CN1196786C

CN1196786C - Methods for preventing graft rejection in transplantation and for producing universal gene therapy host cell

Info

Publication number: CN1196786C
Application number: CNB961805102A
Authority: CN
Inventors: M·比弗尼; R·帕波恩
Original assignee: Applied Research Systems ARS Holding NV
Current assignee: Serono Lab
Priority date: 1996-11-29
Filing date: 1996-11-29
Publication date: 2005-04-13
Anticipated expiration: 2016-11-29
Also published as: CN1234834A

Abstract

The present invention relates to a method for preventing transplantation rejection of transplanted cells, tissues or organs under the condition of no general immunity inhibition, which utilizes proteins LAG-3 newly discovered. When allogeneic or xenogeneic cells are modified by genes to express LAG-3 on the surface and be transplanted, the immunity destruction of the transplanted cells, the transplanted tissues or the transplanted organs can be avoided, and meanwhile, the functions of a parasitifer immune system are kept. The method has one concrete application that universal gene treatment host cells for expressing LAG-3 on the surface are prepared, and thereby, the method has the protection action on preventing transplantation rejection of the parasitifer immune system from occurring.

Description

Resisting transplant rejection cell and uses thereof

Invention field

The present invention relates to prevent that transplanted organ, tissue or cell from transplanting the method that (thing) repels, relate in particular to such method, this method comprises that the pair cell type carries out genetically engineeredly can expressing LAG-3 albumen when making it implant the host.More specifically, the present invention relates to be created in the proteic universal gene therapy host cell of its surface expression LAG-3.

The description of background technology

Lymphocyte activation gene (lymphocyte activation gene) is a member of immunoglobulin superfamily (LAG-3), and it is at people's activating T cell (CD4 ⁺And CD8 ⁺) and the NK cell in optionally transcribed (people such as Triebel, 1990).Its sequence data and through comparing with intron/exon weave construction and chromosomal localization discloses LAG-3 and CD4 be closely related people such as (, 1992) Baixeras.Close relation between LAG-3 and the CD4 has also obtained further confirmation, promptly both to share identical parts be MHC II quasi-molecule people such as (, 1992) Baixeras.Yet opposite with CD4, LAG-3 does not combine (people such as Baixeras, 1992) with human immunodeficiency virus gp120.In vivo, the expression of LAG-3 is not discovery in primary lymphatic organ (as spleen, the Lymphoid tissue relevant with mucous membrane) both, also less than not finding in normal lymphoglandula.Yet, can in the lymphoglandula of infected tonsilla or FH, detect it easily, this phenomenon has been supported such viewpoint: even in vivo, the expression of LAG-3 be after activation (people such as Huard, 1994A).In anti--LAG-3 monoclonal antibody (mAb) down, to the T cell clone carry out the higher and cytokine of expression that the antigen-specific sexual stimulus can cause the increase of thymidine incorporation, activation marker CD25 the output increase (people such as Huard, 1994B).

Therefore, add solubility reorganization LAG-3 and can suppress T cells with antigenic specificity propagation, this hint LAG-3 has regulating and controlling effect (Huard, 1996) on and relates to and extinguish the immune response that is just carrying out at the CD4+T-lymphocyte activator.Recently, show that LAG-3 is also as the collaborative acceptor of NK cell, and define different mode people such as (, 1996) Miyazaki of the killing tumor cell that is subjected to innate immune system control.

The mechanism that the T cell is replied external (allogeneic or xenogenesis) albumen or cell or organ, solve quite clear.Antigen presenting cell (APC) can attracted to inflammation or injured area (this can bring out because of operation transplantation).Can ceaselessly check at whole T cells of periphery and to organize the evidence whether pathogenic agent is arranged or not have external (allogeneic or xenogenesis) tissue.In case discern any warning signal, APC will swallow albumen, digests it and it is the immunity system of passing the host.

Already tumour cell allosome or homologous is carried out genetically engineeredly, expressing viral IL-10, this can induce the local anergy of tumour cell.This processing does not influence the non-transduction tumour in position far away and repels people such as (, 1995) SuZuki.Local I L-10 administration, the whole T cells that are believed to transplanted cells to be reacted change the Th2 phenotype into, and this phenotype does not cause cytolysis and even can shield.

Under no immunosuppressant situation, the cell of natural expression Fas part portable passes through allosome or allos barrier.Host T cell is to the supervision of implantation site, can cause the killing and wounding of T cell people such as (, 1995) Bellgrau when contacting with the Fas part.In addition, the rejection of pancreas islet allogeneic can be homologous by transplanting simultaneously, prevented (people such as Lau, 1996) through the sarcoplast of the genetically engineered Fas of expression part.

Immune well-equipped, can discern the tissue of external, sick or inflammation apace, and be destroyed fast.This is in tissue, organ and the Transplanted cells and the major obstacle in the gene therapy always.Subject matter suppresses, seals with permanent immunity usually or immunity is isolated relevant.The adverse side effect that permanent immunity suppresses comprises: the susceptibility to opportunistic infection and tumour formation rises.

Need not continue under the immunosuppressant situation, wish to accept transplanted tissue for a long time, be target that exists for a long time on the physianthropy.

This paper quotes arbitrary document, and does not mean that and admit that the document is the prior art of being correlated with, and does not also think the material relevant with the patentability of the application's claim.For the statement on interior perhaps date of any document, be based on the applicant in the application obtainable information, thereby the exactness of not representing to admit this statement.

Summary of the invention

Have now found that, transplant and express the proteic cell of LAG-3 in its surface, can shield to avoid the transplant rejection phenomenon of host immune system.

Therefore; the invention provides a kind of cell of genetically engineered mistake; this cell can be the part of tissue or organ to be transplanted; what this cell contained that coding is positioned at the surface strides the proteic DNA of film LAG-3; thereby shield and avoid the transplant rejection effect of host immune system, wherein this DNA can be genomic dna or cDNA.This DNA can be external source or in an object lesson of the present invention, be endogenous, the expression of this DNA can utilize homologous recombination to insert regulating and controlling sequence by orientation and/or amplifiable gene is activated or modifies.LAG-3 albumen is can be by the protein of discerning at the antibody of LAG-3.

When this cell is when treating transplanted tissue or organ a part of, can be directly in the transfection for the treatment of to realize on transplanted tissue or the organ LAG-3 DNA.

Particularly, this cell is general gene therapy host cell, and it is applicable to for example somatic or " sv " gene therapy of any kind of.

In an object lesson, gene therapy host cell also comprises the foreign DNA of the therapeutical agent interested of encoding, thereby the cell of genetically engineered mistake is used as therapeutical agent.As used herein, term " treatment " comprises treating to be controlled and/or prevents.

In another example, the gene of the therapeutical agent interested of encoding is arranged in cellular genome, and this cell also contains the foreign DNA of coding regulating and controlling sequence and/or amplifiable gene, with the expression that activates or modify native gene interested.

Yet genetically engineered cell of the present invention can only contain external source LAG-3 DNA, and unites use with other gene therapy host cells that contain interested therapeutic DNA.

Cell of the present invention should be selected from: sarcoplast, inoblast, hemopoietic stem cell, embryonic stem cell, fetal liver cells, huve cell and Chinese hamster ovary celI.

Aforesaid, derived from the cells of transgenic animal also within the scope of the invention.

Another object of the present invention be on cell surface, express stride the film LAG-3 albumen purposes of (comprising its mutain and variant protein), they are used to make medicine, this medicine can cause the provide protection that prevents host immune system generation transplant rejection.

In addition, the invention provides the purposes of the cell of containing encodes strides film LAG-3 albumen (this albumen is expressed) on cell surface DNA, they are used to make medicine, and this medicine can cause the provide protection that prevents host immune system generation transplant rejection.

Within the scope of the invention, also comprise the purposes of the cell of expressing LAG-3 in its surface, promptly be used to make and cell to be transplanted, tissue or organ blended medicine mutually, this medicine can cause the provide protection that prevents host immune system generation transplant rejection.

The accompanying drawing summary

(primed) mouse boosting cell of Fig. 1-usefulness LAG-3-CHO cell or LH-CHO cell sensitization is for the cytotoxicity of LH-CHO cell.Mean value (± SD) come to carry out shown in the legend freely 5 mouse of sensitization; 2 unsensitized mouse have been assessed in addition.

The mouse boosting cell of Fig. 2-usefulness LAG-3-CHO cell or LH-CHO cell sensitization is for the cytotoxicity of LAG-3-CHO cell.Mean value (± SD) come to carry out shown in the legend freely 5 mouse of sensitization; 2 unsensitized mouse have been assessed in addition.

The structure iron of Fig. 3-D α mammalian expression vector.The abbreviation of using is as follows: DHFR, dehydrofolic acid transcriptional units people such as (, 1981) Subraimani; PML, the derivative of pBR322 (Lusky and Botchan, 1981); HAIVSA, the fragment of the intron A of human glycoprotein hormone α subunit (Fiddes and Goodman, 1981); MMT-1, the promotor of mouse metallothionein(MT) 1 (Hamer and Walling, 1982).

Detailed Description Of The Invention

In every year, thousands of people dies because of the heart, kidney, liver, lung and dying of exhaustion of pancreas. The most effective a kind of therapy is transplanted exactly.

With the therapy of transplanted cells, tissue or correlation among organs, can in the host, bring out the general immunity guard mode for cell, tissue or the organ implanted. Need to set up the repulsion of the specificity protection of graft being avoided host immune system, especially in heteroplastic transplantation, heterograft and gene therapy. In addition, also need to suppress the tolerance to tumor tissues, or otherwise allow host immune system attack tumor tissues.

Therefore, the present invention relates to all said methods, these methods have been utilized the transplanting of the cell or tissue of expressing cross-film LAG-3 albumen, can protect graft to avoid this discovery of host immune system generation graft rejection.

The present invention adopts newfound LAG-3 gene and albumen, and it is normally expressed at the NK cell of the T cell that activates and activation.

As used herein, definition " cross-film LAG-3 albumen " refers to the transmembrane protein in any LAG-3 of containing cytoplasm external structure territory, its salt, functional derivatives, precursor or active part, with and active mutain and its active variation's body, they are all expressed at cell surface.

This definition also refers to such transmembrane protein, it is expressed with its native state, maybe can be blended in by for example genetic engineering any relevant fragment of other albumen (such as glycosyl-phosphatidyl inositol anchor albumen) or other transmembrane proteins (such as TNF acceptor, MPL-part or cross-film immunoglobulin (Ig)).

As used herein, definition " salt " refers to by the carboxylic salts of the compound of known method acquisition or the salt of amido functional group. Carboxylic salts comprises inorganic salts, and for example sodium salt, sylvite, calcium salt, and the salt that forms with organic base are such as the salt that forms with amine such as triethanolamine, arginine, lysines. Amino salt comprises: the salt that for example forms with the inorganic acid example hydrochloric acid, and the salt that forms with organic acid such as acetic acid.

As used herein, definition " function (property) derivative " refers to can be according to functional group or N-or the C-end group derivative that make of known method from the side chain of amino acid moiety, and (they do not destroy protein active when they pharmaceutically can be accepted, do not make the pharmaceutical composition that contains them have toxicity yet), be included in the present invention. Such derivative for example comprises, the acyl derivative of the formed ester of carboxyl or aliphatic acid amides and free amino group or the O-acyl derivative of free hydroxyl, they can by with carboxyl groups for example the reaction of alcanoyl base or aroyl form.

" precursor " is the compound that can be transformed into LAG-3 in human body or animal body.

" active part " as protein, the present invention refers to any fragment or the precursor of the polypeptide chain of compound own, it can be independent also can mixing mutually with correlation molecule or with the residue (for example saccharide residue or phosphate radical residue) of its combination, perhaps when these fragments or precursor have with as the identical aggregation that can refer to peptide molecule when active of the LAG-3 of medicine.

Preferably " active part " is the soluble parts of the outer part of LAG-3 albumen born of the same parents, and the cytoplasm external structure territory that it comprises LAG-3 is the one or more domains in D1, D2, D3 and four domains of D4.

As used herein, definition " active mutain " refers to other protein or polypeptide, wherein the one or more amino acid in the structure are left out or are replaced by other amino acid, one or more amino acid perhaps in sequence, have been added, in order to obtain to have polypeptide or protein with the identical activity of LAG-3. For example, Arg73 and/or Arg75 and/or Arg76 can be replaced by different amino acid (preferably by Glu).

" the active variation's body " of LAG-3 is the different varient of montage mode and all one-level genetic transcription things, and they are to derive because of different montage mechanism at the different cleavage sites of gene to obtain.Preferred varient is to lack the D3 of LAG-3 born of the same parents' outside part and/or the soluble proteins or the transmembrane protein of D4 structural domain, and it can randomly contain some extra amino acid that are positioned at behind D2 or the D3 structural domain.

Striding film LAG-3 albumen can be confirmed with the immune response method in the expression of cell surface.Transmembrane protein can be discerned by for example anti--LAG-3 antibody 11E3 (preserving number No.CNCMI-1612), 17B4 (preserving number No.CNCMI-1240) or 15A9 (preserving number No.CNCMI-1239).

The invention still further relates to the mixture of a kind of aforesaid polypeptide and derivative.

When being used for this specification sheets and claims, that word " LAG-3 ", " LAG-3 albumen ", " LAG-3 molecule " refer to comprise is natural, this polypeptide of synthetic and recombinant forms, and comprises all above-mentioned definition.

Cell of the present invention can be selected from former generation or inferior to cell.As used herein, the term primary cell comprises: be present in cell from the suspension of the isolated cell of vertebrates tissue source (before they are by plating, promptly before being attached to tissue culture such as ware or bottle), be present in derived from the cell in the explant of tissue, above-mentioned two kinds of cells of being inoculated for the first time with derived from the cell suspending liquid of this inoculating cell.Term time refers to cell in all culturing steps subsequently for cell or cell strain.That is, for the first time with the primary cell of inoculation when culture medium takes off and inoculates (going down to posterity), it just is called as time for cell at this paper, all cells in going down to posterity subsequently is also referred to as time for cell.Inferior for cell be by the one or many that goes down to posterity time for cell strain that cell constituted.Cell strain can by with next time for cellularity: (1) gone down to posterity one or many time for cell; (2) in cultivation, show the inferior of limited average population doublings number of times for cell; (3) show contact inhibition, depend on adherent (" depending on adherent " also is not suitable for the cell of breeding in suspension culture) growth time for cell; (4) negation element limit propagationization is inferior to cell." clone's cell strain " is defined as the cell strain derived from single initiating cell (founder cell)." allos cell strain " is defined as the cell strain derived from two or more initiating cells.

The present invention includes former generation and inferior for somatocyte, the for example formed elements of inoblast, keratinocyte, epithelial cell, endotheliocyte, spongiocyte, neurocyte, blood, muscle cell and other somatocyte that can be cultivated and somatocyte precursor, they are by the foreign DNA transfection, and wherein foreign DNA can stably be integrated in the people's gene group or in the episome mode and express in cell.The cell that forms be hereinafter referred to as the primary cell of transfection and transfection time for cell.

When the gene of coding LAG-3 molecule is inserted into mammalian cell, and when implanted allosome of this cell or xenogenesis host, but their can not discerned by host immune system and can cause immunne response.

Host immune system becomes and can not repel these cells, and if these cells are transformed with at surface expression LAG-3 molecule, they can be ostracised originally.LAG-3 also can express on the cell surface of incoherent cell type, mixes with cell or tissue to be transplanted then, and this also can obtain and above-mentioned similar result.Therefore, the present invention relates under first general immunity suppresses pair cell, tissue or organ transplants.

These cells of portable, tissue or organ are to provide protein or to bring into play some function and treat some disease.By using this technology, be about to LAG-3 and be and pass host immune system, graft is accepted by the host.

Thereby can also transform inoblast or other former generations or inferior of expressing LAG-3 by using jointly, prevent transplant rejection specific cells, tissue or organ for cell.This protectiveness state is attributable to and the mechanism of mediation immunne response has been carried out local inhibition or whole body suppresses.This protectiveness state is attributable to cell-mediated Cytotoxic first allergy, disappearance, first responsiveness, tolerance or preventative.In case set up guard mode, it is sustainable long-time, even because of the permanent maintenance of the phenomenon that for example infects tolerance and so on people such as (, 1993) Qin.

The present invention can be used for transplanted cells, tissue, organ or host cell, so that carry gene or gene product for various medical-therapeutic treatment of human body needs.

Can induce the LAG-3 expression of gene by the recombinant DNA technology of standard or by utilizing homologous recombination to activate the technology of endogenous LAG-3 gene.Cell type can obtain from transgenic animal with any method, and these transgenic animal are expressed the LAG-3 gene in its particular organization or irrelevant cell.These these cells can be mixed mutually with the cell that needs protection to prevent transplant rejection then.This prompting, the merocrine secretion of immune protective molecule L AG-3 does not play a role at whole body.With LAG-3 transduction but not have the inoblast of conversion to produce similarly in vivo to reply.Immunoprotection effect with the inoblast inoculum of LAG-3 transduction depends on dosage, but does not rely on the source of LAG-3 molecule.

Use the cell of expressing LAG-3 jointly, can make donor T cell inactivation, prevent the attack of host immune system simultaneously.But the anergia of this treatment inducing specific, tolerance, or otherwise induce provide protection avoiding cytotoxicity cell-mediated in the acceptor, thereby this can cause the long-term change of immune microenvironment can shield to avoid autoreactive T cell.This can realize in the following way: use from a small amount of allograph bone myelocyte of healthy donors jointly and transformed to express people's variant cell of LAG-3.This causes development by microchimerism (microchimerism) to make autoreactive T cell descend people such as (, 1996) Delaney.

The people who suffers from disease specific or defective can be benefited because of the many different cells of heteroplastic transplantation, tissue or organ.For example, organ such as liver, kidney, the heart, pancreas, small intestine is often to transplant, and cell also is applicable to transplanting, pancreas islet for example, can be used for treating the nervous tissue of Parkinson's disease or focal seizure, as the hemopoietic stem cell of the treatment means of chemotherapy or radiotherapy, the normal liver cell of treatment hypercholesterolemia, the heart cell of treatment myocardial infarction, the muscle cell of treatment muscular dystrophy.

For a variety of reasons, simplified marrow transplanting is difficult to realize always.These reasons comprise: transplant rejection, the infection (pollution by graft or immunosuppressive drug causes) or the other reasons that cause because of opportunistic infection.Repulsion is attributable to: and the tendency (being graft versus host disease) that acceptor has the attack acceptor to the resistance and the competitive immunization cell of donor bone marrow implant (graft-versus-host disease, GVHD).By removing the T cell in the graft, perhaps, can control GVHD by using immunosuppressive drug simultaneously.When the T cell was eliminated, graft just restores (reduced).

As the consequence of eliminating the T cell, the graft failure rate is just higher.It is believed that the T cell may play a significant role to transplanting, for example produce cytokine (people such as Kernan, 1987).

Show, only need a small amount of allosome but healthy medullary cell just can reduce even prevent the generation of autoimmune disease in experimental model.Yet the treatment of carrying out has but caused graft versus host disease like this.

Bone marrow transplantation has been used as the method for effecting a radical cure some tumour.This mainly is because tumor tissues can be discerned and kill to allosome T cell, for example in the graft leukemia.

Under all above-mentioned situations, can avoid general immunity to suppress, if transform cell to be transplanted or organ, thereby can express LAG-3 albumen and initiation provide protection when being implanted among the host to graft with the gene of coding LAG-3.

First problem in heteroplastic transplantation is that lack can be for people's tissue of transplanting, at present for the demand of organ considerably beyond supply.Although still be considered to a kind of experimental arrangement, xenotransplantation is considered to the strong alternative form of heteroplastic transplantation.Animal such as pig or baboon just is being considered as organ or cell donor at present.The provide protection of transplant rejection is avoided in foundation, is vital for the organ that successfully uses different plant species clinically.By for example using the antibody of anti-people CD4, CD8, NK cell, zooblast that perhaps will be to be transplanted carries out microencapsulation to be handled, and can overcome host's resistance to small part.According to the present invention, animal can be changed by transgenosis formula ground, so that express the LAG-3 gene in some cell type, for example islet cells can use insulin gene promotor and guidance system or other tissue-specific Mk systems.

It is believed that, in tumor tissues, express LAG-3, in its antagonism host immune system is attacked the cell-mediated formula of tumor tissues, play an important role.

According to a specific embodiment of the present invention,, can transform with antisence LAG-3 molecule or the ribozyme special tumor tissues to LAG-3 courier by using gene therapy or in vitro handling a small amount of tumor tissues and implantation again.Can make immunity system destroy it like this with tumor tissues reaction and association.Also the antibody of available anti-lag-3 is handled a small amount of tumor tissues, and with the T cyto-inhibition that prevents that LAG-3 from being brought out, thereby initiation is at the cell or the humoral immunization of tumor tissues.

At present, be starved of gene therapy and treat various various disease, comprising (but being not limited to): (α-antitripsin) defective disease, disease of brain such as Alzheimer's disease (Alzheimer ' s disease) and other diseases are as growth disease and heart trouble (changing the disease that is caused such as the cholesterol metabolic approach), and immune system defect for adenosine deaminase defective disease (ADA), Dresbach's anemia, thalassemia, hemophilia, diabetes, alpha antitrypsin.

Different cells can be used for individual the transplanting, for example sarcoplast can be used for muscular dystrophy, the cell of material and hemocyte can be used for treating the heredity hemopathy secretion such as TPO, GH, EPO, factors IX or other factors, and other former generation human or animal cells, for example endotheliocyte, epithelial cell, phoirocyte, inoblast, mesenchymal cell, mesothelial cell and parenchyma (parenchymal cell).

Because many problems, the result of gene therapy is also very not satisfactory.Even state-of-the-art test is treated a young girl with adenosine deaminase (ADA) gene in this test, but because fear only also to be not enough to effectively with gene therapy, patient still accepts the PEG-ADA injection weekly.

At present, one of shortcoming of gene therapy scheme is in order to prevent host immune system repulsion cell, need produce host cell individually.Gene therapy must be that treat on individuality on the basis with the individuality.In addition, even in gene therapy, use the cell of self, often find that still genetically modified expression is of short duration, because other viral proteins of expressing can cause the reaction of host immune system.

Other viral proteins of being expressed can use defective adenoviral vector, but still have problem, because still can cause immunne response.The virus of high density is even defective virus also can stimulate inflammatory response and immune attack.The host cell immunity system can be remembered virus vector, and the effectiveness of administration so in the future is just lower.

Lack the replication-defective adenoviral of viral protein E1, be used for the gene therapy scheme routinely.Unfortunately, they are only effectively temporary transient in that grow up and host immunological competence, and this may be (Kozarsky and Wilson, 1993 that immunne response caused at adenovirus or recombinant protein; People such as Barr, 1992; People such as Stratford, 1992; People such as Rosenfeld, 1992; People such as Lemarchand, 1992).Therefore, press for exploitation immunogenicity so not strong novel vector, the method for cell that perhaps can the genetically engineered mistake of immunoprotection.

These carriers can be made into tires up to 10 ¹¹Plaque forming unit/milliliter, and infect the cell of many duplicate stage and non-duplicate stage.Can use replication-defective adenoviral the recombinant protein of physiological concentration to be flowed to the recycle system of whole body.The LAG-3 gene should be in transcribe (people such as Tripathy, 1994) under the control of ubiquitous reactivity cell EFl α promotor and 4F2HC enhanser.

Attempted avoiding this problem by parcel cell or immunosuppressed host.

Use method described herein, xenogenesis or variant cell can be used as the gene therapy host cell of expressing the LAG-3 molecule from the teeth outwards, thereby cause provide protection in order to avoid host immune system generation transplant rejection.They for example comprise, sarcoplast, inoblast, hemopoietic stem cell, embryonic stem cell, fetal liver cells, huve cell and Chinese hamster ovary celI.Gene therapy host cell also can be transformed to express herpes simplex E.C. 2.7.1.20 gene.Can destroy these cells specifically by adding 9-(1,3-dihydroxy-2-third oxygen methyl) guanine (gancylovir).

Tk-" 9-(1,3-dihydroxy-2-third oxygen methyl) guanine " responsive type cell has the clear superiority that is better than non-sensitive type cell, and promptly they can be at any time destroyed people such as (, 1993) Bi.

Therefore, the general host cell that can on its cell surface, express LAG-3 and Hsv-tk gene that makes according to the present invention, can produce a kind of general gene therapy host cell, this cell can be implanted under no immunosuppressant situation, and can no longer need its active whenever destruction.

By using any gene transfer method, allosome of the present invention or heterogenous gene treatment host cell can be transformed with express transgenic and/or LAG-3 gene.These methods for example have (but being not limited to): replication-defective virus method, adeno associated virus method, efficient retrovirus method, dna direct is injected into marrow, electroporation, calcium phosphate transfection method, microinjection, parcel are gone into liposome or erythrocyte ghost method.Express the cell of LAG-3,, can use with the permanent cell of transplanting for the treatment of of expressing proteins of interest as sarcoplast or Chinese hamster ovary celI.If instantaneous to be exposed to LAG-3 enough, can be contained suicide gene (tk gene as described above) by the sarcoplast of LAG-3 transfection or Chinese hamster ovary celI so, thus can be by handling and remove with 9-(1,3-dihydroxy-2-third oxygen methyl) guanine.

This method can be used for replying normal function, promptly by using gene or gene product, or by removing or the insalubrious gene of deactivation (as oncogene).This purpose can realize like this: implant the cell of carrying ribozyme or Antisense cDNA, thereby suppress the generation of unfavorable protein (as HIV albumen or somatomedin).This method can be used for correcting enzyme defect, as in GaucherShi disease or ADA defective disease.

According to another embodiment, the present invention relates to a kind of gene therapy method, it comprises: the gene that (i) inserts required gene of treatment and coding LAG-3 molecule in selected human or animal's cell; (ii) the cell that obtains in the step (i) is introduced among the patient.Perhaps, said gene can be inserted in patient's the histoorgan, and this can carry out in vivo, or carries out by the direct transfection of gene or by the directed carrier at this tissue or organ.

For example, described expression system can be injected directly into naked DNA or virus vector in the cell mass (as muscle cell), perhaps directly is applied in the airway (airway) and (for example treats Cysticfibrosis).Construction not only contains gene of interest and (for example conducts regulon (conductance regulator, CFTR) cDNA), also contain the LAG-3 gene, so that coexpression on this cell type, thereby prevent contingent at the proteic humoral immunoresponse(HI) of for example adenovirus capsid (effectiveness in the time of can limiting repeat administration at the proteic immunne response of adenovirus capsid).Change gene over to hemopoietic stem cell and can be used for carrying multiple drug resistance gene, struggle against mutually so that suppress the side effect of division immunocyte fast during with chemotherapy.Retroviral vector can use to promote stem cell division with combination of cytokines, and these factors have for example steel factor (steel factor), kit part, IL3, GM-CSF, IL-6, G-CSF, LIF, IL12 etc.

Except the LAG-3 gene, the selected cell also gene of other immunosuppressive substances of available code such as IL10, TGF β, Fas part etc. carries out cotransfection.

As an object lesson of the present invention, aforesaid method can be used for treating acceptor, promptly with a small amount of interested and transformed with the cell of expression LAG-3 gene and treat acceptor, this can make the host can tolerate cell, tissue or the organ that next time gives because of the infection tolerance of host immune system.

Only in the elaboration mode, the present invention is described below in conjunction with the following example.

Embodiment 1

Method

Produce and express the Chinese hamster ovary celI of striding film LAG-3

From the pCDM8 plasmid (Invitrogen, San Diego, CA) in, LAG-3 cDNA is downcut with the 1620bp pieces, and carries out purifying with agarose gel electrophoresis.

This fragment subclone is gone into pCLH3AXSV2DHFRh α IVSA (D α) mammalian expression vector (Fig. 3) that digests with Xho.By the calcium phosphate precipitation method, with D α LAG-3 construction transfection CHO-DUKX (DHFR ^-) cell.Cell after the transfection is selecting substratum (the MEM substratum does not contain thymus nucleic acid and Yeast Nucleic Acid, and is added with 10% foetal calf serum of dialysing and 1%L-glutamine+0.02 μ M methotrexate) to go up growth.The expression of LAG-3 is by the Western blotting cracked cell membrane preparation to be checked, and regularly with the monoclonal antibody 17B4 of anti--LAG-3, checks by flow cytometry.

Chinese hamster ovary celI is implanted into mouse

With (wild-type) of untransfected, or, separate from Plastic Bottle with the people LAG-3 of total length or Chinese hamster ovary (CHO) cell of people LHcDNA transfection, and with 1.75 * 10 ⁷The concentration of cells/ml is suspended in the improved minimum essential medium of Dulbecco (DMEM).With 26 mouse is that the C57BL/6 female mice such as the Table I in 7-9 week is divided into 7 groups age, then the cell suspending liquid shown in 200 milliliters (is contained 3.5 * 10 ⁶Cell) subcutaneous injection is in the right side of every animal.In the 3rd, 6 and 7 group, same mouse is accepted the cell of LAG-3 transfection on the right side, accepts control cells (cell of LH-transfection or non-transfected cells) at opposite side.In injection back 4 days, kill mouse with the carbonic acid gas inhalation, cut the skin examination injection site then open.

Table 1-experimental group

Group	The mouse number	The mouse identification number	Inject the Chinese hamster ovary celI on right side	Inject the Chinese hamster ovary celI in left side
Group	The mouse number	The mouse identification number	Inject the Chinese hamster ovary celI on right side	Inject the Chinese hamster ovary celI in left side	1	5	1-5	Wild-type	-
2	5	6-10	LAG-3	-	1	5	1-5	Wild-type	-
2	5	6-10	LAG-3	-	3	5	11-15	LAG-3	Wild-type
4	2	16-17	LH	-	3	5	11-15	LAG-3	Wild-type
4	2	16-17	LH	-	5	5	18-22	LAG-3	-
6	2	23-24	LAG-3	Wild-type	5	5	18-22	LAG-3	-
6	2	23-24	LAG-3	Wild-type	7	2	25-26	LAG-3	LH

Assessment is to the cytotoxicity of Chinese hamster ovary celI

To every group of 5 C57BL/6 female mice subcutaneous injection 4 * 10 ⁵The Chinese hamster ovary celI of individual personnel selection LH or people LAG-3 cDNA transfection.After 14 days, kill mouse, take out spleen, make splenocyte suspension.With nutrient solution (RPMI 1640+10% foetal calf serum+microbiotic) with 10 ⁷The splenocyte suspension (effector) of cells/ml dilutes, and inoculates 3 parts of the same form then under different extent of dilution, to obtain different effector (effector): target cell ratio.For every kind of suspension, prepare 2 blocks of plates.With 5 * 10 ³Cell/100 microlitres are used ⁵¹The target cell that the Cr mark is crossed (by the cell of LH or LAG-3 transfection) adds to (every kind of target cell one flat plate) in the plate.At 37 ℃ after 20 hours, from each hole, take out 20 microlitre supernatant liquors, assess by liquid sudden strain of a muscle method ⁵¹The release conditions of Cr.Calculate cytotoxicity according to following formula, represent with cracked per-cent:

Wherein, " spontaneous " and " maximum " represents nutrient solution hole (the spontaneous release Cr of target cell) and the hole (maximum Cr burst size) of replacing effector suspension with Triton X1% respectively.

The result

Chinese hamster ovary celI is implanted into mouse

Great majority are accepted the mouse of LAG-3 transfection type cell, in injection site adularescent brief summary (nodule), and handle or brief summary does not appear in the animal of accepting LH transfection type Chinese hamster ovary celI with wild-type CHO.In most of mouse, injection wild-type Chinese hamster ovary celI causes diffuse hemorrhage occurring in the injection site.This phenomenon is more not obvious in the animal of accepting LH transfection type Chinese hamster ovary celI.Inject simultaneously in the animal of wild-type and LAG-3 transfection type Chinese hamster ovary celI at different sites, this brief summary only occurs at the position of injection LAG-3 transfection type Chinese hamster ovary celI, and hemorrhage (table 2) occur at the position of injection wild-type.

The previous histologic analysis that carries out in similar experiment shows, has the allos cell in this brief summary.This experimental result is shown in accompanying drawing (identification number for mouse can see Table 1) and is summarized in table 3.

Table 2

The cell of injection	The mouse number	The hemorrhage brief summary of frequency (sum of positive number/injection side)
The cell of injection	The mouse number			LAG-3	10	1/10	9/10
Wild-type	5	3/5	0/5	LAG-3	10	1/10	9/10
Wild-type	5	3/5	0/5	LH	2	1/2	1/2
The LAG-3+ wild-type ^*	7	0/7(a)5/7(c)	7/7(a)1/7(c)	LH	2	1/2	1/2
The LAG-3+ wild-type ^*	7	0/7(a)5/7(c)	7/7(a)1/7(c)	LAG-3+LH ^*	2	0/2(b)1/2(a)	0/2(b)2/2(a)

^*Every kind of cell type is injected in a side separately

(a)=LAG-3 one side

(b)=LH one side

(c)=wild-type one side

Table 3

The mouse of injection	The hemorrhage brief summary of frequency (sum of positive number/injection side)
The mouse of injection			The CHO wild-type	8/12(67％)	0/12(0％)
CHO LH	1/4(25％)	1/4(25％)	The CHO wild-type	8/12(67％)	0/12(0％)
CHO LH	1/4(25％)	1/4(25％)	CHO LAG-3	2/19(11％)	16/19(84％)

Cytotoxicity to Chinese hamster ovary celI

On the Chinese hamster ovary celI surface, express LAG-3, do not influence mouse carries out immune sensitization to heterogenous cell ability.In fact, can make two kinds of target cell cracking from the mouse boosting cell of injection LAG-3-CHO cell, and with same effective and be better than unsensitized mouse with the mouse of LH-CHO cell sensitization (primed).Yet, on cell surface, express LAG-3, be accompanied by pair cell toxicity (the immunity institute to target cell is induced by mouse) decrease in sensitivity, this can be from Fig. 1 and Fig. 2 relatively the finding out of cracking per-cent." natural " cytotoxicity by non-sensitized mice splenocyte is produced does not descend because of surface expression LAG-3.This shows that the surface expression of LAG-3 has reduced the active output of cytotoxic T cell (CTL) (efferent branch).CTL is a kind of in the effector, and it plays a major role in the rejection of transplant organ (G.Berke, 1993), and the function that therefore suppresses them can prolong the survival time of allogeneic.

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Claims

1. the mammalian cell of a genetically engineered mistake; it is characterized in that; what it contained that coding is positioned at this cell surface strides film lymphocyte activation gene 3 proteic DNA; thereby play the provide protection that prevents host immune system generation transplant rejection; described DNA stably is incorporated in the genome of described mammalian cell, and described cell is not an embryonic stem cell.

2. cell as claimed in claim 1 is characterized in that, this encoding lymphocyte activating gene 3 proteic DNA are external sources.

3. cell as claimed in claim 1 is characterized in that, this encoding lymphocyte activating gene 3 proteic DNA are endogenous, thereby and by homologous recombination directed insertion regulating and controlling sequence and/or amplifiable gene, the expression that activates or modify it.

4. cell as claimed in claim 1 is characterized in that, also contains extra immunosuppression thing, and described immunosuppression thing is selected from IL-10, TGF β or Fas part.

5. as claim 1 or 4 described cells, it is characterized in that, also contain the E.C. 2.7.1.20 gene, this gene pairs E.C. 2.7.1.20-9-(1,3-dihydroxy-2-third oxygen methyl) guanine suicide system sensitivity.

6. cell as claimed in claim 1 is characterized in that this is cell-derived from transgenic animal.

7. cell as claimed in claim 1 is characterized in that it is selected from: sarcoplast, inoblast, hemopoietic stem cell, fetal liver cells, huve cell and Chinese hamster ovary celI.

8. that expresses on cell surface strides film lymphocyte activation gene 3 proteic purposes, it is characterized in that it is used to make medicine, and this medicine can cause the provide protection that prevents host immune system generation transplant rejection.

9. the described cell of claim 1 is used to produce the purposes of tissue or organ to be transplanted.

10. the described cell of claim 1 is used to produce the purposes of gene therapy host cell.

11. as claim 9 or 10 described purposes, it is characterized in that described cell also comprises foreign DNA, this foreign DNA therapeutical agent interested of encoding.

12. as claim 9 or 10 described purposes, it is characterized in that described cell also comprises foreign DNA, this dna encoding regulating and controlling sequence and/or amplifiable gene, the expression that activates or modify interested native gene.

13. purposes as claimed in claim 10 is characterized in that, this gene therapy is somatic gene therapy, or " in vitro " gene therapy.

14. the purposes as the described cell of arbitrary claim among the claim 1-7 is characterized in that be used to make medicine, this medicine can cause the provide protection that prevents host immune system generation transplant rejection.

15. purposes as the described cell of arbitrary claim among the claim 1-7; it is characterized in that; be used to make medicine, this medicine mixes mutually with cell, tissue or organ to be transplanted, thereby causes the provide protection that prevents host immune system generation transplant rejection.