" TBP "), but more preferably excretory TIP (but being preferably excretory TBP).

According to the 6th aspect of the present invention, one or more gene targeting tumours that provide gene delivery system will encode TIP (being preferably TBP), described gene delivery system comprises genophore and the vivo gene transfer system of coding TIP (being preferably TBP).

According to the 7th aspect of the present invention, the treatment method for cancer is provided, comprise according to one or more TIP in the gene delivery system of the present invention (preferably TBP) or system or directly give tumor locus.

According to the 8th aspect of the present invention, a kind of gene delivery system is provided, be used for the coding TIP (preferably TBP) one or more genes or body in or the cytophyletic cell of external introducing hematopoiesis (preferably marrow hemopoiesis).

According to the 9th aspect of the present invention, the method for treatment mammalian cancer is provided, comprise and will give individuality that described gene delivery system can be expressed TBP in the cell that derives from hematopoiesis (preferably marrow hemopoiesis) source according to gene delivery system of the present invention.

According to the of the present invention ten aspect, a kind of genophore is provided, it comprises the therapeutic gene of one or more coding TIP (preferably TBP), and selectivity has the expression regulatory element of function functionally to be connected in described therapeutic gene and the cell type that exists in tumor mass.

According to the 11 aspect of the present invention, provide to comprise the extremely genophore of one or more therapeutic genes of described inside tumor to be passed, wherein said therapeutic gene coding TIP (preferably TBP), described TIP also contains one or more effector domains.

According to the 12 aspect of the present invention, the method of treatment mammalian cancer is provided, and this method comprises with a kind of cytokine or cytokine encoding gene with according to the combination of one or more TIP (preferably TBP) gene of the aforementioned either side of the present invention and gives individuality.

According to the 13 aspect of the present invention, provide TIP (preferably TBP) encoding gene is passed to tumor locus.

This carrier preferably comprises described NOI.

Another preferred aspect, the described POI of this vector expression.

Carrier of the present invention can be used in the particularly medical use, uses such as diagnosis or treatment.

Preferably, described NOI is that treatment NOI and/or described POI are treatment POI.

Sometimes in following text, described NS and NOI can be called or be referred to as gene separately.

Described NS and NOI can be any suitable nucleotide sequences.For example, they can be DNA or RNA independently, and they can synthesize preparation, maybe can utilize the recombinant DNA technology preparation, maybe can from natural resource, separate, and maybe can be the combination of these methods.Described NOI has adopted sequence or antisense sequences.

Multiple NS or NOI or its composition can be arranged, and described NS or NOI can directly or indirectly connect each other.Therefore, expressed product can have two or more effector domains (they can be identical or different) and/or two or more TIP territory (they can be identical or different).

Preferably, in use, this carrier can be passed to tumor mass inside with described NOI and/or described POI.

Except that cancer cells, the cell type that is present in the tumor mass includes but not limited to scavenger cell, lymphocyte, tumor infiltrating lymphocyte, epithelial cell etc.

Preferably, described NS and/or described TIP comprise at least one tumour in conjunction with the territory, and this territory can interact with at least a tumour relevant cell surface molecular (" TACSM ").

According to the present invention, described TACSM can include but not limited to cell surface molecule, and it works in growth of tumour cell, migration or in shifting; The adhesion protein acceptor is such as integrin glass connection protein receptor; Growth factor receptors is (such as Urogastron (EGF) acceptor, platelet derived growth factor (PDGF) acceptor, inoblast source somatomedin (FDGF) acceptor, trk C, rhIGF-1 (IGF-1) acceptor; The fibrinolysin activator; Metalloprotease (such as collagenase) 5T4 antigen; Tumour-specific carbohydrate part; Carcinomebryonic antigen; Saliva Orthana; Growth factor receptors; Glycoprotein; With the restricted antigen of its tissue distribution.

Preferably, described TACSM optionally expresses at a kind of cell type or on a limited number of cell type.

Preferably, in use, this carrier can be passed to described NOI and/or described POI the tumor locus of selection.

Described TIP is tumour conjugated protein (" TBP ") or to comprise a kind of tumour conjugated protein preferably.

Described TIP is TBP preferably.

The example of TBP comprises: adhesion molecule, such as isotype (particularly CD44v6, CD44v7-8), the CD56 of adhesion molecule ICAM-1, ICAM-2, LFA-1, LFA-2, LFA-3, LECAM-1, VLA-4, ELAM, N-CAM, N-cadherin, P-selection albumen, CD44 and variation thereof in the born of the same parents; The growth factor receptors part is such as Urogastron (EGF), platelet derived growth factor (PDGF), inoblast source somatomedin (FDGF), nerve growth factor, beta-hypophamine, Regular Insulin, rhIGF-1 (IGF-1), pHGF, nerve growth factor, human growth factor, brain derived growth factor, ciliary neurotrophic factor, glial cell line-derived somatomedin; From the heavy chain of immunoglobulin (Ig) (Ig) variable region and sequence of light chain (from the human and animal source), engineered antibody or from the antibody of phage display library.Phage display library is a kind of technology of expressing immunoglobulin gene in phage, has developed into the means that acquisition has the antibody of required binding specificity.Developed based on the expression system of phage with recently based on the expression system of filobactivirus.Can design described phage expression system, make it allow heavy chain and light chain to form combination at random, test it in conjunction with required antigenic ability.

Described TBP can contain an effector domain, and it is activated when described TBP is incorporated into TACSM.Described one or more effector domain can be activated when described TBP is incorporated into TACSM, causes suppressing propagation, survival or the propagation of tumour cell.Described effector domain can have enzymic activity (such as the prodrug activating enzyme), or this effector domain can comprise a kind of toxin or immunostimulant, such as above listed cytokine/lymphokine.

Preferably, described TBP comprise one or more can with the interactional territory that combines of one or more TACSM on being present in described cancer cells, described TACSM can be identical or different.

Term " interaction " comprises direct combination, because this in conjunction with producing biological effect.

Described TIP preferably a kind of antibody a part or comprise the part of antibody at least.

As everyone knows, antibody plays a crucial role in immunity system.In brief, immunity system operates in three kinds of different substantially modes: by humoral immunization, secretion by cellular immunization and the stimulatory protein(SP) by being called lymphokine.Humoral immunization depends on the albumen that is referred to as immunoglobulin (Ig), about 20% albumen in its formation blood.One immunoglobulin molecules is called an antibody, but that antibody also is used to refer to is many all at the differing molecular of identical target molecule.Humoral immunization also relates to complement, and this is to be activated a next histone that kills and wounds bacterium with binding antibody non-specificly.

In cellular immunization, complete cell is responsible for recognition reaction and is got rid of reaction.The first line of defence of body is identification and kills and wounds microorganism by phagocytic cell that phagocytic cell is the cell that specialization is used to take in and digest undesired material.These cells comprise neutrophilic granulocyte and scavenger cell.The keying action of antibody is to help scavenger cell identification and destroy allogenic material.

In order to carry out these functions, antibody is divided into two zones: with the combining (Fab) territory and transmit signal with initial effect such as phagocytotic process (Fc) territory of AI.Each antibody molecule is made up of two class polypeptide chains: light (L) chain and heavy (H) chain.Single antibody has L chain and two H chains of two identical copies.The N-terminal territory of every chain forms the variable region, and it constitutes antigen binding site.The C-terminal territory is called constant region.H chain (V _H) and L chain (V _L) variable domain form Fv unit, can interact closely, to form strand Fv (ScFv) unit.In most of H chains, find hinge area.Hinge area has flexibility, allows the rest part free movement of Fab land with respect to this molecule.Hinge area also is position the most responsive to the effect of proteolytic enzyme on this molecule, and described proteolytic enzyme can be fractured into antibody antigen binding site (Fab) and effect (Fc) district.

The structure of the structural domain of antibody molecule is suitable for protein engineering, promotes to carry the exchange between the functional domain molecule of antigen-binding activity (Fab and Fv) or effector function (Fc).The structure of antibody also makes it easily to produce to have the antibody that is incorporated into such as the antigen recognition ability of the molecule of toxin, lymphocyte or somatomedin.

Monoclonal antibody is the antibody with the specific homogeneous of same antigen, represents the product of antibody producing cells monospecific polyclonal.People recognize that monoclonal antibody provides the basis of human treatment's product.Yet although mouse antibodies is similar to people's antibody, it is the external source object that their difference is enough to by immune system recognition, produces immunne response thus.This human anti-mouse antibody (HAMA) replys and has limited the availability of mouse antibodies as the human therapy product.

The chimeric antibody technology relates to be transplanted to whole mouse antibodies variable domain on people's antibody constant domain.The immunogenicity of chimeric antibody is lower than mouse antibodies, but they keep its antibodies specific, and demonstrates HAMA and reply reduction.

In chimeric antibody, the variable region keeps the variable region of mouse fully.Yet the structure of this antibody makes may produce the suitable variable region of specificity, and they mainly are human on the source.The antigen binding site of antibody is formed by 6 complementary determining regions (CDR) of heavy chain and light chain variable part.Each antibody structure territory is made up of 7 antiparallel beta sheets, forms the beta sheet bucket with the ring that connects beta chain.CDR district in these rings.It is feasible to another support beta sheet bucket more CDR and relative specific thereof being arranged from a support beta sheet bucket.This is called CDR and transplants.In the CDR grafted antibody research clinical study in early days, its immunogenicity as if do not have or mouse or chimeric antibody strong like that.In addition, beyond CDR, can make sudden change, so that improve it, as in the so-called humanized antibody in conjunction with activity.

Fab, Fv and strand Fv (ScFv) fragment with VH and VL that connects by peptide linker show antigen-specific similar to original monoclonal antibody and affinity.Can be with being connected to or the non-antibody molecule of N-terminal or C-terminal produces the ScFv fusion rotein.In these molecules, the molecular guide that Fv can be used for specifically being connected is expressed the cell of suitable antigen.Also can by with two kinds of different binding specificities engineered be single antibody chain, the generation bifunctional antibody.Difunctional Fab, Fv and ScFv antibody can comprise engineered structural domain, transplant territory or humanization territory such as CDR.

In the enzymotherapy (VDEPT) that virus instructs,,, foreign gene is passed to normal cell or cancer cells such as retrovirus vector by virus vector.Described foreign gene a kind of enzyme of encoding, this enzyme can be converted into toxic metabolites (5 FU 5 fluorouracil) with nontoxic prodrug (for example 5-flurocytosine), and the latter kills and wounds the cell (1994 Ann New YorkAcad Sci 71b:115-124 such as Sikora) of making it.If used promotor is a tumour-specific, then this toxic product is only synthetic in described tumour cell.Research in animal model shows, the treatment of the type can be transmitted than ordinary method and be Duoed prodrug (Connors and Knox 1,995 1995 Stem Cells 13:501-511) up to 50 times.A kind of version of this technology uses the TAA of puting together with the prodrug saccharase, so that the specificity transmission of guiding tumour to be provided.This method is called antibody pacemaker enzyme prodrug therapy (ADEPT) (the 45th page of Maulid S and PatelSD " Molecular Biotechnology " 1997 Wiley-Liss Inc).

Known a large amount of monoclonal antibody and immunoglobulin-like molecular specificity ground are in conjunction with being present in the lip-deep antigen of particular cell types (such as tumour cell).Set up the method for identification, characterized, clone and engineered these molecules well, for example adopted hybridoma, phage display library and the scFv library that derives from mouse or transgenic mice.The gene of coding immunoglobulin (Ig) or immunoglobulin-like molecule can be expressed in multiple heterologous expression system.The big glycosylated protein that comprises immunoglobulin (Ig) is effectively from eukaryotic cell (particularly mammalian cell) secretion and assembling.

Can be such as Fab, Fv or the segmental little non-glycosylated fragment of scFv with functional form production in mammalian cell or bacterial cell.

Immunoglobulin (Ig) or immunoglobulin-like molecule can derive from human antibodies, or derive from engineered humanization rodent animal antibody, such as CDR grafted antibody, maybe can derive from phage display library, maybe can be synthetic immunoglobulin-like molecules.

Antigen binding domain can comprise by the heavy chain of the immunoglobulin (Ig) of isolating genetic expression and light chain, maybe can use a kind of light chain of immunoglobulin (Ig) and a kind of heavy chain of brachymemma, forms Fab or F (ab) ' ₂Fragment.Perhaps, can use the heavy chain and the light chain of clipped form, assembling forms the Fv fragment.Also can use engineered scFv fragment, in this case, only need the individual gene coding for antigens in conjunction with the territory.One preferred aspect, form described antigen binding domain by Fv or scFv.

When pathogenic agent was invaded body, lymphocyte was replied with three types reaction.The lymphocyte of humoral system (B cell) secretion can send signal and make scavenger cell or other cell with this pathogenic agent degraded in conjunction with the antibody of this pathogenic agent.The lymphocyte of cell system (T cell) is carried out the function of two kinds of main types.Cytotoxic T lymphocyte (CTL) develops the ability of the cell that Direct Recognition and kill and wound pathogenic infection.Helper cell (TH cell) is discerned this pathogenic agent independently, and justacrine protein factor (lymphokine) stimulates the growth and the responsiveness of B cell, T cell and scavenger cell, has strengthened the ability of immunne response thus greatly.

Therefore, one preferred aspect, described TIP comprises a kind of immunoglobulin (Ig) or its part or its bioisoster.

In a preferred embodiment, described TIP comprises IgG and/or IgE or its part or its bioisoster.

In a more preferred, described TIP comprises IgE or its part or its bioisoster.

Described TIP preferably discerns trophocyte's surface antigen.

Described TIP preferably discerns 5T4 antigen.

Trophocyte's surface antigen limits (Hole and Stern1988 Br.J.Cancer 57:239-246) with singly filling grand antibody 5T4 at first, high level expression on miscellaneous human cancer cell (1994 J.Biol.Chem.269:9319-9324 such as Myers), but in the individual healthy tissues of non-gestation, basically be limited to low expression level in minority specialization epithelium (Myers etc., ibid and the reference of wherein quoting).Related to 5T4 antigen aspect the metastatic potential causing developing, so the antibody of this molecule of specific recognition is expressed in this antigenic tumour in treatment and may be had clinical cognation.

Also can be by multiple technology known in the art, with the variable domain humanization of 5T4 monoclonal antibody, described technology comprises to be transplanted to the CDR region sequence on the human skeleton.These technology are used for making up complete humanized antibody or Humanized single chain antibody (Sab) then, such as the ScFv that is coupled to the Fc district (referring to Antibody Engingeering:a practical approach, McCafferty etc. edit 1996 OUP).

Here, term Sab is not limited to the only mankind or Humanized single chain antibody.Yet the preferably human single-chain antibody of described Sab or Humanized single chain antibody or its part are such as the ScFv that is coupled to the Fc district.

Described NS and NOI and/or described TIP and POI preferably link together.

Described TIP and POI preferably directly link together.

Any among described NS, NOI, TIP and the POI or multiplely preferably also comprise at least a other function ingredients.

Preferably described at least TIP and/or POI also comprise at least a other function ingredients.

Following any or multiple of preferably described other function ingredients: signal entity (such as signal peptide), immunostimulant, toxin or bioactive enzyme.

One preferred aspect, described POI is secretor type POI.Therefore, aspect this, described other function ingredients is preferably at least a can to cause described POI excretory entity, such as the signal entity of the present invention.

Another kind of preferred component in addition is a promotor.

Term " promotor " uses with the normal implication of this area, for example is the RNA polymerase binding site in the Jacob-Monod genetic expression theory.

This carrier preferably comprises a tumor-specific promoters enhanser.

Other preferred component in addition comprise can the described POI of effective expression entity.For example, described other component can be an enhanser.Here, the term enhanser comprise with other protein ingredient of transcription initiation mixture in conjunction with, and promote its promotor that links to each other to instruct the dna sequence dna of transcription initiation thus.

This carrier preferably be used for described NOI and/or POI is external and/or body in be passed to described tumour.

Carrier of the present invention can be used in the gene therapy, is used for described NOI and/or described POI are passed to the selection site.

Gene therapy comprises any or multiple: one or more nucleotide sequences such as adding in one or more target sites (such as target cell), displacement, disappearance, interpolation, operation for example.If described target site is a target cell, then described cell can be the part of tissue or organ.In Molecular Biology (Robert Meyers edits, and Pub VCH is such as the 556-558 page or leaf), find the general content of gene therapy.

As an example again, gene therapy also provides a kind of means, utilizes this means, and is any or multiple: nucleotide sequence (such as gene) can be used for displacement or add a kind of defective gene; Can eliminate a kind of Disease-causing gene or gene product; Can add a kind of new gene, for example the phenotype that is more suitable for generation; Can be on molecular level manipulating cells, with treatment cancer (Schmidt-Wolf and Scmidt-Wolf, 1994, Annals of Hematology 69:273-279) or other illness, such as immune, cardiovascular, neural, inflammation or communicable disease; Can operate and/or introduce antigen, to elicit immunne response, such as gene vaccine.

Carrier of the present invention can be virus vector or non-virus carrier.Non-viral system includes but not limited to the DNA infection protocol.Here, transfection comprises that the employing non-virus carrier is passed to gene the method for Mammals target cell.Typical infection protocol comprise the transfection of electroporation, the biological emission of DNA (biolistics), lipid mediation, fine and close (compacted) DNA mediation transfection, liposome, immunoliposome, lipofectin reagent, cationics mediation, cationic surface amphiphile (CFA) (Natue Biotechnology 1996 14:556) and their combination.The viral system includes but not limited to adenovirus carrier, adeno associated virus (AAV) carrier, herpesvirus vector, retrovirus vector, lentiviral vectors, baculovirus vector.The example of other carrier comprises external transfer system, and they include but not limited to the DNA infection protocol, such as the transfection of the transfection of the biological emission of electroporation, DNA, lipid mediation, fine and close DNA mediation).

This carrier is virus vector preferably.

This carrier is retrovirus vector preferably.

In the nearest several years, proposed retrovirus is used for gene therapy.Retrovirus is RNA viruses basically, and its life cycle is different from lytic virus.Aspect this, when the retroviral infection cell, its genome is converted into dna form.Say slightly at length that more retrovirus is a kind of virus that contains geneome RNA, when entering host cell, its geneome RNA is converted into dna molecular by ThermoScript II.This DNA copy produces the albumen of the essential encoding viral of new rna gene group and infectious virus particle assembling as template.Therefore, retrovirus is the infectivity entity that duplicates by the DNA intermediate.

Many retroviruss are arranged, and the example comprises: muroid leukosis virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney muroid leukosis virus (Mo-MLV), FBR muroid osteosarcoma virus (FBR MSV), Moloney muroid sarcoma virus (Mo-MSV), Abelson muroid leukosis virus (A-MLV), birds myelocytomatosis virus-29 (MC29) and birds protoerythrocyte hyperplasia virus (AEV).

In (" Retroviruses " 1997 Cold Sprig Harbour LaboratoryPress edit: JM Coffin, SM Hughes, HE Varmus 758-763 page or leaf) such as Coffin, can find the table look-up that retrovirus is detailed.

Can find the details of some retrovirus genome structure in the art.As an example, can find the details of HIV from NCBI Genbank (being genome registration number AF033819).

All retroviruss contain three main encoding domains, i.e. gag, pol, env, the essential virosome albumen of they codings.Yet retrovirus is divided into two classes widely: i.e. " simply " and " complexity ".The difference of these classifications is its genome structure.Simple retrovirus only carries this essential information usually.On the contrary, Fu Za retrovirus is also encoded and is derived from the other adjusting albumen of multiple montage information.

Retrovirus even can also be divided into 7 groups.Wherein represent retroviruss for 5 groups with carcinogenic potential.All the other 2 groups is slow virus and spumavirus.The summary of these retroviruss comes across " Retroviruses " (1997 Cold Spring Harbour Laboratory Press edit: JM Coffin, SM Hughes, HE varmus 1-25 page or leaf).

All carcinogenic members except that human T-cell leukemia virus-bovine leukemia virus group (TLV-BLV) are simple retrovirus.HTLV, BLV and slow virus and spumavuruses are complicated retroviruss.The most carcinogenic retrovirus of some research is Rous sarcoma virus (RSV), mouse mammary tumour virus (MMTV) and muroid leukosis virus (MLV) human T-cell leukemia virus (HTLV).

Slow virus group even be further divided into " primates " and " non-human primate " slow virus.The example of primates slow virus comprises human immunodeficiency virus (HIV) (being the virulence factor of human autoimmune deficit syndrome (AIDS)) and simian immunodeficiency virus (SIV).The non-human primate Slow virus group comprises prototype " slow virus " evil spirit enemy's virus (VMV) and relevant arthritis-Encephalitis virus (CAEV), equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV) and the bovine immunodeficiency virus of describing recently.

Difference between slow virus family and other type retrovirus is that slow virus has the ability (1992 EMBO.J 11:3053-3058.Lewis such as Lewis and Emerman 1994 J.Virol.68:510-516) that infects somatoblast and Unseparated Cell.On the contrary, can not infect Unseparated Cell such as other retrovirus of MLV, such as the cell of configuration example such as muscle, brain, lung and liver organization.

Between period of infection, retrovirus is attached to specific cell surface receptor at first.When entering the susceptibility host cell, the ThermoScript II of the encoding viral that retrovirus rna gene group is then carried by parental virus copy is DNA.This DNA is transported to host cell nuclear, is integrated into host genome subsequently in nuclear.In this stage, it is commonly referred to provirus.During cell fission, provirus and is transcribed with other cell protein is the same stably in host genome.The provirus coding is made required albumen and the packaging machine of more viruses, and provirus can leave cell by the process that is sometimes referred to as " blastogenesis ".

As what shown, each retrovirus genome comprises the gene that is called gag, pol and env, their coding virosome albumen and enzymes.The flank at these gene two ends is for being called the zone of long terminal repeat (LTR).LTR is responsible for proviral integration and transcribes.They also work as enhanser-promoter sequence.In other words, LTR can control the expression of virogene.The dressing shell of retrovirus RNA takes place by means of the psi sequence that is positioned at viral genome 5 ' end.

LTR itself is identical sequence, and they can be divided into three kinds of elements, and they are called U3, R and U5.U3 derives from described RNA 3 ' and holds distinctive sequence.R is obtained from the tumor-necrosis factor glycoproteins at described RNA two ends, holds distinctive sequence and U5 derives from described RNA 5 '.In different retroviruss, the size of these three kinds of elements can noticeable change.

For easy to understand, Fig. 5 has provided the genomic simple general diagram of retrovirus (drawing not in scale) that shows LTR, gag, pol and env essential characteristic.

For viral genome, transcription initiation site is arranged in the boundary between left hand end LTR (as implied above) U3 and the R, and poly A interpolation (termination) site is arranged in the boundary between right hand end LTR (as implied above) R and the U5.U3 contains the most transcriptional control element of provirus, and they comprise promotor and pair cell albumen and a plurality of enhancer sequence of in some cases the virus transcription activator being replied.Some retrovirus has any or multiple following gene: tat, rev, tax and rex, their codings participate in the albumen of the adjusting of genetic expression.

Shown in above diagram, the basic molecular structure of retrovirus rna gene group is (5 ') R-U5-gag, pol, env-U3-R (3 ').In the retrovirus vector genomic gene, gag, pol and env do not exist or do not have a function.The Zone R at these RNA two ends is tumor-necrosis factor glycoproteinss.On behalf of rna gene group 5 ' and 3 ', U5 and U3 hold distinctive sequence respectively.This three group ends sequence forms the long terminal repeat (LTR) in the proviral DNA, and proviral DNA is the genome form that is integrated in institute's cells infected genome.The LTR of wild-type retrovirus is made up of (5 ') U3-R-U5-(3 '), so U3 and U5 all contain for provirus and integrate important sequence.Must sequence comprise the primer binding site of the first chain reverse transcription, the primer binding site and the packaging signal of the second chain reverse transcription for required other of correct function in the genome.

For structure gene gag, pol and env itself and more detailed slightly details, the internal structure albumen of gag coding virus.Gag is processed as maturation protein MA (matrix), CA (capsid), NC (nucleocapsid) by proteolysis.The gene pol ThermoScript II (RT) of encoding, it contains archaeal dna polymerase and the relevant active and intergrase (IN) of RNA enzyme H, the duplicating of its mediated gene group.Surface (SU) glycoprotein of gene env coding virosome and stride film (TM) albumen, their form the mixture with the special looks mutual effect of cell receptor protein.This interaction finally causes the fusion of viromembrane and cytolemma.

Envelope protein is a kind of viral protein, and it wraps by virion, and plays a major role allowing virus to enter in the target cell.The envelope glycoprotein mixture of retrovirus comprises two peptide species: a kind of outside glycosylation hydrophobic polypeptides (SU) and a kind of transmembrane protein (TM).These albumen form oligomeric " protuberance " or " projection of protuberance " on the virosomal surface together.Two peptide species are encoded by env, and synthetic with the form of polyprotein precursor, and described polyprotein precursor is cut by proteolysis during it is transported to cell surface.Although uncut Env albumen can be incorporated into described acceptor, the cutting incident itself is that to activate this proteic fusion potentiality necessary, and that fusion is that virus enters host cell is necessary.Usually, SU and TM albumen all in a plurality of sites by glycosylation.Yet, in some virus, be example with MLV, TM is not glycosylated.

Although the assembling of the Virosome particles of coating does not always need SU and TM albumen, with regard to this point, they play basic role really in the process of entering.Aspect this, the SU territory is incorporated into the acceptor molecule on target cell surface, normally the specific receptors molecule.It is believed that this binding events activates the TM protein film and merges inducing potential, after this viromembrane and cytolemma merge.In some virus, MLV particularly, it is believed that cutting incident that the short part that causes TM kytoplasm tail is removed in exposing these proteic whole fusion-activity, play a crucial role (1994 J.Virol.68:4602-4627 such as Brody, 1994 J.Virol.68:1773-1781 such as Rein).Still on the viromembrane inboard, its its length in different retroviruss is significantly different for this kytoplasm " tail " in TM TMD distally.

Therefore, the specificity of SU/ acceptor interaction can limit the host range and the tissue tropism of retrovirus.In some cases, this specificity can limit the transduction potentiality of recombinant retrovirus carrier.Here transduction comprises the method that non-viral gene is passed to target cell with virus vector.For this reason, MLV has been used in many gene therapy experiments.Specific MLV with the envelope protein that is called 4070A is called as amphophilic virus, and it also can infect the human cell, because its envelope protein " docks " with the phosphoric acid translocator of guarding in people and mouse.This translocator is ubiquitous, so these viruses can infect many cell types.Yet from the angle of security, this is useful for limit target cell specifically in some cases.For this reason, engineered array mouse parent preferendum retrovirus, they infect specific human cell specifically with normally only its amphophilic retrovirus of infecting mouse cell is different.The erythropoietin section replaces the envelope protein segment, produce recombinant Retroviruses, described recombinant Retroviruses is incorporated into the human cell of its surface expression erythropoietin receptor specifically, such as red corpuscle precursor (the 45th page of Maulik and Patel 1997 " MolecularBiotechnology:Therapeutic Applications and Strategies " 1997.Wiley-Liss Inc.).

Except that gag, pol and env, complicated retrovirus also contains " assisting " gene, and they edit accessory protein.Accessory protein be defined as by beyond common replicator or structure gene gag, pol and the env encoded protein, by the auxiliary gene encoded protein.These accessory proteins are different from the albumen of the adjusting that participates in genetic expression, as by tat, rev, tax and rex encoded protein.The example of auxiliary gene comprises one or more among vif, vpr, vpx, vpu and the nef.These auxiliary genes for example can be found in HIV (referring to editors such as for example " Retroviruses " Coffin, Pub.CSHL 1997 the 802nd and 803 page).Nonessential accessory protein can work in the cell type of specialization, provide that cell protein provides to the small part repeat function.Usually, auxiliary gene just in the env downstream, comprises U3 district or env and the lap each other of LTR between pol and env.

The regulation mechanism of complicated retrovirus has been evolved, and described regulation mechanism is used the transcription activating protein and the cell transcription factor of encoding viral.The viral protein of these trans-actings is as the activator of the rna transcription that is instructed by LTR.The trans-activator that slow virus is transcribed is by viral tat genes encoding.Tat is incorporated into stable stem-ring RNA secondary structure, is called TAR, and its a kind of function is to make the apparent best orientation that goes up of Tat, transcribes with trans-activation.

As discussed previously, proposed that retrovirus as transfer system (perhaps expressing as transmitting carrier), particularly is passed to one or more purposes site with NOI or a plurality of NOI.This transfer can be in vivo, external generation, or in vivo or external combination take place.When using in this mode, retrovirus is commonly referred to retrovirus vector or recombinant retrovirus carrier.Retrovirus vector has been used for studying the retrovirus all respects of life cycle, comprises acceptor use, reverse transcription and RNA packing (by Miller summary, 1992 Curr TopMicrobiol Immunol 158:1-24).

Be used for the typical recombinant retrovirus carrier of gene therapy, can removing one or more the part of gag, pol and env protein-coding region at least from virus.This makes that retrovirus vector is a replication defect type.The part of removing even can replace with a kind of NOI so that produce the virus that its genome conformity can be gone into host genome, but the viral genome of wherein modifying itself is irreproducible owing to lack structural protein.In the time of in being integrated into host genome, the expression of described NOI taking place, causes for example treating effect.Therefore, pass through usually: NOI is integrated in the recombinant viral vector; The virus vector of modifying is packaged in the virosome shell; And the transduction in permission purpose site (such as target cell or targeted cell population), and described NOI is transferred to the purpose site.

Breeding also separates a large amount of retrovirus vectors (for example to prepare the retrovirus vector of suitable titre), to adopt the combination of package cell line or auxiliary cell line and recombinant vectors, is used for the transduction in follow-up for example purpose site, and this is possible.

In some cases, breeding and separates gag, pol and the env gene isolation that may need retrovirus and is introduced it respectively in the host cell, with generation " package cell line ".Described package cell line produces the required albumen of packing retrovirus DNA, but can not cause the dressing shell owing to lack the psi district.Yet when the recombinant vectors that will carry NOI and psi district was introduced package cell line, accessory protein can comprise the positive recombinant vectors of psi, produces the recombinant virus original seed.This can be used for cells infected, so that described NOI is introduced in the cellular genome.Its genome lacks the recombinant virus of making all required genes of viral protein, only can infect once, and is irreproducible.Therefore, described NOI is introduced the host cell gene group, and do not produce potential deleterious retrovirus.In " Retroviruses " (1997 Cold Spring HarbourLaboratory Press edit: JM Coffin, SM Hughes, the 449th page of HE Varmus), summarized available packing system.Yet, be not always in its titre level on the meaning of satisfactory level and say that this technology has problem.Yet, developed the design of retrovirus package cell line, to solve the problem of the spontaneous generation helper virus that particularly previous design runs into usually.Because homology promotes reorganization greatly, so reduce or the homology eliminated between this carrier and the helper virus genome has reduced the problem that helper virus produces.

Recently, develop packing cell, wherein in independently being transfected into the isolating expression plasmid of package cell line, carried gag, pol and env encoding viral district, made the generation of wild-type virus need three recombination event.This strategy is sometimes referred to as three plasmid transfection methods (1995 Nucl.Acids Res.23:628-633 such as Soneoka).

When the development carrier, transient transfection also can be used for measuring the generation of carrier.Aspect this, transient transfection avoids producing the required long period of stable carrier production clone, if this carrier or retrovirus packing composition pair cell are toxic, then uses transient transfection.The component that is commonly used to produce retrovirus vector comprises the proteic plasmid of coding Gag/Pol, the coding proteic plasmid of EnV and contains the plasmid of NOI.The generation of carrier comprises to be gone into to contain in the cell of other required component with one or more transient transfections in these components.If the gene that this vector encoded virulent gene or coded interference host cell duplicate, gene such as cell cycle inhibitor or coding cell death inducing, then may be difficult to produce stable carrier production clone, but transient transfection can be used for producing carrier before necrocytosis.In addition, utilize transient transfection, developed the carrier titre level of generation and the clone of being on close level (Pear etc. 1993, PNAS 90:8392-8396) of the carrier production clone that gets self stabilization.

The proteic toxicity of some HIV may make to be difficult to produce the stable packing system based on HIV that because this toxicity, the transient transfection by carrier and helper virus prepares the HIV carrier usually.Some worker has used the Env albumen of vesicular stomatitis virus (VSV) to replace the Env albumen of HIV.The proteic insertion promotion of VSV Env carrier concentrates as the HIV/VSV-G carrier, and by transient transfection, its titre is 5 * 10 ⁵(after concentrating is 10 ⁸) (1996Science 272:263-267 such as Naldini).Therefore, the transient transfection of HIV carrier can provide the strategy of usefulness, with the carrier (1994 PNAS.91:9564-9568 such as Yee) that produces high titre.

If described retrovirus component comprises the env nucleotide sequence, then this sequence all or part of can be randomly with all or part of replacement of another env nucleotide sequence.Replacing this env gene with allos env gene is to be called the technology of pseudotyping (Pseudotyping) and an example of strategy.Pseudotyping is not a kind of new phenomenon, can find the example among the 841-847 at 1997 Cell 90 such as WO-A-98/05759, WO-A-98/05754, WO-A-97/17457, WO-A-96/09400, WO-A-91/00047 and Mebatsion.

Pseudotyping can provide one or more advantages.For example, for lentiviral vectors, may limit these carriers based on the env gene product of the carrier of HIV and only infect and express the proteic cell that is called CD4.If but the env gene in these carriers has used the env sequence from other RNA viruses to replace, then they have the scope that infects widely (Verma and Somia 1997Nature 389:239-242).As an example, the worker used will be based on the carrier pseudotyping (ibid for Verma and Somia 1997) of HIV from the glycoprotein of VSV.Perhaps, can modify env, with its specificity of influence (such as changing).

Therefore, a kind of entity (such as dna molecular) described in term " recombinant retrovirus carrier ", this entity contains enough retrovirus sequences, with the rna transcription thing that allows this carrier in the presence of essential retrovirus is proteic, be packaged as can target cell infection counter-transcription-ing virus particle.The infection of target cell comprises reverse transcription and is integrated in the target cell genome.

Term " recombinant retrovirus carrier " also comprises the counter-transcription-ing virus particle that contains by the rna gene group of described dna molecule encode.Described retrovirus vector also contains non-viral gene, by this carrier described non-viral gene is passed to target cell.Recombinant retrovirus carrier can not duplicate independently and produce the infectious retrovirus particle.Usually, the retrovirus vector of reorganization lacks other gene that functional gag-pol and/or env gene or coding duplicate desirable proteins.

Term " directed retrovirus vector " is meant that its cells infected or the ability of expressing are limited to the recombinant retrovirus carrier of some cell type of host living beings in target cell.An example of directed retrovirus vector is the directed retrovirus vector of genetic modification envelope protein, and the envelope protein of wherein said genetic modification is incorporated into the cell surface molecule of only finding in the finite population cell type of host living beings.Another example of directed retrovirus vector is a kind of like this carrier, and it contains one or more retrovirus transcripts of permission only expression promoter and/or enhancer element in a certain proportion of cell type of host living beings.

Therefore, the invention provides a kind of useful transfer system.This transfer system can be with NOI and/or POU guiding tumour, and NOI and/or POI are gone back to the nest in the tumour.

This carrier can be used for directly giving entity (such as biological or its cell), such as giving in external or the body.On this meaning, this carrier can directly be passed to for example tumor locus.Perhaps, this carrier can pass through a kind of carrier, such as giving a kind of entity in external or the body.An example of carrier is the liposome or the cell that wherein can contain this carrier.An example of stable carrier cell is a hematopoietic cell, such as medullary cell.These carrier cells can improve the another specificity of this carrier of the present invention.

To further describe these aspects of the present invention and others.

The visible potentiality based on monoclonal antibody therapy that are used for the treatment of tumor disease are not recognized (to summarize in Scheinberg and Chapman 1995 Monoclonal anitibodies (Birth and Lennox edit) the 2.1st chapter as yet fully; George etc., 1994 Immunol.Today 15:559-561).As a result, monoclonal antibody and radio isotope, cytotoxic drug or toxin are puted together, render a service to attempt to improve.Yet, produce disappointed result usually with the clinical experiment of this class conjugate.Antibody and antibody conjugates lack one of them major cause of rendeing a service in the treatment solid tumor, be immunoglobulin (Ig) and such as other albumen of high-molecular weight immunotoxin to the penetrance of solid tumor poor (Juweid etc. 1992 for example, Cancer Res.52:5144-5153; 1986 Cancer Res.46:3183-3191 such as Epenetos).Other reason of lack rendeing a service comprises the many immunotoxins in the drawing-in system circulation and non-specific toxicity, immunogenicity and inappropriate pharmacokinetics (summarizing in Scheinberg and Chapman 1995 Monoclonal anitibodies (Birth and Lennox edit) the 2.1st chapter) of antibody-radionuclide conjugate.

Opposite with effectiveness in the general shortage body, in some external test, many monoclonal antibodies demonstrate the ability (summarizing in Sandlie and Michaelsen1996 Anitibody engineering:a practical approach, McCafferty grade in an imperial examination 9 chapters) of outstanding inhibition growth of tumour cell.People have established well, and antibody can cause activating the multiple effector function that the Fc by heavy chain of antibody partly mediates to the combination of specific antigen.The different effector function of Fc district mediation of inhomogeneity immunoglobulin (Ig), the Fc acceptor on comprising the activating complement cascade and being incorporated into different immune effector cells (1988 Nature 332:563 such as Duncan and 738).In external test, be present in the antibody that Fc acceptor on the immune effector cell is incorporated into the tumour target cell and take, by the multiple mechanism that is referred to as the cytotoxicity (ADCC) of antibody dependent cellular, can cause destroying target cell.For example the antibody of IgG1 and IgG3 and at the Fc acceptor that the lower IgG2 of degree and IgG4 subclass take IgG on human monocyte and scavenger cell, neutrophilic granulocyte and NK cell (NK) cell stimulates ADCC (1991 Cancer Res.51:1117-1123 such as Munn; Primus etc., 1993 Cancer Res.53:3355-3361).Yet, destroy the ability relative mistake of tumour in this antibody-like body, prompting ADCC in many current therapies, do not play an important role (1994 Immunol.Today 15:559-561 such as George) based on antibody.This there are several possible reasons, comprise that antibody penetrates poor (the 1995 Cancer Res.55:3752-3756 such as Yuan) of penetrance and the following fact of solid tumor: taken the competitiveness of specific antibody poor (1991 Cancer Res.51:1117-1123 such as Munn) under most of high-affinity receptors (FcgRI) molecule normal circumstances on the scavenger cell by serum IgG.

Before showed, can participate in the cell-mediated ADCC of allos NK reaction (1993 Cancer Res.53:3355-3361 such as Primus) external with the tumour cell of the gene transfer of coding monoclonal antibody.Yet the NK cell destroys in the tumour cell in vivo and works hardly, and this part is owing to existence on autologous tumor cell self MHC I class restricted (Correa and Raulet 1995 Immunity 2:61-71) because of its killing ability.

Suppose that also tumor infiltrating lymphocyte (TIL) can be used as carrier, with antibody gene is passed to tumour, with at this tumor locus secretion anti-tumour antibody (1993 J.Immunother.13:143-152 such as Tsang).Yet, the external transduction back of TIL adopts marker gene to carry out autotransplantation, having shown that isolating TIL does not demonstrate allows it to return the specific mechanism of going back to the nest (1996 J.Clin.Invest.97:515-521 such as Economou) of tumour place of deposition (deposits), so this method has limited value.The present invention is opposite with these discoveries, can or be passed to the carrier at tumor mass (or position) with NOI and/or POI guiding because the invention provides.

Reported that also the gene transfer of the monochain immunotoxin of will encoding goes into human lymphokine activated T cells (LAK cell) (1997 Nature 385:78-80 such as Chen).Except that the LAK cell being introduced again the problem of described tumor locus, this method also has the latent defect relevant with being limited to external use.These shortcomings comprise must cultivate described T cell in high-caliber cytokine (such as IL-2), producing the LAK cell, and have contingency question aspect being used for the treatment of producing enough cells.

On the one hand, the present invention relates to use genophore, the gene (such as therapeutic gene) of excretory tumour conjugated protein (TBP) that will encode is passed to tumor mass inside, and determines the lead method of described inside tumor of the expression of TBP.The encode expression of one or more genes in described tumor mass of described TBP then causes the local TBP of generation, subsequently since number of mechanisms make tumor growth, survival rate or send out reduction.Because secrete described TBP, thus the cell of transduction produce TBP can be not only to working on the cell of being transduceed, and, therefore obtain bystander effect to working on the adjacent tumour cell.

Except that cancer cells, in tumor mass, have many cell types.The immunocyte that these cell types can comprise the cell (for example endotheliocyte) of tumor vascular system and soak into tumour is (such as tumor infiltrating lymphocyte (TIL) and scavenger cell (Normann 1985 CancerMetastasis Re.4:277-291; 1996 Cancer Res.56:4625-4629 such as Leek).Any of these cell type all can be directed to, expressing described TBP, and can be as the local factory that produces BP in the tumour.The cell that is used for producing described TBP in the described tumor mass is cancer cells, endotheliocyte or scavenger cell preferably.Perhaps, the progenitor cell of can lead monocyte or endotheliocyte is such as the positive peripheral blood lymphocytes (1997 Science275:964-967 such as Asahara) of CD34.

Described TBP preferably comprises one or more in conjunction with the territory, describedly can be incorporated into one or more TACSM that are present on the cancer cells in conjunction with the territory.Therefore, the TBP by one or more cell type productions in the described tumor mass is secreted also owing to its affinity to described TACSM is directed to cancer cells.Described TACSM can optionally be present on a limited number of cell type.Therefore, the projecting tissue of amount of the TACSM that exists on the most of cancer cells in the described tumor mass.Described TACSM preferably can exist only in tumour cell with detecting and have in individual interior a limited number of other types of organization of this tumour.More preferably, described TACSM is tumour-specific basically in having the individuality of described tumour.

The one or more of described TBP can be made up of the fragment of the native ligand of the binding affinity of TACSM native ligand (this native ligand can be adhesion molecule or growth factor receptors part (for example Urogastron)) or the reservation of for example TACSM in conjunction with the territory.Perhaps, the described heavy chain and the sequence of light chain that can derive from immunoglobulin (Ig) (Ig) variable region in conjunction with the territory.This variable region can derive from natural human antibody or from the antibody (such as rodent animal antibody) of another species.Perhaps, described variable region can derive from engineering reform antibody, such as humanized antibody, or from the phage display library of immune animal or non-immune animal or the phage display library of sudden change.As second kind of system of selection, described variable region can derive from single chain variable fragment (scFv).Described TBP can contain other sequence, with reach poly and or as in conjunction with the transcribed spacer between the territory, or contain in the gene owing to the described TBP of coding and insert other sequence that restriction site produces, comprise Ig hinge sequence or new transcribed spacer and engineered joint sequence.

Described TBP is except that containing one or more immune globulin variable regions, can also contain all or part of Ig CH, all can contain a natural full Ig, an engineered Ig, an engineered Ig sample molecule, a strand Ig or a strand Ig sample molecule.Perhaps or in addition, described TBP can contain the one or more structural domains from another albumen (such as toxin).

In one aspect of the invention, provide the gene delivery system of gene targeting tumour that is used for one or more codings TBP, comprise a kind of TBP of coding genophore and one individual in gene delivery system.Described gene delivery system can be such as with the non-viral gene transfer system of the DNA of DNA compact agent (compactionagent) fine and close (compact) or contain liposome or the immunoliposome of the fine and close DNA of useful DNA compact agent (such as polylysine).This carrier can be the plasmid DNA carrier.Perhaps, this carrier can be a recombinant viral vector, and such as adenovirus carrier, adeno associated virus (AAV) carrier, herpesvirus vector or retrovirus vector, in these cases, the gene transmission is mediated by the virus infection target cell.This carrier is recombinant retrovirus carrier preferably, and it can be directed retrovirus vector.This retrovirus vector is preferably for example by producing and anti-human complement in human cell system.

Usually, this carrier will contain the described gene of guidance or each gene (such as therapeutic gene) expression promoter, and can contain TBP gene effective expression or be regulated the other gene element of expression, comprise enhanser, translation initiation signal, internal ribosome entry site (IRES), splicing signal and polyadenylation signal.The activity of described promotor and/or enhanser can be a tissue limitations.Can use for example tumor-specific promoters-enhanser, such as the promoter-enhancer of 5T4 antigen gene or the promoter-enhancer of CEA gene.Perhaps or in addition, can there be the one or more elements that are used to be regulated expression, the enhanser of regulating such as hypoxemia.A binding member that example is transcription factor HIF1 of the Expression element (HRE) that hypoxemia is regulated.Provide one or more enhancer elements of being regulated expression to exist with multiple copied.The expression of this gene or a kind of gene (such as therapeutic gene) preferably can be induced by the hypoxemia that can find in tumor mass (or hypoxemia supply).More preferably, the expression of instructing this gene (such as therapeutic gene) expression promoter and/or enhanser to contain hypoxia responsive element and reaching in tumour cell is higher than the element of adjacent non-tumor cell.

Other carrier component provides the others of function vector, such as adopting element well known in the art to carry out keeping, appraise and decide the position, duplicating the integration when suitable of carrier.

In the preferred embodiment of the present invention aspect this, retrovirus vector is provided, be used for being passed to described tumour in one or more genosomes with the described TBP of coding.Suitable retrovirus vector is that known in the art (referring to for example Gunzberg and Salmons 1996Gene Therapy, Lemoine and Cooper Bios edit; With 1995 J.Virol.69:7430-7436 such as Cosset).In particularly preferred embodiments, by comprise the gene element that hypoxemia is regulated in this retrovirus vector, the expression of described TBP can be strengthened in the hypoxemia district of this tumour.In this case,, described hypoxemia regulatory element is inserted among one or two LTR of retrovirus, replace the LTR enhanser, or insert in the another location of this carrier by standard molecular biological technique.Encode described TBP-kind or several genes can express by promoter-enhancer, described promoter-enhancer causes the expression in tumour cell to be higher than adjacent non-tumor cell, maybe this expression preferably is tumour-specific basically.The example of suitable promotor comprises the promoter-enhancer of promoter-enhancer, MUC1 gene or the CEA gene of 5T4 antigen gene.

In another aspect of this invention, provide the treatment method for cancer, this method comprises one or more described TBP genes in the gene delivery system of first aspect present invention or system or directly gives tumor locus.

In another aspect of this invention, be provided in coding one or more genes of TBP or the body or the cytophyletic gene delivery system of external introducing hematopoiesis (preferably marrow hemopoiesis).Described hematopoiesis (preferably marrow hemopoiesis) cell preferably monocyte-scavenger cell pedigree cell or such as the cell precursors of the positive stem cell of CD34.For external transmission, described gene can be inserted in the plasmid vector, and transmit by multiple DNA transfection method, described DNA transfection method comprises the transfection of electroporation, the biological emission of DNA, lipid mediation or the transfection of fine and close DNA mediation.Perhaps, can come external transduction hematopoiesis (preferably marrow hemopoiesis) cell or the positive stem cell of CD34 with virus vector, such as adenovirus carrier, retrovirus vector or lentiviral vectors.This carrier will contain the startup of instructing this gene or each gene (such as therapeutic gene) to express to give, and can contain the other gene element that is useful on effective expression or is regulated expression, comprise enhanser, translation initiation signal, internal ribosome entry site (IRES), splicing signal or polyadenylation signal.Described promotor or enhanser or splicing signal can be tissue limitations, preferentially in the monocyte scavenger cell such as scavenger cell activity are arranged.Described promotor and/or enhanser can contain the element that is useful on the expression of modulated joint, such as the enhanser of hypoxemia adjusting.The example of the Expression element that hypoxemia is regulated is a HIF1 transcription factor response element.This element can exist with multiple copied.The promotor that hypoxemia is regulated and the example of enhanser comprise promotor and the enhanser (Semenza etc., 1994 J.Biol.Chem 269:23757-23763) from the gene (such as pgk gene) of enolase gene, erythropoietin gene and coding glycolytic ferment.Isolating HRE can poly, to improve replying hypoxemia.Other carrier component can provide the others of function vector, such as adopting component known in the art to carry out keeping, appraise and decide the position, duplicating the integration when suitable of carrier.

Behind the described cell of the external introducing of this carrier, can directly introduce described cell in patient's body again, or before introducing the patient again, can with the suitable combination of stimulation of cytokine and somatomedin they so that it is along monocyte-scavenger cell differentiation pathway differentiation.Can stimulate the differentiation of CD34 positive cell with the cytokine that comprises IL-3, GMCSF and MCSF.Perhaps by the cultivation of adhesion plastics, perhaps use or independent GMCSF or in conjunction with other cytokine (comprising MCSF), make the monocyte differentiation.

For introducing hematopoiesis (preferably marrow hemopoiesis) cell or the positive stem cell of CD34 in gene (such as the therapeutic gene) body, can use the interior transfer system of suitable body to transmit above-mentioned transcription unit.Described gene delivery system can be such as with the non-viral gene transfer system of the DNA of DNA compact agent densification or contain liposome or the immunoliposome of the DNA of useful DNA compact agent densification.Perhaps, this carrier can be a recombinant viral vector, such as targeted adenovirus vectors, adeno associated virus (AAV) carrier, herpesvirus vector or such as the retrovirus vector of slow virus.The preferably directed recombinant retrovirus carrier of this carrier, it is preferably for example by preparing this carrier and anti-human complement from human package cell line.

CD34 is positive, and stem cell also can be divided into endotheliocyte (1997 Science275:964-967 such as Ashara).Except that being differentiated to form monocyte and scavenger cell, design contains this differentiation pathway according to the positive stem cell of CD34 of TBP encoding gene of the present invention.

Other carrier component can provide the others of function vector, such as adopting component known in the art to carry out keeping, appraise and decide the position, duplicating the integration when suitable of carrier.

In the preferred embodiment of the present invention aspect this, can carry in the plasmid vector or retrovirus vector introducing self peripheral blood lymphocytes of TBP encoding gene, wherein said TBP encoding gene is in hypoxemia adjusting promotor or under preferentially promoters active is controlled in scavenger cell.The monocyte of described transfection is introduced in this patient's body again, and they migrate to the hypoxemia district of tumour, make that the generation at the inner TBP of this tumor mass strengthens.Before being injected into described patient again, can randomly handle described scavenger cell with cytokine.Perhaps or in addition, this carrier can comprise can express cell factor gene (such as IFNg, CSF-1 or GM-CSF gene) dna sequence dna so that induce the differentiation of institute's transfectional cell.Described cytokine gene also can be subjected to show at tumor locus the adjusting of the gene element of increased activity.

In another aspect of this invention, the treatment mankind or non-human mammal method for cancer are provided, this method comprises takes out a certain amount of blood from the individuality of suffering from cancer, be rich in the cellular preparations of monocyte and scavenger cell or its stem cell precursor by this blood preparation, adopt the gene delivery system of third aspect present invention that the TBP gene is introduced in the cellular preparations, so that produce the transfection or the transduction of monocyte and scavenger cell or its stem cell precursor with described TBP gene, and with the cell or the system of institute's transfection or transduction or directly introduce tumor locus again.Before described cellular preparations is introduced again, can randomly handle, so that initiation is to the differentiation of activated macrophage with cytokine.

In another aspect of this invention, provide the method for treatment mammalian cancer, this method comprises and gives individual gene delivery system of the present invention that TBP can express in described system in the cell that derives from hematopoiesis (preferably marrow hemopoiesis) source.

Provide the genophore of one or more genes (such as therapeutic gene) that comprise the TBP that encodes again on the one hand of the present invention, selectivity has the expression regulatory element of function functionally to be connected in wherein said gene and the cell type in tumor mass.The TBP of this aspect of the present invention suppresses the tumour function by be incorporated into the TACSM that basic role is arranged in tumour cell survival or diffusion.The TACSM of this aspect of the present invention can be an effective cell surface molecule at growth of tumour cell, migration or in shifting, and is present on other cell type in cancer cells or the tumor mass.Described TACSM preferably is present on cancer cells or tumor vascular system or the scavenger cell, for such as growth factor receptors, plasminogen activator, metalloprotease or the antigenic molecule of 5T4.By any approach of describing in two aspects more than the present invention, one or more genes of the described TBP of coding can be passed to described inside tumor.Described TBP is incorporated into corresponding TACSM, has blocked the function of described TACSM, and causes suppressing described growth of tumor, migration or transfer thus.

In still another aspect of the invention, the genophore that will comprise a kind of gene (such as therapeutic gene) is passed to this inside tumor, wherein said gene (such as therapeutic gene) coding TBP, and it also contains one or more effector domains.Described one or more effector domain can be activated when described TBP is incorporated into TACSM, causes suppressing propagation, survival or the diffusion of tumour cell.The TACSM of this aspect of the present invention is a kind of cell surface molecule that obtains specificity T BP, such as tumour-specific carbohydrate part, carcinomebryonic antigen, Saliva Orthana, growth factor receptors or another kind of glycoprotein.Described TACSM is the limited antigen of its tissue distribution preferably, finds on the main most cells in described tumour cell or described tumour.Perhaps, described TACSM is present on tumour scavenger cell or the tumor vascular system.In some cases, described TACSM can not be shed to the circulation from described cell surface on fine degree.Yet, come off and may take place.As an example, 5T4 antigen is shed in the matrix, can be used for further described NOI and/or described POI being positioned to described tumor environment.

Effector domain of the present invention can have enzymic activity, can be prodrug activation enzyme for example, or it can be non-enzyme territory.The TBP example that contains the effector domain with enzymic activity comprises antibody-enzyme conjugate or syzygy.Described antibody-enzyme conjugate, comprised and alkaline phosphatase (Senter etc., 1988 Proc Natl.Acad.Sci.85:48424846); Carboxypeptidase G 2 (1988 Br.J.Cancer 58:700703 such as Bagshawe); P-lactamase (1991 Bioorg.Med.Chem.Left.1:21-26 such as Shepherd); And the conjugate of penicillin-V-Ntn hydrolase (1990 Cancer Immunol.Immunother.31:202-306 such as Kerr).Antibody-enzyme syzygy (1993 Cancer Res.53:2123-2127 such as Goshorn have also been described; 1992 Bio/Tehcnologv 10:1128-1132 such as Wles).In these examples each all can be used for this aspect of the present invention.Can be contained in TBP enzyme syzygy in addition or other enzyme comprise human carboxypeptidase A 1 or its mutant (1997 J.Biol.Chem.272:15804-15816 such as Smith); Isocytosine deaminase (1994 Cancer Res.54:1503-1506 such as Mullen); HSV thymidine kinase (1988 Proc.Natl.Acad.Sci.85:7572-7576 such as Borrelli); Nitroreductase (nitroreductase); P450-reductase enzyme and P450.

The C-terminal to immunoglobulin (Ig) is merged in the described best heredity in one or more prodrug activation enzymes territory, or merges to immunoglobulin domains, such as scfv or single-chain antibody and Fab fragment.In the particularly preferred embodiment of the present invention aspect this, described one or more immunoglobulin domains are human or humanized, and described enzyme is people's fermentoid, such as carboxypeptidase P450 or P450 reductase enzyme.Described enzyme can be a mutant enzyme, and it more effectively transforms prodrug than natural people's fermentoid.According to the present invention, can use any enzyme useful in the ADEPT strategy.

In all cases, the suitable precursor medicine is used for treat this patient in conjunction with suitable precursor medicine activating enzymes.The example of prodrug comprises phosphoric acid Etoposide (using Senter etc. with alkaline phosphatase, 1988 Proc.Nat.Acad.Sci.85:4842-4846); 5-flurocytosine (using 1994 Cancer Res.54:1503-1506 such as Mullen with Isocytosine deaminase); Zorubicin-N-para hydroxybenzene oxygen yl acetamide (uses (1990 Cancer Immonol.Immunother.31:202-206 such as Kerr) with penicillin-V Ntn hydrolase; Two (2 chloroethyl) aminobenzoyl L-glutamic acid of right-N-(using) with carboxypeptidase G 2; Carboxylamine cynnematin mustargen (using) with the P-lactamase; SR4233 (using) with the P450 reductase enzyme; Gancyclovir (is used with the HSV thymidine kinase, 1988 Proc.Natl.Acad.Sci.85:7572-7576 such as Borrelli) mustard seed prodrug that uses with nitroreductase (1997 J Med Chem 40:1270-1275 such as Friedlos) and endoxan (using 1996 Cancer Res 56:1331-1340 such as Chen with P450).

Perhaps, described effector domain can be non-enzyme territory.The example in non-enzyme effect territory comprises such as from the endotoxic toxin of pseudomonas, such as all or part of of the cytokine of IL-2 or IFN γ or from the effector domain of heavy chain immunoglobulin.

In the preferred embodiment of the present invention aspect this, described TBP contains one can activating macrophage FcgR I, II or the effector domain of III acceptor.When described TBP is incorporated into antigen on the described tumour cell, scavenger cell in the described tumour hypoxemia district is activated, directly destroy described tumour cell by phagolysis or ADCC, or the described scavenger cell secretion pro-inflammatory cytokine that is activated, the natural immunity that described pro-inflammatory cytokine is used for strengthening described tumour is replied.Described TBP can contain the Fc district of a Fc district from immunoglobulin (Ig), a sudden change, the receptors bind fragment in a Fc district, maybe can contain another FcR in conjunction with the territory.

Described TBP preferably contains an entity, effector domain entity preferably, and it gives proteinic external and/or body internal stability.

According to the present invention, described TBP can comprise the complete Fc district of IgG (such as human IgG1 or IgG3), preferably from IgE (such as human IgG E) or its part.

In the preferred embodiment of the present invention aspect this, described TBP contains human IgG1's constant region and the identification antigenic Sab in conjunction with the territory of 5T4 (single-chain antibody).

In the particularly preferred embodiment of the present invention aspect this, described TBP contains a people IgE constant region and the identification antigenic Sab in conjunction with the territory of 5T4 (single-chain antibody).

Described effector domain can be by the part of cDNA coding, the part of described cDNA at the frame endomixis to the DNA of codes for tumor in conjunction with the territory.Perhaps, can use the genomic fragment that contains intron, such as human IgG1's weight chain constant area gene group fragment.

Here term " intron " is with its ordinary meaning use-for example intragenic DNA intervening sequence, and it is removed by the RNA montage, therefore be not present in the ripe messenger RNA(mRNA), and coded protein not.Intron can condition montage or alternative montage in different cell types.

The gene of coding TBP is introduced monocyte or scavenger cell can treat with other and combine, to bring out the scavenger cell differentiation and to activate.For example, in introducing described patient again before, the external cell of keeping can be used such as the cytokine of IFN γ, CSF-1 or GM-CSF and handle.Perhaps, in can body or externally introduce the gene of these cytokines of coding to described monocyte/macrophage with the identical or different carrier that derives from described TBP gene.Therefore, provide the method for treatment mammalian cancer more on the one hand of the present invention, this method comprises and gives the individual cells factor or cytokine encoding gene and one or more compositions according to the TBP gene of the arbitrary aforementioned aspect of the present invention.

According to the present invention, can use standard molecular biological technique well known by persons skilled in the art.This class technology has been described in the literature all sidedly.Referring to (1989) Molecular Cloning:a laboratory manual such as for example Sambrook; Hames and Glover (1985-1997) DNA Cloning:a practical aprroach, I-IV rolls up (second edition).Particularly in (1996) Antibody engineering:a practical approach such as McCafferty, provided the method for engineering immunoglobulins gene.

One preferred aspect, the present invention relates to the TBP encoding gene is passed to tumor locus.This (uses such as treatment) aspect medical use has considerable advantage, and in described medical use, because TBP walks around many and the relevant problem of systems communicate protein in the mankind, so TBP is suitable for.

Opposite with the problem of generation and transferrin qualitative correlation, method of the present invention allows gene is passed to described tumor locus, therefore walks around many production problems.Therefore described TBP produces in original position in body people cell, the described local factory that produces as the medicine of producing based on gene (such as treatment) from body people cell.This is reducing to have significant advantage aspect the system toxicity.This proteic activity is maximum, and therefore this proteic glycosylation shows the people's quasi-mode that is suitable for individuality to be treated.

Can go into tumor locus or for example targeting vector or engineered hematopoiesis (preferably marrow hemopoiesis) cell or the systems communicate of its precursor in conjunction with direct direct projection, use method of the present invention.Systems communicate may be particularly advantageous in many indications, particularly aspect the treatment transmissible disease.In these cases, described gene delivery system or engineered cell can give by large bolus injection or with appropriate formulation infusion intravenously.Medicinal acceptable preparation can comprise etc. ooze salts solution, slow in salts solution or tissue culture medium (TCM).Can comprise other preparaton, such as sanitas or stablizer.

Therefore, the present invention also comprises the medicinal compositions for the treatment of one or more individualities by gene therapy, wherein said composition comprise the treatment significant quantity according to carrier of the present invention or its expression product.Described medicinal compositions can be used for the people or be used for animal.Usually, the doctor can determine to be suitable for most the accurate dosage of curee's individuality, and this dosage will change with age, body weight and the reaction of particular patient.

Described composition can randomly comprise medicinal acceptable carrier, thinner, vehicle or auxiliary.Can planned route of administration and the pharmacy practice of standard, select pharmaceutical carrier, vehicle or thinner.Described medicinal compositions can comprise or comprise other carrier agent (such as the lipid transfer system) that any suitable binder, lubricant, suspension agent, Drug coating, solubilizing agent and auxiliary or enhanced virus enter target site except that described carrier, vehicle or thinner.

When suitable, the route of administration of described medicinal compositions can be for following any or multiple: suck, with suppository or vaginal suppository form, with lotion, solution, emulsifiable paste, ointment or dusting form topical administration, with skin patch use, oral or give to contain with the capsule of independent or fusion vehicle or ovulum or with elixir, solution or the form of suspension that contains seasonings or tinting material such as the tablet form of the vehicle of starch or lactose, or they can parenteral injection, for example in the cavity, intravenously, muscle or subcutaneous injection.Give for parenteral, described composition preferably gives with the aseptic aqueous solution form, and the described aqueous solution can contain other material, and for example enough salt or monose are so that this solvent and blood etc. ooze.Give for cheek or hypogloeeis, described composition can be giving with the tablet of conventionally form preparation or the form of lozenge.

Therefore, a preferred aspect of the present invention relates to a kind of carrier, and it comprises the NS of (a) coding TIP and the NOI of the POI that (b) encodes; Wherein said TIP can discern tumour, makes that in use this carrier can be passed to described tumour with described NOI and/or described POI.

In an example of the present invention embodiment, TIP is IgG or IgE or its part.

In another example embodiment of the present invention, TIP is EGF or its part.

In another example embodiment of the present invention, TIP identification trophocyte surface antigen, and at least one described effector domain is a kind of excretory co stimulatory molecule.Below be about further background content of this embodiment and details.

One aspect of mentioning, back of the present invention relates to and is used for activated lymphocyte and uses activated lymphocyte treatment method for cancer.It also relates to the fusion rotein that is used for activated lymphocyte, relate to the nucleic acid of encoding said fusion protein and carry the carrier of described nucleic acid.

Lymphocyte needs at least two kinds of different signals, by activation effect thing function antigen is replied (Bretscher and Cohn 1970 Science 169:1042-1049; Crabtree 1989Science 243:355-361).Described first signal is specific to antigen.For bone-marrow-derived lymphocyte, the three-dimensional epi-position on the many macromole of described B cell antigen receptor (surface immumoglobulin) identification.For the T lymphocyte, TXi Baoshouti (TCR) is identified on the antigen presenting cell peptide antigen (1986 Ann.Rev.Immnol.4:593-619 such as Weiss) that the albumen by main histocompatibility (MHC) family presents.

The activation of first signal in the separation causes lymphocytic apoptosis (apoptosis) usually, or causes setting up lasting unresponsiveness or anergy (Weiss etc. see above-mentioned).In order to reach activated lymphocyte, need subsidiary signal, it can or go up the cell surface that exists by antigen presenting cell (APC) by cytokine stimulates the part transmission jointly.

Identifying now has many these class co stimulatory molecules, comprises adhesion molecule LFA-3, ICAM-1, ICAM-2.The main co stimulatory molecule of the last existence of APC is the member of B7 family, comprises B7-1 (CD80), B7-2 (CD86) and B7-3.These molecules are parts of common costimulatory receptor on the lymphocyte, comprise CD28 (WO92/00092), and it may be the most important common costimulatory receptor of tranquillization T cell.The different members of B7 family glycoprotein can be passed to unlike signal T cell (1996 J.Biol.Chem.271:1591-1598 such as Nunes) subtly.

Although the tumour of setting up is abnormal expression antigen in its surface usually, their immunogenicity is poor.Previous supposed that the method that is used for the immune stimulatory tumor cell offers enhancement antigen and the common lymphocyte that stimulates at the environment of tumour antigen.The gene of coding B7-1 and B7-2 is separately or in conjunction with the transfection of cytokine, shown that enhancing is to the development of experimental tumor immunity in the animal model (1997 Int.J.Cancer 71:476-482 such as Leong for example; 1996 Eur.J.Immunol.26:1335-1341 such as Zitvogel; 1997 J.Immunol158:2834-2841 such as Cayeux).Yet in the actual therapeutic that these results is translated as to human cancer, there have many important problem to have to be to be solved.A subject matter in this class research is to be passed to a large amount of tumour cells in need be with the B7 genosome, renders a service obtaining.Second problem is that the expression guiding tumour cell with B7 is important, activates to avoid unsuitable immunocyte at other cell type.

This aspect of the present invention has the gene of the co stimulatory molecule (" SCM ") of binding affinity by transmitting the coding excretory to tumour antigen, has solved these particular problems.Like this, the transfectional cell of relatively small amount is as the local factory that produces co stimulatory molecule in the tumour, and described molecule is from producing cell detachment, and is incorporated into other cell in the tumour.The additional advantage of this aspect of the present invention is that tumour cell needs not be the target of transfection.

SCM of the present invention is a kind of new engineered fusion rotein, and it comprises one and is used for from mammalian cell excretory signal peptide, at least one antigen binding domain and at least one other structural domain as the common stimulus signal of immune system cell from immunoglobulin (Ig) or immunoglobulin-like molecule.Also can design to use and contain the different common compositions that stimulate the SCM in territory.By expression SCM encoding gene in the individual autogenous cell of this treatment, and produce described SCM, any posttranslational modification that is added in this albumen by this host cell all is real thus, and the albumen of full functionality and suitable pharmacokinetics are provided.

WO-A-92/00092 has described the B7-1 of clipped form, and they are by placing translation stop codon before the membrane-spanning domain, and secretion is come out from mammalian cell.Under this specific situation, use allos signal peptide from oncostatin M gene.WO-A92/00092 has also described fusion rotein, and they contain the extracellular domain of the B7-1 that is blended in immunoglobulin fc region.This quasi-molecule can be incorporated into the CD28 on the T cell, is used for stimulating T cell proliferation.Yet this stimulation only takes place on moderate, unless described B7 or B7 derivative are fixed in solid surface.

Gerstmayer etc. (1997 J.Immol.158:4584-4590) seedling has been stated the syzygy of a kind of B7-2, this syzygy is blended in the specific scFv to ErbB2, after connect a kind of myc epi-position mark and a polyhistidine mark, described polyhistidine mark is secreted out when expressing in yeast pichia pastoris phaff (Pichia pastoris).This molecule keeps antigenic combination and stimulating proliferation jointly with PMA and the pre-T cell that stimulates of IL-2.Yet the glycosylation of this molecule is the glycosylation of yeast type, may cause pharmacokinetics improperly in the mankind.

According to the present invention, can use any suitable common stimulation territory.As an example, stimulate the territory can be selected from following proteic born of the same parents' outside part jointly: cell surface glycoprotein B7 family comprises B7-1, B7-2 and B7-3; Or other common irritation cell surface glycoprotein, such as, but not limited to common costimulatory receptor ligand molecular, comprise CD2/LFA-3, LFA-1/ICAM-1 and ICAM-3.Study verifiedly, monocyte depends on each (1991 Eur J Immunol 21:1711-1718 such as Van Seventer) among two kinds of receptors ligand approach CD2/LFA-3 and the LFA-1/ICAM-1 to the common stimulation of T cell.In addition, show, the third LFA-1 counter receptor ICAM-3 be a kind of tranquillization with the lymphocytic co stimulatory molecule of activated T (1993 Eur J Immunol 23:2799-2806 such as Hemandez-Caselles).

Other possible co stimulatory molecule can comprise a new glycoprotein receptor, called after SLAM, identified that it works as when occupied, strengthen the amplification of T cell in the mode that does not rely on CD28, and induce the Th0/Th1 cytokine to produce profile (1995 Nature376:260-263 such as Cocks).

Show that a kind of cell surface glycoprotein CD6 is as the common stimulation on the T cell and adhere to acceptor and work.Four kinds of CD6 isotypes (Cd6a, b, c, d) (1997 Eur J Immunol 27:2971-2980 such as Kobarg) have been described.The effect (1997 Eur J Immunol 27:2757-2764s such as Silvy) of antigen (VLA-4) integrin in human mind B cell-stimulating in described very late period also proposed.Endotheliocyte also provides distinctive common stimulus signal, and they influence the phenotype (1996 Eur JImmunol 26:610-617 such as Karmann) of activated CD4+T cell.The B3 albumen that exists on the lipopolysaccharides activated B cell surface has been described, this albumen can provide the common stimulation to tranquillization T cell, causes mainly discharging the IL-2 and the interferon-gamma (1995 J BiolChem 270:23429-23436 such as Vinay) of interleukin (IL)-4 and IL-5 and negligible quantity.A kind of co expression of common stimulation T cell antigen (A6H) has newly been pointed out the possible function relevant with these cell denominators (1995 Int Immunol 7:1425-1432 such as Labuda) on T cell and the tumour cell.

In a preferred embodiment of the invention, described common stimulation territory is the part of B7-1 or B7-2, is more preferably complete born of the same parents' outside part of B7-1 or B7-2.

By expressing the new gene of a kind of fusion rotein of coding, form described SCM, described fusion rotein contains one or more antigen binding domains and one or more common stimulations territory.If described antigen binding domain is made up of a heavy chain and a light chain, then described common stimulation territory and one or another immunoglobulin chain fusion are preferably merged with described heavy chain.If described antigen binding domain is scFv, then described common stimulation territory and this scFv merge.Can with described territory in the following sequence (N-terminal is to C-terminal) arrange: connect common stimulation territory behind the antigen binding domain; Or connect antigen binding domain after stimulating the territory jointly.Preferably, described common stimulation territory places N-terminal, after connect described antigen binding domain.Signal peptide is contained in N-terminal, can be the natural signals peptide in for example described common born of the same parents' external stimulus territory.Different structural domains can be by several other sequences separately, its reason may be to comprise easily the Restriction Enzyme restriction enzyme site so that help its structure in this new gene, or it is as the peptide spacer between the described structural domain, or it is used as flexible peptide linker or other function is provided.Described structural domain is preferably separated by flexible joint.

Can use the different genes of two or more different SCM that encode, obtain improved common stimulation, or obtain the common stimulation of natural T cell and inducing of anamnestic response.For example, coding can be contained the gene of the SCM of B7-1 extracellular domain, the gene that contains the SCM of B7-2 extracellular domain with coding gives.

Therefore, in one aspect of the invention, one or more genophores are provided, they can express one or more excretory co stimulatory molecules in mammalian cell, each excretory co stimulatory molecule comprises the structural domain of at least one antigen binding domain and at least a born of the same parents' outside part from the cell surface co stimulatory molecule.Described common stimulation territory can get the molecule of expressing on the comfortable antigen presenting cell surface, such as the B7 family member.Described common stimulation territory is preferably from B7-1, B7-2 or B7-3.It most preferably is made up of B7-1 amino-acid residue 1 to about 215 (being described in WO-A-96/00092) of the residue of ripe B7-1 molecule, or is made up of amino acid/11 to about 225 (being described in 1997 J.Immunol.158:4584-4590 such as Gerstmeyer) of the B7-2 of mature cell format surface.

Comprise at least one according to the genophore of this aspect of the present invention and be used for mammalian cell expression promoter and enhanser and a polyadenylation site.Suitable promotor and enhanser comprise from human cytomegalovirus's MIE promoter-enhancer or are present in preferential expression promoter in the cell in this tumour.This class promoter-enhancer comprises the promoter-enhancer from MUC1 gene, CEA gene or 5T4 antigen gene.If express two kinds or a plurality of SCM, then the coding region of these SCM can be inserted in two isolated vectors, maybe can use single carrier to express described two or more genes.Under latter event, every kind of gene provides the promotor of a copy separation, or uses an internal ribosome entry site (IRES) to separate described two kinds of encoding sequences.

The present invention also comprises mutant, varient, homologue or the segmental use of the open sequence of this paper.

Term " varient ", " homologue " or " fragment " that relates to nucleotide sequence comprises any displacement, variation, modification, replacement, the disappearance of this sequence or adds (or a plurality of) nucleic acid, nucleotide sequence coded maybe can the coding that is produced that provides has the entity of the identical function that this paper proposes, and preferably is identical at least on biological activity.Specifically, term " homologue " is included in the homology of structure and/or function aspects, the nucleotide sequence coded or identical entity that can encoding function be proposed with this paper of the gained that provides.About sequence homology, preferably be at least 75% with the homology of sequence shown in this paper, more preferably be at least 85%, more preferably be at least 90%.More preferably be at least 95% with the homology of sequence shown in this paper, more preferably be at least 98%.

Specifically, term used herein " homology " can be equal to term " identity ".Can determine relative sequence homology (being sequence identity) by commercially available computer program, described computer program can calculate the homology percentage between two or more sequences.A representative instance of this computer program is CLUSTAL.

Term " varient ", the allelic variation synonym of " homologue " or " fragment " and described sequence.

Term " varient " also comprise with can with the sequence complementary sequence of nucleotide sequence hybridization shown in this paper.Preferably, term " varient " comprise with can be under stringent condition the sequence complementary sequence of nucleotide sequence hybridization shown in (for example 65 ℃ and 0.1 * SSC{1 * SSC=0.15M NaCl, 0.015 Trisodium Citrate pH 7.0}) and this paper.

The present invention also comprise can with the nucleotide sequence of nucleotide sequence of the present invention (comprising and the complementary of sequence shown in this paper sequence) hybridization.One preferred aspect, the present invention includes can be under stringent condition (for example 65 ℃ and 0.1 * SSC) with the nucleotide sequence of the hybridization of nucleotide sequence (comprising and sequence complementary sequence described herein) shown in this paper.

Term " varient ", " homologue " or " fragment " that relates to aminoacid sequence comprises any displacement, variation, modification, replacement, the disappearance of this sequence or adds (or a plurality of) amino acid, the gained amino acid that provides has the identical function that this paper proposes, and preferably is identical at least on biological activity.Specifically, term " homologue " is included in the homology of structure and/or function aspects, and the gained aminoacid sequence that provides has the identical functions that is proposed with this paper.About sequence homology, preferably be at least 75% with the homology of sequence shown in this paper, more preferably be at least 85%, more preferably be at least 90%.More preferably be at least 95% with the homology of sequence shown in this paper, more preferably be at least 98%.

Generally, the present invention relates to comprise NS and the optional b of (a) coding TIP) carrier of the NOI of coding POI; Wherein said TIP can discern tumour, makes that in use this carrier can be passed to described tumour with described NOI and/or described POI.

A preferred aspect of the present invention relates to the NS that comprises (a) coding TIP and (b) carrier of NOI of coding POI; Wherein said TIP can discern tumour, makes that in use this carrier can be passed to described tumour with described NOI and/or described POI.

A preferred aspect more of the present invention relates to the NS that comprises (a) coding TIP and (b) carrier of NOI of coding POI; Wherein said TIP can discern tumour, makes that in use this carrier can be passed to described tumour with described NOI and/or described POI; And wherein said TIP and POI merge mutually.

This aspect of the present invention is favourable, because it allows to produce the fusion product that for example comprises an effect component and a guiding component, and with its transmission.

A preferred aspect more of the present invention relates to the NS that comprises (a) coding TIP and (b) carrier of NOI of coding POI; Wherein said TIP can discern tumour, makes that in use this carrier can be passed to described tumour with described NOI and/or described POI; Wherein said TIP and POI merge mutually; And wherein said POI can be secreted.

This aspect of the present invention is highly favourable, because it provides a kind of means, is used for for example producing POI by a few cell original position, is passed at least one flanking cell with a part of inciting somebody to action the POI that be produced at least subsequently.Therefore, only need to infect a few cell to obtain useful result of treatment.

Therefore, change a kind of saying, the invention provides according to the purposes of carrier of the present invention as any or multiple produced in situ factory among described NS, NOI, POI and the TIP.

In addition, the invention provides when being present in the cell purposes that NOI and/or POI is passed to flanking cell according to carrier of the present invention.

More preferably aspect of the present invention relates to the NOI of the NS, (b) coding POI that comprise (a) coding TIP and the carrier of the nucleotide sequence of the secretion entity of (c) encoding; Wherein said TIP can discern tumour, makes that in use this carrier can be passed to described tumour with described NOI and/or described POI; Wherein said TIP and POI merge mutually; And wherein said POI can be secreted.

Further describe the present invention by embodiment now, these embodiment implement the present invention with helping those skilled in the art, the scope that does not limit the present invention in any way.With reference to the following drawings:

The dna sequence dna of Fig. 1 a-code displaying 5T4 scFv, this sequence called after 5T4scFv.1.Provided the sequence of described ripe secretory protein.

The cDNA sequence of Fig. 1 b-code displaying 5T4Sabl.This sequence starts from a HindIII restriction site, after connect a translation initiation signal and a signal peptide.

Fig. 2 shows the sequence of B7-1.5T4.1.

Fig. 3 shows the diagram that stimulates two SCM in territory jointly based on B7-1; Fig. 3 a shows SCM B7-1.5T4.1, and Fig. 3 b shows the B7-1.5T4.2 that wherein stimulates territory and tumour to reverse in conjunction with the order in territory jointly.The Sp=signal peptide; The extracellular domain of B7 ec=B7-1; The light chain variable territory of V1=5T4; The heavy chain variable domain of Vh=5T4.

Fig. 4 shows the sequence of people B7-2 extracellular domain, comprises signal peptide sequence.Maturation protein starts from amino acid/11 7.Meet a flexible joint gly-gly-gly-gly-ser behind the B7-2 derived sequence.

The retrovirus genomic simple general diagram (not in scale drawing) of Fig. 5 for showing LTR, gag, pol and env essential characteristic.

Embodiment

The structure of embodiment 1-5T4 Sab and retrovirus vector are to the transmission of tumour

With standard technique (Antibody engineering:a practical approach McCafferty etc. edit 1996 OUP), the cDNA of clones coding muroid 5T4 monoclonal antibody and to its order-checking.Use the variable region sequences of this antibody to make up panimmunity sphaeroprotein sample molecule, comprise scFv.The encoding sequence of 5T4 scFv, 5T4scFv.1 is shown in Fig. 1 a.In this molecule, this dna sequence encoding is from the Vh of mouse 5T4 monoclonal antibody, after meet the V1 district of 15 amino acid whose flexible joints and mouse 5T4 antibody.The aminoacid sequence gly-gly-gly-gly-ser of 3 copies of described flexible joint coding, and the dna sequence dna similarity between the described tumor-necrosis factor glycoproteins has been reduced, with the risk of avoiding recombinating between the described tumor-necrosis factor glycoproteins when the plasmid that contains described sequence is produced in intestinal bacteria.

The dna sequence dna that is shown in Fig. 1 a also can be used for merging formation frame endomixis body by the sequence with scFv encoding sequence and coding Fc district, and makes up multiple single-chain antibody (Sab).Make up Sab with a series of DNA boxes that can independently change to be suitable for specific purpose.

Box 1-translation initiation signal and signal peptide.

In order to obtain correct translation initiation and, to use following sequence from the mammalian cell secretion:

aagcttCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGC

AACAGCTACAGGTGTCCACTCC

This contains one and is used to fill and swells into the restriction site of HindIII easily (lower case) of expression vector, the total translation initiation signal (ANNATGPu) of mammalian cell and the encoding sequence of immunoglobulin gene signal peptide sequence.

Box 2-scFv

5T4scFv.1 the sequence of secretion part be shown in Fig. 1 a.This molecule can be expressed as Vh-(gly ₄-ser) ₃Joint-V1.

5T4 scFv2 is made up of the 5T4 variable region sequences that connects with order V1-flexible joint Vh.In this case, 20 amino acid whose peptide (gly of described joint coding ₄-ser) ₄When trivial section of V was this order, long joint improved the assembling of described scFv.(Antibody Engineering:a practical approach such as Pluckthun, McCafferty etc. edit 1996OUP).

Box 3-CH

In 1982 Nucl.Acids res.10:4071-4079 such as Ellison, provided the sequence of people gl constant region genomic clone.This sequence also contains the constant region intron except that containing this encoding sequence.3 ' the end that this sequence meets one of scFv sequence of frame ground and box 2 merges.Carrier (the 1993 Nucl.Acids Res.21:2921-2929 such as Walls that are used for this formation object space civilian dress and join have been described.

The cDNA of the 5T4 Sab of called after 5T4Sabl is shown in Fig. 1 b, and it contains box 1,2 and 3.

In order in people's cell, to express 5T4 specificity scFv or Sab, this encoding sequence is inserted among the carrier pCIneo (Promega), place under the control of strong promoter and polyadenylation signal.Described translation initiation signal and from box 1, be positioned at the immunoglobulin (Ig) leader sequence (signal peptide) of described coding region 5 ' end, guarantee described scFv or Sab effectively secretion from mammalian cell.

For the Ig of The expressed, make up two isolating translation boxes, one is used for heavy chain, and one is used for light chain.These boxes are separated by the internal ribosome entry site (IRES) (1996 Nucl.Acids Res.24:2697-2700 such as Ramesh) from picornavirus FMDV.Perhaps, (Ward and Bebbington1995 Monoclonal Antibodies Birth and Lennox edit, Wiley-Liss) from every kind of cDNA of hCMV promoter expression of copy separation.

Can express retrovirus in order to produce with the specific 5T4 antibody of 5T4 or immunoglobulin-like molecule, coding is inserted in the retrovirus based on the gene of the Sab of 5T4 or the bicistronic mRNA information of encoding heavy chain and light chain, and wherein retrovirus genome transcript is produced by strong promoter (such as the hCMV-MIE promotor).A suitable plasmid is pHIT111 (1995 Nucl.Acids Res.23:628-633 such as Soneoka), inserts required gene with standard technique, to replace the LacZ gene.Then, the plasmid pHIT-5T4.1 of gained is transfected into FLYRD18 or FLYA13 package cell line (1995 J.Virol.69:7430-7436 such as Cosset), selects transfectant according to the resistance to 1mg/ml G418.G418 resistance packing cell produce high titre can the infected person cell recombinant Retroviruses.Then, with this virus prepared product infected person cancer cells, and can be injected into tumour in the body.Express described 5T4Sab then, and from described tumor cell secretion.

In pHIT111, MoMLV LTR promoter-enhancer is used for expressing described therapeutic gene in described target cell.Also can modify this carrier, make described therapeutic gene transcribe from an internal promoter-enhanser, described internal promoter-enhanser such as main in described tumour cell promoters active-enhanser or contain the promoter-enhancer of hypoxemia regulatory element.A suitable promotor is the HSVTK promotor (1994 Proc.Natl.Acad.Sci.91:6496-6500 such as Firth) with brachymemma of 3 copy mouse PGK HRE.

The embodiment 2-expression vector transfection monocytes/macrophages of coding TBP

With standard technique method (editors such as Sandlie and Michaelsen 1996 Antibody engineering:a practical approach.McCafferty, the 9th chapter) with laboratory scale, and by elutriation (for example Ceprate of CellPro) with on a large scale, from the human peripheral separating periphery blood monocytic cell.Be attached to plastics by spending the night, enrichment attached cell (being monocyte basically) can allow cell break up along the scavenger cell differentiation pathway by cultivating attached cell 1-3 week.

With expression vector transfection monocyte and the scavenger cell that can in people's cell, express TBP.In order to carry out the composing type high level expression, in utilizing the carrier pCI (Promega) of hCMV-MIE promoter-enhancer, express described TBP.In order to carry out the expression of hypoxia inducible, replace the hCMV promotor with the promotor that contains at least one HRE.Suitable promotor is HSV TK promotor with brachymemma of 3 copy mouse PGK HRE 1994Proc.Natl.Acad.Sci.91:6496-6500 such as () Firth.

Can carrier be introduced monocyte and scavenger cell with multiple infection protocol, described infection protocol comprises that the transfection that the DNA of particle mediation transmits (biological emission), electroporation, cationics mediation (for example uses Superfect, Qiagen).In these methods each all carried out according to manufacturer's explanation, and specifies according to each manufacturer, considers that parameter to be changed is to obtain optimum.Perhaps, can use virus vector such as defective adenoviral vector (Microbix Inc or QuantumBiotechnologies Inc.).

The mensuration of the ADCC that embodiment 3-is macrophage-mediated

Will be with the cell from primary human tumor or tumor cell line of the retrovirus transduction of expressing TBP, and by the mixing of embodiment 2 described preparations, be used to analyze the ADCC activity that mediates by described TBP from body or allogenic human scavenger cell.Perhaps, engineeredly can be used for the lead tumour cell of non-transduction of ADCC by embodiment 2 is described with the scavenger cells that produce TBP.

According to standard step (editors such as Sandlie and Michaelsen 1996 Antibody engineering:apractical approach.McCafferty, the 9th chapter), suitably revise, carry out this mensuration.In brief, with effector cell's (scavenger cell or new isolating monocyte) with 3 * 10 ⁶Cell/ml is suspended in the suitable tissue culture medium (TCM) (DMEM/Hepes derives from LifeTechnologies, contains 1% foetal calf serum).To use ⁵¹3 * 10 of Cr mark ⁵The tumour target cell places the 0.1ml substratum in the every hole of round bottom microtiter plate.(noticing that substratum can comprise from the exhausted substratum of producing described TBP cell).50ml effector cell is added in each hole, with this plate centrifugal 2 minutes with 300g, and in incubator for tissue culture in 37 ℃ of incubation different times (for example 4 hours).Then, centrifugal collection supernatant liquor, and in gamma counter, count.With with respect to from representing the result with the cracking percentage of the total chromium that discharges the identical target cell sample of 0.1%Tween-20 cracked.In this is measured, can change the effector cell: target cell ratio, to produce the titre curve.

For previous scavenger cell differentiation or the stimulation that causes, cytokine is added in the culture.The IFNg (Sigma) that adds 100-5000U/ml.Also can add CSF-1 or GM-CSF (Santa Cruz Biotechnology) with suitable concentration.

The analysis of the effectiveness of embodiment 4-in animal model

According to the technology of fine foundation (referring to 1997 Cancer Res.57:1228-1232 such as for example Strobel; 1997 Pancreas 14:237-248 such as McLeod), culturing in vivo people tumour derived cell system and tissue in " naked " mouse of inherited immunity defective.Also can use the homology mouse model of homology tumour system being introduced immunocompetent mouse species.These are as suitable animal model, to assess gene delivery system of the present invention.With carrier or engineered cell system or directly give in the described tumour, in treatment or untreated animal, monitor growth of tumor.This system is used for limiting the effective dosage ranges and the suitableeest route of administration of the present invention's treatment.

The structure of embodiment 5-B7-scFv fusion rotein

The extracellular domain of B7-1 is limited by the proteic amino-acid residue 1-215 of natural human B7-1.This sequence is used for making up the excretory fusion rotein with its signal peptide sequence, and this fusion rotein also contains the scFv that derives from the 5T4 monoclonal antibody.The sequence of described 5T4 scFv provides in Fig. 1 a.

Use standard molecular biological technique, make up the dna encoding sequence of encoding fusion protein, in described fusion rotein, the N-terminal of 5T4 scFv merges in amino acid 215 backs of people B7-1.The sequence of this encoding sequence B7-1.5T4.1 is shown in Fig. 2.This fusion rotein contains a flexibility (gly-gly-gly-gly-ser) transcribed spacer between described B7-1 and 5T4 scFv sequence.Insert terminal (starting from Nucleotide 733) in this joint and introduce a BamH1 restriction site easily, other joint also is provided, screen with optimum expression according to bifunctional fusion proteins.Fig. 3 illustrates this fusion rotein.May make up described scFv equally and be N-terminal and B7-1.5T4.2 (Fig. 3 b) that described B7 extracellular domain is a C-terminal.In this case, the encoding sequence that only needs ripe B7-1 (not having signal peptide).In this case, the signal peptide such as the immunoglobulin (Ig) leader sequence can be added to the N-terminal of described scFv.

For using B7-2 to stimulate the fusion rotein of extracellular domain jointly, use the signal peptide of B7-2 and the sequence that extracellular domain replaces B7-1.Fig. 4 shows that SCM B7-2.5T4.1 stimulates the encoding sequence in territory jointly.Connect preceding 225 amino acid of people B7-2 and a flexible joint (gly4-ser) of its signal peptide before its coding.Can insert the structural domain (referring to Fig. 3) in 5T4scFv.1 downstream with the BamHI site of this sequence end.This sequence comprises described B7-2 signal peptide, and it can be used for allowing this fusion rotein secretion, and in this fusion rotein, described B7-2 territory is positioned at the N-terminal of this fusion rotein.

Every kind of engineered cDNA is inserted among the mammalian expression vector pCI, in the mammalian tissues culturing cell, express with permission.For this reason, a joint sequence is added to this encoding sequence 5 ' end, this joint sequence is introduced a restriction site easily that is used to insert the pCI polylinker, and is close to first ATG codon adding translation initiation signal CCACC.Formation thing among the pCI is transfected into suitable mammalian host cell line, such as COS-1, to confirm the secretion of described SCM.To fill to swell and treat in the expression vector as the gene delivery system of therepic use from transcribe box or the described suitable section Asia of transcribing box of pCI subsequently.

The embodiment 6-expression vector transfection monocytes/macrophages of coding SCM

With standard technique method (editors such as Sandlie and Michaelsen 1996 Antibody engineering:a practical approach.McCafferty, the 9th chapter) with laboratory scale, and by elutriation (for example Ceprate of CellPro) with on a large scale, from the human peripheral separating periphery blood monocytic cell.Be attached to plastics by spending the night, enrichment attached cell (being monocyte basically) can allow cell scavenger cell differentiation pathway break up by cultivating attached cell 1-3 week.

With expression vector transfection monocyte and the scavenger cell that can in people's cell, express SCM.In order to carry out the composing type high level expression, in utilizing the carrier pCI (Promega) of hCMV-MIE promoter-enhancer, express described SCM.In order to carry out the expression of hypoxia inducible, replace the hCMV promotor with the promotor that contains at least one HRE.Suitable promotor is HSV TK promotor with brachymemma of 3 copy mouse PGK HRE 1994Proc.Natl.Acad.Sci.91:6496-6500 such as () Firth.

Can carrier be introduced monocyte and scavenger cell with multiple infection protocol, described infection protocol comprises that the transfection that the DNA of particle mediation transmits (biological emission), electroporation, cationics mediation (for example uses Superfect, Qiagen).In these methods each all carried out according to manufacturer's explanation, and specifies according to each manufacturer, considers that parameter to be changed is to obtain optimum.Perhaps, can use virus vector such as defective adenoviral vector (Microbix Inc or QuantumBiotechnologies Inc.).

Embodiment 7-SCM is incorporated into the analysis of expressing CTLA-4 and the antigenic cell of 5T4

Expect that described B7-1 or B7-2 territory are incorporated into the human T-cell specifically and go up CD28 and the CTLA-4 that exists.Use following facs analysis, measure and the combining of the Chinese hamster ovary cell of T cell or choose CTLA-4 or CD28 transfection.With 5 * 10 ⁵The target cell of expression CTLA-4 or the use by oneself culture supernatant one of COS-1 cell of SCM gene transient transfection of the suitable cell (Chinese hamster ovary celI of untransfected) that lacks CTLA-4 and 0.1m1 arise from 4 ℃ of incubations 1 hour.Wash described cell, and with 1mg to the specific monoclonal antibody in described B7 territory (for example Mab 9E10) incubation, then with the anti-mouse IgG of FITC labelled goat (Pharbingen) incubation, and pass through facs analysis.

With expressing antigenic target cell of 5T4 (the A9 cell of 5T4 transfection) or control cells (A9), estimate scFv equally and combine with 5T4 is antigenic.

The analysis of the common stimulating activity of embodiment 8-

With the antigenic cDNA of coding people 5T4 (1994 J.Biol.Chem.269:9319-9324 such as Myers) that inserts expression vector pCIneo, the mouse cell lines in the Balb/c source that transfection is set up is such as the HC11 cell.

(Johnstone and Thorpe 1996 Immunochemistry inPractice.Blackwell. the 4th chapter) separates the splenic t-cell from the Balb/c mouse by standard method.By in the substratum that contains 10ng/ml PMA (Sigma) and 100U/ml people IL-12 (Boehringer Mannheim), cultivating 1-2 days, stimulate the T cell in advance.With in the 96 hole tissue culture dishes 10 ⁴The HC11-5T4 cell of cells/well with up to 0.1ml with the supernatant liquor incubation of the COS cell of SCM gene transfection 2 hours.Will be up to 10 ⁵The pre-T cell that stimulates adds in every hole, uses the 0.25mCi/ hole ³Handle described cell in the H-thymidine arteries and veins, after 24 hours, measure with the liquid flashing counting device ³Mixing of H-thymidine.

Expection ³Mixing of H-thymidine will be strengthened because of the existence of SCM.

The common analysis that stimulates in the embodiment 9-animal model

The HC11 cell of personnel selection 5T4 antigen gene (embodiment 4) transfection is cultivated as the tumour in the Balb/c mouse.Before implantation, the combination of SCM gene B7-1.5T4.1 or B7-2.5T4.1 or these two kinds of genes is introduced in the described tumour cell, and monitored the growth of not expressing the control tumor of SCM gene in described tumour and the body.

Believe that the SCM expression of gene causes the remarkable reduction of tumor growth.

The structure of embodiment 10-B7-1/ScFv (specific) fusion rotein to people 5T4

Use standard molecular biological technique, make up a fusion rotein, this fusion rotein by by flexible joint with to the V of the specific muroid Mab 5T4 of people 5T4 _HAnd V _LB7-1 leader sequence that merges and extracellular domain are formed.

By having two homology oligonucleotide annealing with engineered 5 ' SmaI of following oligonucleotide and 3 ' Spe I site, and make up the flexible joint of the extracellular domain and the described scFv that are used for connecting B7.1, described oligonucleotide is:

The upstream

5’GGG?GGT?GGT?GGG?AGC?GGT?GGT?GGC?GGC?AGT?GGC?GGC

GGC?GGA?A?3’

And downstream

5’CTA?GTT?CCG?CCG?CCG?CCA?CTG?CCG?CCA?CCA?CCG?CTC

CCA?CCA?CCC?CC?3’。

This joint filled by Sma I and Spe I swell, produce pLINK into pBluescript (Stratagene).By PCR, from the primer of pLK444-mB7.1 (by R.Germain NIH, the USA supply) by introducing 5 ' EcoRI and 3 ' Sma I site, signal peptide (sp) and the extracellular domain of amplification muroid B7.1, described primer is:

Forward

5’C?TCG?AAT?TCC?ACC? ATG?GCT?TGC?AAT?TGT?CAG?TTG?ATG?C

3’

Oppositely

5’CTC?CCC?GGG?CTT?GCT?ATC?AGG?AGG?GTC?TTC?3’。

Described B7.1PCR product is cloned into pLINK by Eco RI and Sma I, forms pBS/B7Link.

By following primer from the increase V of described 5T4 specificity ScFv of pHEN1-5T4ScFv _HAnd V _L:

Forward primer

5’CTC?ACT?AGT?GAG?GTC?CAG?CTT?CAG?CAG?TC?3’

Reverse primer

5’CTC?GCG?GCC?GCT?TAC?CGT?TTG?ATT?TCC?AGC?TTG?GTG

CCT CCA CC 3 ', described primer is introduced 5 ' Spe I and 3 ' Not I site.PBS/B7Link digests with Spe I and Nor I, and is connected to form the OBM 233 that is made up of the sequence that is expressed as SEQ ID No.5:B7 Link scFv sequence with described ScFv.

This syzygy is used for making up recombinant vectors, for example retrovirus, slow virus, adenovirus, poxvirus, vaccinia virus, baculovirus.This class carrier can be used for direct injection patient's tumour.For described fusion rotein is passed to tumour cell, monocytes/macrophages/CD34+ cell is before injecting back the patient, with the external transduction of described recombinant vectors.These cells will be transported to tumour.Described ScFv will be incorporated into the specific tumour antigen of expressing on the tumor cell surface, such as 5T4 (1994 JBC such as Myers).On special antigen presenting cell (for example scavenger cell, dendritic cell and B cell) surface, find B7.Part CD28 and CTL-A4 that it is positioned at it on CD4 and the cd8 cell interact.Interact in the time of B7/CD28/CTL-A4 and MHC-peptide/TXi Baoshouti, cause the remarkable increase of IL-2, this promotes amplification (the Linsley PS of CD8 (cytotoxic T cell), Brady W, Grosmaire L, Aruffo A, Damle NK, Ledbetter JA J Exp Med on March 1st, 1991; 173 (3): 721-730, the accumulation that B cell-stimulating antigen B7 and combining of CD28 stimulate T cell proliferation and I1-2 mRNA jointly).In animal model, the tumour cell with the B7 transfection has demonstrated retardation growth (Townsend SE, Allison JP Science 1,993 15; 259 (5093): 368-370).

The transient expression of embodiment 11-B7-1/ScFv and LScFv and purifying

In order to carry out the transient expression of B7-1/ScFv, end user CMV expression plasmid pCIneo (Promega).Downcut B7/ScFv by digesting from OBM 233, and it is filled the pCIneo that swells into using EcoRI/Not I digestion with EcoR I/Not I.(Profectin Promega), by using relevant plasmid transfection 293T cell, carries out the transient expression of recombinant protein to adopt calcium phosphate.The conditional likelihood of used condition and manufacturer's suggestion.Pollute in order to reduce bovine serum, use serum-free optimen substratum (Gibco BRL).After 36-48 hour, results transfection supernatant liquor, (Amicon, Glos.UK) 10 filter membranes (filter) (purifying/concentrated all albumen greater than 10kDa) and Centricon (Amicon) 10 filter membranes are centrifugal by Centriprep.About 30 times of supernatant concentration.

For bioactive B7-1, it must show with one of its native ligand (perhaps CTLA-4 or CD28) of finding on T cell special group (for example CD4+) surface and combine.Interaction when analyzing the A9 cell of bioactive B7-1/ScFv fusion rotein and its native ligand CTLA-4 (Ancell, MN, the CTLA4-Ig form of USA supply) and expressing human 5T4.In brief, in 96 orifice plates at the bottom of the U-shaped, with about 5 * 10 ⁵A9-h5T4 cell and 100ul or B7.1/ScFv or LScFv supernatant liquor were in 4 ℃ of incubations 1 hour.After the washing, cell was with CTLA4-Ig (Ancell) incubation 1 hour.After the washing, the anti-mouse Ig (Dako) that puts together with FITC detects bonded CTLA4-Ig.

The result shows that CTLA4-Ig combines with the obvious of B7-1 extracellular domain, is bonded to the positive A9 cell surface of people 5T4 by described ScFv.Shortage is active with combining of the negative A9 cell of 5T4, and the interaction that further specifies B7 and CTLA4-Ig and ScFv and 5T4 is specific.

Embodiment 12-ScFv-IgG fusion rotein example

The structure of ScFv-IgG

The sequence of coding translation initiation sequence and human normal immunoglobulin κ light chain signal peptide is synthetic as two complementary single stranded oligonucleotides, when described single stranded oligonucleotide is annealed, also contain an inner Xho I site of 5 ' end and stay 5 ' overhang of next Xba I coupling in addition and 3 ' overhang of a Pst I coupling, described oligonucleotide is:

ctagactcgagCCACC?ATG?GGA?TGG?AGC?TGT?ATC?ATC?CTC?TTC

TTG?GTA?GCA?ACA?GCT?ACA?GGT?GTC?CAC?TCC?GAG?GTC?CAG

ctgca

With

g?CTG?GAC?CTC?GGA?GTG?GAC?ACC?TGT?AGC?TGT?TGC?TAC

CAA?GAA?GAG?GAT?GAT?ACA?GCT?CCA?TCC?CAT

GGTGGctcgagt。

Then, this sequence clone is gone among the pBluescript II (Stratagene) that limits with Xba I and Pst I, produce pBSII/Leader.

By PCR, with the following oligonucleotide that mixes a Pst I site and mix a Hind III in 3 ' end in this product 5 ' end from the pHEN1 described 5T4 scFv that increases:

GTC?CAG?CTG?CAG?CAG?TCT?GG

With

CG?TTT?GAT?TTC?AAG?CTT?GGT?GC。

Then it is limited with those enzymes, and insert among the pBRII/Leader that limits with same enzyme, produce pBRII/Leader/scFv.

By PCR, with the following oligonucleotide that mixes a Hind III site and mix an Xho I site in 3 ' end in 5 ' end from the cloned genes amplification HIgG of institute 1 constant region:

gcgc?AAG?CTT?gaa?atc?aaa?cgg?GCC?TCC?ACC?AAG?GGC?CCA

With

gcgc?ctcgag?TCA?TTT?ACC?CGG?AGA?CAG?GG。

Then it is limited with those enzymes, and insert among the pBRII/Leader/scFv that limits with same enzyme, produce pBRII/Leader/scFv/HG1.This sequence that constitutes thing is shown in the drawings.

This syzygy is used for making up recombinant vectors, for example retrovirus, slow virus, adenovirus, poxvirus, vaccinia virus, baculovirus.This class carrier can be used for direct injection patient's tumour.For described fusion rotein is passed to tumour cell, monocytes/macrophages/CD34+ cell is before injecting back the patient, with the external transduction of described recombinant vectors.These cells will be transported to tumour.Described ScFv will be incorporated into the specific tumour antigen of expressing on the tumor cell surface, such as 5T4 (1994 JBC such as Myers).Bonded IgG will promote the destruction (ibid for Can res 1991 such as Munn, and ibid for 1993 Cancer Res such as Primus) of specific tumors by the cytotoxicity that set is referred to as antibody dependent cellular.

The structure of embodiment 13-ScFv-IgE1 (people IgE1 CH)

Preparation constitutes thing by the fusion of the similar 5T4 scFv-people IgE CH of forming as the sequence shown in the SEQ ID No.6.

Pass through PCR, also use following the end to mix a Hind III site and mix the oligonucleotide in an Xho I site subsequently by RT in 3 ' end in 5 ', amplification derives from the people IgE1 CH cDNA of human B cell RNA, prepares this fusion and constitutes thing, and described oligonucleotide is:

gcgc?AAG?CTT?gaa?atc?aaa?cgg?GCC?TCC?ACA?CAG?AGC?CCA

With

gcgc?ctcgag?TCA?TTT?ACC?GGG?ATT?TAC?AGA。

Then it is limited with those enzymes, and insert among the pBSII/Leader/scFv that limits with same enzyme, produce pBSII/Leader/scFv/HE1.

As mentioned above, described ScFv-IgE can be constituted thing and add in the recombinant viral vector, to be used for the gene therapy of cancer, for example direct injection patient tissue, or external transduction derives from monocytes/macrophages/CD34+ cell of patient.This fusion rotein will be secreted, and is incorporated into and has described ScFv to its specific antigenic tumour cell.IgE is incorporated into tumour cell should promote strong histamine response by activating mastocyte.This will cause strong inflammatory reaction and destroy tumour cell, as the IgE that reports destroys the cytotoxicity of parasite (for example worm larva) (eosinophilic granulocyte in Capron M 1988 diseases: acceptor and medium. be stated from: the 6th page of transformation reactions and clinical immunology progress (progress in allergy and clinical immunology) (Proc.13th Int.Congress of Allergy and Clinical Immunology) Hogrefe and Huber Toronto).This inflammation and tumor destruction should start raising of other immune effector cell.The report in past is pointed out, handles with MMTV antigen-specific IgE Mab, causes resisting protection (Nagy E Istanvan B, SehonAH 1991 Cancer Immunol.Immunotherapy the 34th volume: 63-69) of expressing the antigenic tumour of MMTV.

The structure of embodiment 14-B7/EGF

The B7-EGF synthetic gene

By inserting pBS/B7 Link from the gene region amplification PCR products of encoding mature EGF peptide (referring to registration number X04571), the fusion of preparation B7-EGF constitutes thing.This formation thing has as the sequence shown in the SEQ ID No.7.

By obtaining cDNA to carrying out RT from the isolated RNA of clone (ATCC:CRL1573) such as 293 people's kidneys system, adopt this cDNA to pass through PCR, contain a Spe I restriction enzyme site and contain the oligonucleotide in a terminator codon and a Not I site in C-terminal in N-terminal with following, the described DNA that increases, described oligonucleotide is:

GG?ACT?AGT?AAT?AGT?GAC?TCT?GAA?TGT?CCC

With

ATT?AGC?GGC?CGC?TTA?GCG?CAG?TTC?CCA?CCA?CTT?C。

Products therefrom is with the restriction of those enzymes, and connects among the pBS/B7Link with the same enzyme restriction, produces pBS/B7 Link EGF.Downcut described B7Link EGF box with Eco RI and Not I then, be inserted in the derivative (Soneoka etc., 1995, Nucl Acid Res 23:628) of the pHIT111 that no longer carries the LacZ gene.

Use the other method of ScFv to be to use its corresponding acceptor is had the somatomedin of high-affinity, Urogastron for example, it is incorporated into several acceptors, comprises the erb-2 with tumour generation height correlation.

As mentioned above, described fusion can be constituted thing and add in the recombinant viral vector, to be used for gene therapy, for example direct injection patient tissue, or external transduction derives from monocytes/macrophages/CD34+ cell of patient.This fusion rotein will be secreted, and is incorporated into and has the antigenic tumour cell of described erb-2.

Urogastron (EGF) will be incorporated into its part erb-2 (a kind of EGF acceptor), eliminate the needs of ScFv thus.Erb-2 and tumour cell height correlation (Hynes NE SeminCancer Biol 1993 Feb; 4 (1): 19-26, erb-2 gene amplification and the overexpression in people's tumour: its participates in tumor development, as the importance of prognostic factor with as the potentiality of the target of cancer therapy).On special antigen presenting cell (for example scavenger cell, dendritic cell and B cell) surface, find B7.It interacts with its part CD28 and the CTL-A4 that are positioned on CD4 and the cd8 cell.Interact in the time of B7-CD28/CTL-A4 and MHC-peptide/TXi Baoshouti, cause the remarkable increase of IL-2, this promotes amplification (the Linsley PS of CD8 (cytotoxic T cell), Brady W, Grosmaire L, Aruffo A, Damle NK, LedbetterJA J Exp Med on March 1st, 1991; 173 (3): 721-730, the accumulation that B cell-stimulating antigen B7 and combining of CD28 stimulate T cell proliferation and interleukin-2 mRNA jointly).In animal model, the tumour cell with the B7 transfection has demonstrated retardation growth (Townsend SE, Allison JP Science 1,993 15; 259 (5093): the directly common tumor rejection that stimulates the melanoma cells of back B7 transfection of 368-370 B7 CD8+T cell).Reported that the CTL that B7 will strengthen the tumour antigen of tumor cell specific replys, caused the destruction of all these cells thus.

Embodiment 15-expresses the generation of merging the clone that constitutes thing

By Xho I digestion, downcut described ScFV-IgG gene from pBSII/L/ScFv/hIgG1, it is cloned into pLXSN by Xho I site, produce pLXSN/ScFv-IgG, make that it is in transcribing under the control of described LTR behind the chromosomal integration.By adopting three plasmid HIT systems (Landau and Littman 1992 J Virol 66 5510,1995NAR 23:628-633 such as Soneoka Y), common transfection contains the plasmid (pCIEGPPD) of described MLV gap-pol gene and contains the plasmid (pRV67) of VSV G coating, is preparation virus among the 293T in human kidney cells.Results virus after 48 hours, BHK-21 cell (ATCC#CCL-10) is used for transduceing.The cell of transduction by 1mg/ml G418 (Gibco BRL) is added in the substratum, is selected in about 24 hours of transduction back.Results are from the supernatant liquor of positive bacterium colony, by Centriprep (Amicon, Glos.UK) centrifugal the concentrating of 10 filter membranes (purifying/concentration is greater than all albumen of 10kDa) Centricon (Amicon) 10 filter membranes.About 30 times of supernatant concentration.

Adopt similar scheme, other fusion rotein is cloned into pLXSN by Xho I site, express and concentrate.

Fusion rotein and the cell bonded facs analysis of expressing specific ligand

In order to determine that whether described ScFv-IgG fusion rotein is specific to its antigen people 5T4, be that A9-neo carries out facs analysis to human bladder cancer's tumour system (EJ) or stable muroid clone A9-h5T4 (1994 JBC such as Myers) and the 5T4 feminine gender of expressing h5T4.In round bottom 96 orifice plates (Falcon), with about 5 * 10 ⁵1: 5 diluent one of A9 or EJ cell and 100ul concentrated supernatant (as mentioned above) arises from 4 ℃ of incubations 1 hour.After the washing, the antibody of puting together with anti-human IgG/FITC (Dako) detects bonded albumen.Analysis of cells on Becton Dickinson FACS instrument.FACS result shows, constitutes thing with the negative control of only being made up of described ScFv albumen and compares, and is constituting in those 5T4 positive cells of thing processing with described ScFv-IgG, and fluorescence activity changes 10 times at least.A9 neo FACS shows, not the non-specific binding of this fusion rotein ScFv component.

With the above facs analysis that similarly carries out ScFv-IgE, only be to use anti human IgE-FITC (Dako) to monitor the combination of this fusion rotein.

With FACS and HC11-erb-2 positive cell (Hynes etc. 1990), analyze the combination of described B7/EGF fusion rotein.With CTLA4-Ig (Ancell, USA) biological activity of the B7 component of analysis bonded fusion rotein.Show the combination of CTLA-4 with anti-mouse IgG-FITC.Sum up

Therefore, the invention provides and be passed to the means of tumor locus for example treating compound.

All publications of mentioning in above specification sheets all are attached to herein by reference.Do not depart from the scope of the present invention and the method for the invention of spirit and the various modifications and variations of system, it will be apparent to those skilled in the art that.Although described the present invention, should be appreciated that claimed the present invention should excessively not be limited to this class specific embodiments in conjunction with concrete preferred embodiment.In fact, the various modifications of implementing described pattern of the present invention are conspicuous to biology field or those skilled in the relevant art, and they will belong to the scope of following claims.

Sequence table

SEQ?ID?NO.1

Referring to Fig. 1 a.

SEQ?ID?NO.2

Referring to Fig. 1 b.

SEQ?ID?NO.3

Referring to Fig. 2.

SEQ?ID?NO.4

Referring to Fig. 4.

SEQ?ID?NO.5

ATGGCTTGCAATTGTCAGTTGATGCAGGATACACCACTCCTCAAGTTTCCATGTCCAAGGCTCATTCTTCTCTTTGTGCT

80GCTGATTCGTCTTTCACAAGTGTCTTCAGATGTTGATGAACAACTGTCCAAGTCAGTGAAAGATAAGGTATTGCTGCCTT

160GCCGTTACAACTCTCCGCATGAAGATGAGTCTGAAGACCGAATCTACTGGCAAAAACATGACAAAGTGGTGCTGTCTGTC

240ATTGCTGGGAAACTAAAAGTGTGGCCCGAGTATAAGAACCGGACTITATATGACAACACTACCTACTCTCTTATCATCCT

320GGGCCTGGTCCTTTCAGACCGGGGCACATACAGCTGTGTCGTTCAAAAGAAGGAAAGAGGAACGTATGAAGTTAAACACT

400TGGCTTTAGTAAAGTTGTCCATCAAAGCTGACTTCTCTACCCCCAACATAACTGAGTCTGGAAACCCATCTGCAGACACT

480AAAAGGATTACCTGCTTTGCTTCCGGGGGTTTCCCAAAGCCTCGCTCTCTTGGTTGGAAAAATGGAAGAGAATTACCTGG

560CATCAATACGACAATTTCCCAGGATCCTGAATCTGAATTGTACACCATTAGTAGCCAACTAGATTTCAATACGACTCGCA

640ACCACACCATTAAGTGTCTCATTAAATATGGAGATGCTCACGTGTCAGAGGACTTCACCTGGGAAAAACCCCCAGAAGAC

720CCTCCTGATAGCAAGCCCGGGGGTGGTGGGAGCGGTGGTGGCGGCAGTGGCGGCGGCGGAACTAGTGAGGTCCAGCTTCA

800GCAGTCTGGACCTGACCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATICACTGGCT

880ACTACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGACGTATTAATCCTAACAATGGTGTTACT

960CTCTACAACCAGAAATTCAAGGACAAGGCCATATTAACTGTAGACAAGTCATCCACCACAGCCTACATGGAGCTCCGCAG

1040CCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCTACTATGATTACGAACTATGTTATGGACTACTGGGGTC

1120AAGTAACTTCAGTCACCGTCTCTTCAGGTGGTGGTGGGAGCGGTGGTGGCGGCACTGGCGGCGGCGGATCTAGTATTGTG

1200ATGACCCAGACTCCCACATTCCTGCTTGTTTCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAG

1280TAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTACACTGCTCATATCCTATACATCCAGTCGCTACGCTG

1360GAGTCCCTGATCGCTTCATTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTTTGCAGGCTGAAGACCTG

1440GCAGTTTATTTCTGTCAGCAAGATTATAATTCTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGTAA?1518

SEQ?ID?NO.6

CTCGAGCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCGAGGTCCAGCTG

80CAGCAGTCTGGACCTGACCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGG

160CTACTACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGACGTATTAATCCTAACAATGGTGTTA

240CTCTCTACAACCAGAAATTCAAGGACAAGGCCATATTAACTGTAGACAAGTCATCCACCACAGCCTACATGGAGCTCCGC

320AGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCTACTATGATTACGAACTATGTTATGGACTACTGGGG

400TCAAGTAACTTCAGTCACCGTCTCTTCAGGTGGTGGTGGGAGCGGTGGTGGCGGCACTGGCGGCGGCGGATCTAGTATTG

480TGATGACCCAGACTCCCACATTCCTGCTTGTTTCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTG

560AGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTACACTGCTCATATCCTATACATCCAGTCGCTACGC

640TGGAGTCCCTGATCGCTTCATTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTTTGCAGGCTGAAGACC

720TGGCAGTTTATTTCTGTCAGCAAGATTATAATTCTCCTCCGACGTTCGGTGGAGGCACCAAGCTTGAAATCAAACGGGCC

800TCCACACAGAGCCCATCCGTCTTCCCCTTGACCCGCTGCTGCAAAAACATTCCCTCCAATGCCACCTCCGTGACTCTGGG

880CTGCCTGGCCACGGGCTACTTCCCGGAGCCGGTGATGGTGACCTGGGACACAGGCTCCCTCAACGGGACAACTATGACCT

960TACCAGCCACCACCCTCACGCTCTCTGGTCACTATGCCACCATCAGCTTGCTGACCGTCTCGGGTGCGTGGGCCAAGCAG

1040ATGTTCACCTGCCGTGTGGCACACACTCCATCGTCCACAGACTGGGTCGACAACAAAACCTTCAGCGTCTGCTCCAGGGA

1120CTTCACCCCGCCCACCGTGAAGATCTTACAGTCGTCCTGCGACGGCGGCGGGCACTTCCCCCCGACCATCCAGCTCCTGT

1200GCCTCGTCTCTGGGTACACCCCAGGGACTATCAACATCACCTGGCTGGAGGACGGGCAGGTCATGGACGTGGACTTGTCC

1280ACCGCCTCTACCACGCAGGAGGGTGAGCTGGCCTCCACACAAAGCGAGCTCACCCTCAGCCAGAAGCACTGGCTGTCAGA

1360CCGCACCTACACCTGCCAGGTCACCTATCAAGGTCACACCTTTGAGGACAGCACCAAGAAGTGTGCAGATTCCAACCCGA

1440GAGGGGTGAGCGCCTACCTAAGCCGGCCCAGCCCGTTCGACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGTG

1520GTGGACCTGGCACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGCCAGTGGGAAGCCTGTGAACCACTCCACCAG

1600AAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCACGTCCACCCTGCCGGTGGGCACCCGAGACTGGATCGAGGGGG

1680AGACCTACCAGTGCAGGGTGACCCACCCCCACCTGCCCAGGGCCCTCATGCGGTCCACGACCAAGACCAGCGGCCCGCGT

1760GCTGCCCCGGAAGTCTATGCGTTTGCGACGCCGGAGTGGCCGGGGAGCCGGGACAAGCGCACCCTCGCCTGCCTGATCCA

1840GAACTTCATGCCTGAGGACATCTCGGTGCAGTGGCTGCACAACGAGGTGCAGCTCCCGGACGCCCGGCACAGCACGACGC

1920AGCCCCGCAAGACCAAGGGCTCCGGCTTCTTCGTCTTCAGCCGCCTGGAGGTGACCAGGGCCGAATGGGAGCAGAAAGAT

2000GAGTTCATCTGCCGTGCAGTCCATGAGGCAGCGAGCCCCTCACAGACCGTCCAGCGAGCGGTGTCTGTAAATCCCGGTAA

2080ATGAGAGCTC?2090

SEQ?ID?NO.7

ATGGCTTGCAATTGTCAGTTGATGCAGGATACACCACTCCTCAAGTTTCCATGTCCAAGGCTCATTCTTCTCTTTGTGCT

80GCTGATTCGTCTTTCACAAGTGTCTTCAGATGTTGATGAACAACTGTCCAAGTCAGTGAAAGATAAGGTATTGCTGCCTT

160GCCGTTACAACTCTCCGCATGAAGATGAGTCTGAAGACCGAATCTACTGGCAAAAACATGACAAAGTGGTGCTGTCTGTC

240ATTGCTGGGAAACTAAAAGTGTGGCCCGAGTATAAGAACCGGACTTTATATGACAACACTACCTACTCTCTTATCATCCT

320GGGCCTGGTCCTTTCAGACCGGGGCACATACAGCTGTGTCGTTCAAAAGAAGGAAAGAGGAACGTATGAAGTTAAACACT

400TGGCTTTAGTAAAGTTGTCCATCAAAGCTGACTTCTCTACCCCCAACATAACTGAGTCTGGAAACCCATCTGCAGACACT

480AAAAGGATTACCTGCTTTGCTTCCGGGGGTTTCCCAAAGCCTCGCTTCTCTTGGITGGAAAATGGAAGAGAATTACCTGG

560CATCAATACGACAATTTCCCAGGATCCTGAATCTGAATTGTACACCATTAGTAGCCAACTAGATTTCAATACGACTCGCA

640ACCACACCATTAAGTGTCTCATTAAATATGGAGATGCTCACGTGTCAGAGGACTTCACCTGGGAAAAACCCCCAGAAGAC

720CCTCCTGATAGCAAGCCCGGGGGTGGTGGGAGCGGTGGTGGCGGCAGTGGCGGCGGCGGAACTAGTAATAGTGACTCTGA

800ATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCATGTATATTGAAGCATTGGACAAGTATGCATGCA

880ACTGTGTTGTTGGCTACATCGGGGAGCGATGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGC?945

Claims (39)

1. carrier, it comprises the nucleotide sequence (" NS ") of codes for tumor interact protein (" TIP ") and can randomly comprise purpose nucleotide sequence (" NOI "), described NOI coding purpose product (" POI "); Wherein said TIP can discern tumour, makes that in use described carrier can be passed to described tumour with described NOI and/or described POI.

2. according to the carrier of claim 1, wherein said carrier comprises described NOI.

3. according to the carrier of claim 2, wherein said NOI is treatment POI for treating NOI and/or described POI.

4. according to the carrier of arbitrary aforementioned claim, wherein in use described carrier can be passed to tumor mass inside with described NOI and/or described POI.

5. according to the carrier of arbitrary aforementioned claim, wherein said TIP is a tumour conjugated protein (" TBP ") or to comprise tumour conjugated protein.

6. according to the carrier of arbitrary aforementioned claim, wherein said TIP is TBP.

7. according to the carrier of arbitrary aforementioned claim, wherein said NS and/or described TIP comprise at least a tumour in conjunction with the territory, and described tumour can interact with at least one tumour relevant cell surface molecular (" TACSM ") in conjunction with the territory.

8. according to the carrier of claim 7, wherein said TACSM optionally expresses on a kind of cell type or a limited number of cell type.

9. according to the carrier of arbitrary aforementioned claim, wherein in use described carrier can be passed to described NOI and/or described POI the tumor locus of selection.

10. according to the carrier of arbitrary aforementioned claim, wherein said TIP is the part of antibody or a part that comprises antibody at least at least.

11. according to the carrier of arbitrary aforementioned claim, wherein said TIP discerns a kind of trophocyte's surface antigen.

12. according to the carrier of claim 11, wherein said TIP identification 5T4 antigen.

13. according to the carrier of arbitrary aforementioned claim, wherein said NS and NOI and/or described TIP and POI interconnect.

14. according to the carrier of claim 13, wherein said TIP and POI directly link together.

15. according to the carrier of arbitrary aforementioned claim, any among wherein said NS, NOI, TIP and the described POI or multiplely also comprise at least a other function ingredients.

16. according to the carrier of arbitrary aforementioned claim, wherein described at least TIP and/or POI also comprise at least a other function ingredients.

17. according to the carrier of claim 15 or 16, wherein said other function ingredients is selected from following any or multiple: signal entity (such as signal peptide), immunostimulant, toxin or bioactive enzyme or coding be the sequence of arbitrary above-mentioned substance wherein.

18. according to the carrier of arbitrary aforementioned claim, wherein said retrovirus vector comprises a kind of tumor-specific promoters enhanser.

19. according to the carrier of arbitrary aforementioned claim, wherein said carrier is a retrovirus vector.

20. be passed to the method for tumour with purpose nucleotide sequence (" NOI ") and/or by the purpose product (" POI ") of NOI coding, wherein utilize the carrier that comprises described NOI and/or express described POI that described NOI and/or POI are passed to described tumour; Wherein said NOI and/or described POI can discern tumour; Wherein described NOI and/or described POI are passed to described tumour; And wherein said carrier is the carrier of arbitrary aforementioned claim.

21. according to the method for claim 20, wherein said carrier be used for described NOI and/or POI is external and/or body in be passed to described tumour.

22. carrier is passed to the purposes of tumour with purpose nucleotide sequence (" NOI ") and/or by the purpose product (" POI ") of NOI coding, wherein utilizes the carrier that comprises described NOI and/or express described POI that described NOI and/or POI are passed to described tumour; Wherein when described NOI and/or described POI were passed to tumour, described NOI and/or described POI can discern described tumour; And wherein said carrier is the carrier of arbitrary aforementioned claim.

23. according to the purposes of claim 22, wherein said carrier be used for described NOI and/or POI is external and/or body in be passed to described tumour.

24. treatment needs the curee's of treatment method, described method comprises with purpose nucleotide sequence (" NOI ") and/or by the purpose product (" POI ") of NOI coding and is passed to tumour, wherein utilizes the carrier that comprises described NOI and/or express described POI that described NOI and/or POI are passed to described tumour; Wherein said NOI and/or described POI can discern tumour; Wherein described NOI and/or described POI are passed to described tumour; And wherein said carrier is the carrier of arbitrary aforementioned claim.

25. according to the method for claim 24, wherein said carrier be used for described NOI and/or POI external/or body in be passed to described tumour.

26. the purposes of genophore, but soon the therapeutic gene of coding excretory TIP (preferably TBP) is passed to tumor mass inside.

27. gene delivery system, be used for the coding TIP (preferably TBP) one or more gene targeting tumours, described system comprise the coding TIP (preferably TBP) genophore and vivo gene transfer system.

28. the treatment method for cancer comprises according to (preferably at least a TBP) gene or the system of at least a TIP in the gene delivery system of claim 27 or directly give tumor locus.

29. gene delivery system, be used for the coding TIP (preferably TBP) one or more genes or body in or the cytophyletic cell of external introducing hematopoiesis (preferably marrow hemopoiesis).

30. the method for treatment mammalian cancer comprises giving individuality with the gene delivery system according to claim 29, described gene delivery system can be expressed TIP (preferably TBP) in the cell that derives from hematopoiesis (preferably marrow hemopoiesis) source.

31. genophore, it comprises the therapeutic gene of one or more coding TIP (preferably TBP), and selectivity has the expression regulatory element of function functionally to be connected in described therapeutic gene and the cell type that exists in tumor mass.

32. genophore, it comprises one or more therapeutic genes to described inside tumor to be passed, wherein said therapeutic gene coding TIP (preferably TBP), and described TIP also contains one or more effector domains.

33. the method for treatment mammalian cancer, described method comprise a kind of cytokine or cytokine encoding gene and one or more TIP (preferably TBP) gene in conjunction with giving individuality.

34.TIP (preferably TBP) encoding gene is to the transmission of tumor locus.

35. comprise NS and the b of (a) coding TIP) carrier of NOI of coding POI; Wherein said TIP can discern tumour, makes that in use described carrier can be passed to described tumour with described NOI and/or described POI; And wherein said TIP and POI merge mutually.

36. comprise the NS of (a) coding TIP and the carrier of the NOI of the POI that (b) encodes; Wherein said TIP can discern tumour, makes that in use described carrier can be passed to described tumour with described NOI and/or described POI; Wherein said TIP and POI merge mutually; And wherein said POI can be secreted.

37. according to the purposes of the carrier of arbitrary aforementioned claim, promptly as any or multiple produced in situ factory among described NS, NOI, POI and the TIP.

38. according to the purposes of the carrier of arbitrary aforementioned claim, in the time of promptly in being present in cell with any or multiple flanking cell that is passed among described NS, NOI, POI and the TIP.

39. carrier roughly as described herein.

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